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Techniques and 
Instrumentation for Structure 
Determination 



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2 



Contents 

Articles 

I.C. Baianu, Ph.D.,M.Inst.P., Editor (with listed contributors) 
Principles 

Crystal 2 

Diffraction 5 

Uniform theory of diffraction 16 

Crystallography 17 

Paracrystalline 22 

Quantum optics 24 

X-rays 26 

Electron 42 

Neutron 65 

Muon 75 

X-ray diffraction 80 

Electron diffraction 82 

Neutron diffraction 86 

Neutron scattering 90 

Inelastic neutron scattering 9 1 

Ionization cooling 93 

Deep inelastic scattering 94 

Timeline of microphysics 95 

Automatic calculation of particle interaction or decay 100 

S -matrix 105 

List of materials analysis methods 108 

List of neutrino experiments 115 

Instruments 119 

Optical microscope 119 

Confocal microscope 130 

Atomic force microscope 133 

Electron microscope 140 

Synchrotron 148 

X-ray microscope 154 



Field emission microscope 156 

Scanning tunneling microscope 157 

Transmission Electron Aberration-corrected Microscope 164 

ISIS 166 

ISIS neutron source 169 

Sudbury Neutrino Observatory 171 

ATLAS experiment 176 

Techniques 185 

Microscopy 185 

X-ray crystallography 200 

X-ray scattering techniques 225 

Fourier transform spectroscopy 227 

Hyperspectral imaging 23 1 

2D-FT NMRI and Spectroscopy 235 

NMR microscopy 241 

Chemical imaging 242 

Fluorescence microscopy 249 

Fluorescence correlation spectroscopy 254 

Fluorescence cross-correlation spectroscopy 263 

Circular dichroism 264 

Vibrational spectroscopy 27 1 

Vibrational circular dichroism 275 

Raman spectroscopy 285 

Microscope image processing 291 

Electron microscopy 293 

Diagnostic electron microscopy 301 

HiRISE 302 

Scanning confocal electron microscopy 307 

Acronyms in microscopy 309 

Nanoscience 316 

Nanotechnology 329 

Surface science 342 

Electron tomography 345 

Virtopsy 346 

Xenon-enhanced CT scanning 347 

X-ray microtomography 347 

Positron emission tomography 35 1 



Raman microscopy 360 

Neutron spin echo 367 

References 

Article Sources and Contributors 370 

Image Sources, Licenses and Contributors 378 

Article Licenses 

License 383 



LC. Baianu, Ph.D.JVLInst.P., Editor (with 

listed contributors) 



Principles 



Crystal 



A crystal or crystalline solid is a solid material whose constituent 
atoms, molecules, or ions are arranged in an orderly repeating pattern 
extending in all three spatial dimensions. The scientific study of 
crystals and crystal formation is known as crystallography. The process 
of crystal formation via mechanisms of crystal growth is called 
crystallization or solidification. The word crystal is derived from the 
Ancient Greek word KpiiotaWoi; (krustallos), meaning "rock-crystal" 
but also "ice", from Kpiioq (kruos), "icy cold, frost". The word 

once referred particularly to quartz, or "rock crystal". 

Most metals encountered in everyday life are poly crystals. Crystals are 
often symmetrically intergrown to form crystal twins. 




Quartz crystal. The individual grains of this 
polycrystalline mineral sample are clearly visible. 



Crystal structure 

The process of forming a crystalline structure from a fluid or from 
materials dissolved in the fluid is often referred to as the 
crystallization process. In the old example referenced by the root 
meaning of the word crystal, water being cooled undergoes a phase 
change from liquid to solid beginning with small ice crystals that grow 
until they fuse, forming a polycrystalline structure. The physical 
properties of the ice depend on the size and arrangement of the 
individual crystals, or grains, and the same may be said of metals 
solidifying from a molten state. 

Which crystal structure the fluid will form depends on the chemistry of 
the fluid, the conditions under which it is being solidified, and also on 
the ambient pressure. While the cooling process usually results in the 
generation of a crystalline material, under certain conditions, the fluid 
may be frozen in a noncrystalline state. In most cases, this involves 
cooling the fluid so rapidly that atoms cannot travel to their lattice sites 
before they lose mobility. A noncrystalline material, which has no 
long-range order, is called an amorphous, vitreous, or glassy material. 
It is also often referred to as an amorphous solid, although there are 
distinct differences between crystalline solids and amorphous solids: 
most notably, the process of forming a glass does not release the latent 
heat of fusion. 




Insulin crystals grown in outer space 




Halite (sodium chloride) - a single, large crystal. 



Crystalline structures occur in all classes of materials, with all types of 

chemical bonds. Almost all metal exists in a polycrystalline state; amorphous or single-crystal metals must be 



Crystal 



produced synthetically, often with great difficulty. Ionically bonded crystals can form upon solidification of salts, 
either from a molten fluid or upon crystallization from a solution. Covalently bonded crystals are also very common, 
notable examples being diamond, silica, and graphite. Polymer materials generally will form crystalline regions, but 
the lengths of the molecules usually prevent complete crystallization. Weak van der Waals forces can also play a role 
in a crystal structure; for example, this type of bonding loosely holds together the hexagonal-patterned sheets in 
graphite. 

Most crystalline materials have a variety of crystallographic defects. The types and structures of these defects can 
contain a profound effect on the properties of the materials. 

Crystalline phases 

• Polymorphism is the ability of a solid to exist in more than one crystal form. For example, water ice is ordinarily 
found in the hexagonal form Ice I , but can also exist as the cubic Ice I , the rhombohedral ice II, and many other 
forms. 

• Amorphous phases are also possible with the same molecule, such as amorphous ice. In this case, the 
phenomenon is known as polyamorphism. 

• For pure chemical elements, polymorphism is known as allotropy. For example, diamond, graphite, and fullerenes 
are different allotropes of carbon. 




A large monocrystal of potassium dihydrogen 

phosphate grown from solution by Saint-Gobain 

for the megajoule laser of CEA. 



Special cases 

Since the initial discovery of crystal-like individual arrays of atoms 
that are not regularly repeated, made in 1982 by Dan Shechtman, the 
acceptance of the concept and the word quasicrystal have led the 
International Union of Crystallography to redefine the term crystal to 
mean "any solid having an essentially discrete diffraction diagram", 
thereby shifting the essential attribute of crystallinity from position 
space to Fourier space. Within the family of crystals one distinguishes 
between traditional crystals, which are periodic, or repeating, at the 
atomic scale, and aperiodic (incommensurate) crystals which are not. 
This broader definition adopted in 1996 reflects the current 
understanding that microscopic periodicity is a sufficient but not a 
necessary condition for crystals. 

While the term "crystal" has a precise meaning within materials 
science and solid-state physics, colloquially "crystal" refers to solid 
objects that exhibit well-defined and often pleasing geometric shapes. 
In this sense of the word, many types of crystals are found in nature. 
The shape of these crystals is dependent on the types of molecular 
bonds between the atoms to determine the structure, as well as on the 
conditions under which they formed. Snowflakes, diamonds, and table 
salt are common examples of crystals. 

Some crystalline materials may exhibit special electrical properties 

such as the ferroelectric effect or the piezoelectric effect. Additionally, 

light passing through a crystal is often refracted or bent in different 

directions, producing an array of colors; crystal optics is the study of 

these effects. In periodic dielectric structures a range of unique optical properties can be expected as seen in photonic 

crystals. 




Gallium, a metal that easily forms large single 
crystals 



Crystal 



Crystalline rocks 

Inorganic matter, if free to take that physical state in which it is most 
stable, tends to crystallize. There is no practical limit to the size a 
crystal may attain under the right conditions, and selenite single 
crystals in excess of 10 m are found in the Cave of the Crystals in 



Naica, Mexico 



[4] 



Crystalline rock masses have consolidated from aqueous solution or 
from molten magma. The vast majority of igneous rocks belong to this 
group and the degree of crystallization depends primarily on the 
conditions under which they solidified. Such rocks as granite, which 
have cooled very slowly and under great pressures, have completely 
crystallized, but many lavas were poured out at the surface and cooled 
very rapidly; in this latter group a small amount of amorphous or 
glassy matter is frequent. Other crystalline rocks, the evaporites such 
as rock salt, gypsum and some limestones have been deposited from 
aqueous solution, mostly owing to evaporation in arid climates. Still 
another group, the metamorphic rocks which includes the marbles, 
mica-schists and quartzites; are recrystallized, that is to say, they were 
at first fragmental rocks, like limestone, shale and sandstone and have 
never been in a molten condition nor entirely in solution. The high 
temperature and pressure conditions of metamorphism have acted on 
them erasing their original structures, and inducing recrystallization in the solid state 




Ice crystals 




Fossil shell with calcite crystals 



[] 



Properties 



Crystal 


Particles 


Attractive forces 


Melting 
point 


Other properties 


Ionic 


Positive and negative 
ions 


Electrostatic attractions 


High 


Hard, brittle, good electrical conductor in molten state 


Molecular 


Polar molecules 


London force and dipole-dipole 
attraction 


Low 


Soft, non-conductor or extremely poor conductor of 
electricity in liquid state 


Molecular 


Non-polar molecules 


London force 


Low 


Soft conductor 



See also 

• Atomic packing factor 

• Colloidal crystal 

• Crystal growth 

• Crystal habit 

• Crystal oscillator 

• Crystal system 

• Crystallite 

• Crystallographic database 

• Liquid crystal 

• Quasicrystal 



Crystal 



References 

[1] KpijaxaAAoi; (http://www. perseus. tufts. edu/hopper/text?doc=Perseus:text: 1999. 04. 0057:entry=kru/stallos), Henry George Liddell, 

Robert Scott, A Greek-English Lexicon, on Perseus Digital Library 
[2] Kpuoc; (http://www. perseus. tufts. edu/hopper/text?doc=Perseus:text: 1999. 04. 0057:entry=kru/os), Henry George Liddell, Robert Scott, A 

Greek-English Lexicon, on Perseus Digital Library 
[3] "kreus-" (http://www.bartleby.com/61/roots/IE243.html). The American Heritage Dictionary of the English Language: Fourth Edition: 

Appendix I: Indo-European Roots. 2000. . 
[4] National Geographic, 2008. Cavern of Crystal Giants (http://ngm.nationalgeographic.eom/2008/l 1/crystal-giants/shea-text) 

Further reading 

• Howard, J. Michael; Darcy Howard (Illustrator) (1998). "Introduction to Crystallography and Mineral Crystal 
Systems" (http://www.rockhounds.com/rockshop/xtal/index.html). Bob's Rock Shop. Retrieved 2008-04-20. 

• Krassmann, Thomas (2005—2008). "The Giant Crystal Project" (http://giantcrystals.strahlen.org). Krassmann. 
Retrieved 2008-04-20. 

• Various authors (2007). "Teaching Pamphlets" (http://www.iucr.ac.uk/iucr-top/comm/cteach/pamphlets. 
html). Commission on Crystallographic Teaching. Retrieved 2008-04-20. 

• Various authors (2004). "Crystal Lattice StructuresTndex by Space Group" (http://cst-www.nrl.navy.mil/ 
lattice/spegrp/). U.S. Naval Research Laboratory, Center for Computational Materials Science. Retrieved 
2008-04-20. 

• Various authors (2010). "Crystallography" (http://www.xtal.iqfr.csic.es/Cristalografia/index-en.html). 
Spanish National Research Council, Department of Crystallography. Retrieved 2010-01-08. 



Diffraction 



Diffraction refers to various phenomena which occur when a wave 
encounters an obstacle. In classical physics, it is described as the 
apparent bending of waves around small obstacles and the spreading 
out of waves past small openings. Similar effects occur when light 
waves travel through a medium with a varying refractive index or a 
sound wave through one with varying acoustic impedance. Diffraction 
occurs with all waves, including sound waves, water waves, and 
electromagnetic waves such as visible light, x-rays and radio waves. 
As physical objects have wave-like properties (at the atomic level), 
diffraction also occurs with matter and can be studied according to the 
principles of quantum mechanics. 




The intensity pattern formed on a screen by 
diffraction from a square aperture 



While diffraction occurs whenever propagating waves encounter such 

changes, its effects are generally most pronounced for waves where the 

wavelength is on the order of the size of the diffracting objects. If the 

obstructing object provides multiple, closely-spaced openings, a complex pattern of varying intensity can result. This 

is due to the superposition, or interference, of different parts of a wave that traveled to the observer by different paths 

(see diffraction grating). 

The formalism of diffraction can also describe the way in which waves of finite extent propagate in free space. For 
example, the expanding 



Diffraction 



profile of a laser beam, the beam shape of a radar antenna and the field 
of view of an ultrasonic transducer are all explained by diffraction 
theory. 




Generation of an interference pattern from 
two-slit diffraction 




Colors seen in a spider web are partially due to 
diffraction, according to some analyses. 



Examples 

The effects of diffraction are regularly seen in everyday life. The most 
colorful examples of diffraction are those involving light; for example, 
the closely spaced tracks on a CD or DVD act as a diffraction grating 
to form the familiar rainbow pattern seen when looking at a disk. This 
principle can be extended to engineer a grating with a structure such 
that it will produce any diffraction pattern desired; the hologram on a 
credit card is an example. Diffraction in the atmosphere by small 
particles can cause a bright ring to be visible around a bright light 
source like the sun or the moon. A shadow of a solid object, using light 
from a compact source, shows small fringes near its edges. The speckle 
pattern which is observed when laser light falls on an optically rough 
surface is also a diffraction phenomenon. All these effects are a 
consequence of the fact that light propagates as a wave. 




Solar glory at the steam from hot springs. A glory 
is an optical phenomenon produced by light 
backscattered (a combination of diffraction, 

reflection and refraction) towards its source by a 
cloud of uniformly-sized water droplets. 



Diffraction can occur with any kind of wave. Ocean waves diffract 

around jetties and other obstacles. Sound waves can diffract around 

objects, which is why one can still hear someone calling even when hiding behind a tree. Diffraction can also be a 

concern in some technical applications; it sets a fundamental limit to the resolution of a camera, telescope, or 

microscope. 



Diffraction 



History 

The effects of diffraction of light were first carefully observed and 
characterized by Francesco Maria Grimaldi, who also coined the term 
diffraction, from the Latin diffringere, 'to break into pieces', referring 
to light breaking up into different directions. The results of Grimaldi' s 
observations were published posthumously in 1665. Isaac 

Newton studied these effects and attributed them to inflexion of light 
rays. James Gregory (1638—1675) observed the diffraction patterns 
caused by a bird feather, which was effectively the first diffraction 
grating to be discovered. Thomas Young performed a celebrated 



, >!,' V 




Thomas Young's sketch of two-slit diffraction, 
which he presented to the Royal Society in 1803 



[7]- 



experiment in 1803 demonstrating interference from two closely spaced slits. Explaining his results by interference 
of the waves emanating from the two different slits, he deduced that light must propagate as waves. Augustin-Jean 
Fresnel did more definitive studies and calculations of diffraction, made public in 1815 and 1818, and thereby 



gave great support to the wave theory of light that had been advanced by Christiaan Huygens 
by Young, against Newton's particle theory. 



[10] 



and reinvigorated 



The mechanism of diffraction 




Photograph of single-slit diffraction in a circular 
ripple tank 



Diffraction arises because of the way in which waves propagate; this is 
described by the Huygens— Fresnel principle. The propagation of a 
wave can be visualized by considering every point on a wavefront as a 
point source for a secondary radial wave. The subsequent propagation 
and addition of all these radial waves form the new wavefront. When 
waves are added together, their sum is determined by the relative 
phases as well as the amplitudes of the individual waves, an effect 
which is often known as wave interference. The summed amplitude of 
the waves can have any value between zero and the sum of the 
individual amplitudes. Hence, diffraction patterns usually have a series 
of maxima and minima. 



The form of a diffraction pattern can be determined from the sum of the phases and amplitudes of the Huygens 
wavelets at each point in space. There are various analytical models which can be used to do this including the 
Fraunhofer diffraction equation for the far field and the Fresnel diffraction equation for the near field. Most 
configurations cannot be solved analytically, but can yield numerical solutions through finite element and boundary 
element methods. 



Diffraction systems 

It is possible to obtain a qualitative understanding of many diffraction phenomena by considering how the relative 
phases of the individual secondary wave sources vary, and in particular, the conditions in which the phase difference 
equals half a cycle in which case waves will cancel one another out. 

The simplest descriptions of diffraction are those in which the situation can be reduced to a two-dimensional 
problem. For water waves, this is already the case, water waves propagate only on the surface of the water. For light, 
we can often neglect one direction if the diffracting object extends in that direction over a distance far greater than 
the wavelength. In the case of light shining through small circular holes we will have to take into account the full 
three dimensional nature of the problem. 

Some of the simpler cases of diffraction are considered below. 



Diffraction 



Single-slit diffraction 

A long slit of infinitesimal width which is illuminated by light diffracts 
the light into a series of circular waves and the wavefront which 
emerges from the slit is a cylindrical wave of uniform intensity. 

A slit which is wider than a wavelength has a large number of point 
sources spaced evenly across the width of the slit. The light at a given 
angle is made up of contributions from each of these point sources and 
if the relative phases of these contributions vary by 2jt or more, we 
expect to find minima and maxima in the diffracted light. 




Numerical approximation of diffraction pattern 

from a slit of width four wavelengths with an 

incident plane wave. The main central beam, 

nulls, and phase reversals are apparent. 



Singk-siit diffraction pattern 




Graph and image of single-slit diffraction 



We can find the angle at which a first minimum is obtained in the diffracted light by the following reasoning. The 
light from a source located at the top edge of the slit interferes destructively with a source located at the middle of 
the slit, when the path difference between them is equal to XI2. Similarly, the source just below the top of the slit will 
interfere destructively with the source located just below the middle of the slit at the same angle. We can continue 
this reasoning along the entire height of the slit to conclude that the condition for destructive interference for the 
entire slit is the same as the condition for destructive interference between two narrow slits a distance apart that is 

half the width of the slit. The path difference is given by i—Lso that the minimum intensity occurs at an angle 

2 

6 . given by 

mm 

d sin# m i n = A 
where d is the width of the slit. 

A similar argument can be used to show that if we imagine the slit to be divided into four, six, eight parts, etc., 
minima are obtained at angles 8 given by 

n 

d sin 9 n = nX 
where n is an integer other than zero. 

There is no such simple argument to enable us to find the maxima of the diffraction pattern. The intensity profile can 
be calculated using the Fraunhofer diffraction integral as 

1(6) = I Q sine 2 (d sin 6/ A) 



Diffraction 



where the sine function is given by sinc(x) = sin(jtx)/(jtA:) if x *■ 0, and sinc(0) = 1. 

This analysis applies only to the far field, that is, at a distance much larger than the width of the slit. 



Diffraction grating 

A diffraction grating is an optical component with a regular pattern. 
The form of the light diffracted by a grating depends on the structure of 
the elements and the number of elements present, but all gratings have 
intensity maxima at angles 6 which are given by the grating equation 






2-slit (top) and 5-slit diffraction of red laser light 




Diffraction of a red laser using a diffraction 
grating 




A diffraction pattern of a 633 nm laser through a 
grid of 150 slits 



d (sin 9 m + sin #,) = m\. 
where 8. is the angle at which the light is incident, d is the separation of grating elements and m is an integer which 
can be positive or negative. 

The light diffracted by a grating is found by summing the light diffracted from each of the elements, and is 
essentially a convolution of diffraction and interference patterns. 

The figure shows the light diffracted by 2-element and 5-element gratings where the grating spacings are the same; it 
can be seen that the maxima are in the same position, but the detailed structures of the intensities are different. 



Diffraction 



10 



Diffraction by a circular aperture 

The far-field diffraction of a plane wave incident on a circular aperture 
is often referred to as the Airy Disk. The variation in intensity with 
angle is given by 




A computer-generated image of an Airy disk 




Computer generated light diffraction pattern from 

a circular aperture of diameter 0.5 micrometre at 

a wavelength of 0.6 micrometre (red-light) at 

distances of 0. 1 cm — 1 cm in steps of 0. 1 cm. 

One can see the image moving from the Fresnel 

region into the Fraunhofer region where the Airy 

pattern is seen. 



1(8) = I 



2Ji(kasm9) 



ka sin 6 J 

where a is the radius of the circular aperture, k is equal to 2jtA and J is a Bessel function. The smaller the aperture, 
the larger the spot size at a given distance, and the greater the divergence of the diffracted beams. 



Propagation of a laser beam 

The way in which the profile of a laser beam changes as it propagates is determined by diffraction. The output mirror 
of the laser is an aperture, and the subsequent beam shape is determined by that aperture. Hence, the smaller the 
output beam, the quicker it diverges. Diode lasers have much greater divergence than He— Ne lasers for this reason. 

Paradoxically, it is possible to reduce the divergence of a laser beam by first expanding it with one convex lens, and 
then collimating it with a second convex lens whose focal point is coincident with that of the first lens. The resulting 
beam has a larger aperture, and hence a lower divergence. 



Diffraction 



11 



Diffraction-limited imaging 

The ability of an imaging system to resolve detail is ultimately limited 
by diffraction. This is because a plane wave incident on a circular lens 
or mirror is diffracted as described above. The light is not focused to a 
point but forms an Airy disk having a central spot in the focal plane 
with radius to first null of 



The Airy disk around each of the stars from the 

2.56 m telescope aperture can be seen in this 

lucky image of the binary star zeta Bootis. 



d = 1.22XN, 

where X is the wavelength of the light and N is the f-number (focal length divided by diameter) of the imaging optics. 
In object space, the corresponding angular resolution is 

smO = 1.22 — , 

where D is the diameter of the entrance pupil of the imaging lens (e.g., of a telescope's main mirror). 

Two point sources will each produce an Airy pattern — see the photo of a binary star. As the point sources move 
closer together, the patterns will start to overlap, and ultimately they will merge to form a single pattern, in which 
case the two point sources cannot be resolved in the image. The Rayleigh criterion specifies that two point sources 
can be considered to be resolvable if the separation of the two images is at least the radius of the Airy disk, i.e. if the 
first minimum of one coincides with the maximum of the other. 

Thus, the larger the aperture of the lens, and the smaller the wavelength, the finer the resolution of an imaging 
system. This is why telescopes have very large lenses or mirrors, and why optical microscopes are limited in the 
detail which they can see. 



Diffraction 



12 



Speckle patterns 

The speckle pattern which is seen when using a laser pointer is another diffraction phenomenon. It is a result of the 
superpostion of many waves with different phases, which are produced when a laser beam illuminates a rough 
surface. They add together to give a resultant wave whose amplitude, and therefore intensity varies randomly. 



Common features of diffraction patterns 

Several qualitative observations can be made of diffraction in general: 

• The angular spacing of the features in the diffraction pattern is 
inversely proportional to the dimensions of the object causing the 
diffraction. In other words: The smaller the diffracting object, the 
'wider' the resulting diffraction pattern, and vice versa. (More 
precisely, this is true of the sines of the angles.) 

• The diffraction angles are invariant under scaling; that is, they 
depend only on the ratio of the wavelength to the size of the 
diffracting object. 

• When the diffracting object has a periodic structure, for example in 
a diffraction grating, the features generally become sharper. The 
third figure, for example, shows a comparison of a double-slit 
pattern with a pattern formed by five slits, both sets of slits having 
the same spacing, between the center of one slit and the next. 

Particle diffraction 

Quantum theory tells us that every particle exhibits wave properties. In 
particular, massive particles can interfere and therefore diffract. 
Diffraction of electrons and neutrons stood as one of the powerful 
arguments in favor of quantum mechanics. The wavelength associated 
with a particle is the de Broglie wavelength 

x = h - 

P 
where h is Planck's constant and p is the momentum of the particle (mass x velocity for slow-moving particles). For 

most macroscopic objects, this wavelength is so short that it is not meaningful to assign a wavelength to them. A 

sodium atom traveling at about 30,000 m/s would have a De Broglie wavelength of about 50 pico meters. 

Because the wavelength for even the smallest of macroscopic objects is extremely small, diffraction of matter waves 
is only visible for small particles, like electrons, neutrons, atoms and small molecules. The short wavelength of these 
matter waves makes them ideally suited to study the atomic crystal structure of solids and large molecules like 
proteins. 

Relatively larger molecules like buckyballs were also shown to diffract. 




The upper half of this image shows a diffraction 

pattern of He-Ne laser beam on an elliptic 

aperture. The lower half is its 2D Fourier 

transform approximately reconstructing the shape 

of the aperture. 



Diffraction 



13 



Bragg diffraction 

Diffraction from a three dimensional periodic structure such as atoms 
in a crystal is called Bragg diffraction. It is similar to what occurs 
when waves are scattered from a diffraction grating. Bragg diffraction 
is a consequence of interference between waves reflecting from 
different crystal planes. The condition of constructive interference is 
given by Bragg' s law: 




Following Bragg's law, each dot (or reflection), in 

this diffraction pattern forms from the 

constructive interference of X-rays passing 

through a crystal. The data can be used to 

determine the crystal's atomic structure. 



m\ = 2dsm9 

where 

X is the wavelength, 

d is the distance between crystal planes, 

6 is the angle of the diffracted wave. 

and m is an integer known as the order of the diffracted beam. 

Bragg diffraction may be carried out using either light of very short wavelength like x-rays or matter waves like 
neutrons (and electrons) whose wavelength is on the order of (or much smaller than) the atomic spacing . The 
pattern produced gives information of the separations of crystallographic planes d, allowing one to deduce the crystal 
structure. Diffraction contrast, in electron microscopes and x-topography devices in particular, is also a powerful tool 
for examining individual defects and local strain fields in crystals. 



Coherence 

The description of diffraction relies on the interference of waves emanating from the same source taking different 
paths to the same point on a screen. In this description, the difference in phase between waves that took different 
paths is only dependent on the effective path length. This does not take into account the fact that waves that arrive at 
the screen at the same time were emitted by the source at different times. The initial phase with which the source 
emits waves can change over time in an unpredictable way. This means that waves emitted by the source at times 
that are too far apart can no longer form a constant interference pattern since the relation between their phases is no 
longer time independent. 

The length over which the phase in a beam of light is correlated, is called the coherence length. In order for 
interference to occur, the path length difference must be smaller than the coherence length. This is sometimes 
referred to as spectral coherence, as it is related to the presence of different frequency components in the wave. In the 
case of light emitted by an atomic transition, the coherence length is related to the lifetime of the excited state from 
which the atom made its transition. 



Diffraction 14 

If waves are emitted from an extended source, this can lead to incoherence in the transversal direction. When looking 
at a cross section of a beam of light, the length over which the phase is correlated is called the transverse coherence 
length. In the case of Young's double slit experiment, this would mean that if the transverse coherence length is 
smaller than the spacing between the two slits, the resulting pattern on a screen would look like two single slit 
diffraction patterns. 

In the case of particles like electrons, neutrons and atoms, the coherence length is related to the spatial extent of the 
wave function that describes the particle. 

See also 

Atmospheric diffraction 

Bragg diffraction 

Bracken spectre 

Cloud iridescence 

Diffraction formalism 

Diffraction grating 

Diffraction limit 

Diffractometer 

Dynamical theory of diffraction 

Electron diffraction 

Fraunhofer diffraction 

Fresnel diffraction 

Fresnel imager 

Fresnel number 

Fresnel zone 

Neutron diffraction 

Prism 

Powder diffraction 

Refraction 

Schaefer— Bergmann diffraction 

Thinned array curse 

X-ray scattering techniques 

References 

[1] Dietrich Zawischa. "Optical effects on spider webs" (http://www.itp.uni-hannover.de/~zawischa/ITP/spiderweb.html). . Retrieved 

2007-09-21. 
[2] Andrew Norton (2000). Dynamic fields and waves (http://books. google. com/?id=XRRMxjr24pwC&pg=PA102&dq=sound+wave+ 

diffraction+behind+tree). CRC Press, p. 102. ISBN 9780750307192. . 
[3] Francesco Maria Grimaldi, Physico-mathesis de lumine, coloribus, et iride, aliisque adnexis... [The physical mathematics of light, color, and 

the rainbow, and other things appended...] (Bologna ("Bonomia"), Italy: Vittorio Bonati, 1665), pages 1-11 (http://books.google.com/ 

books ?id=FzYVAAAAQAAI&pg=PAl&source=gbs_toc_r&cad=3#v=onepage&q&f=false): "Propositio I. Lumen propagatur seu 

diffunditur non solum directe, refracte, ac reflexe, sed etiam alio quodam quarto modo, diffracte." (Proposition 1. Light propagates or spreads 

not only in a straight line, by refraction, and by reflection, but also by another, certain fourth way: by diffraction.) 
[4] lean Louis Aubert (1760). Memoires pour Vhistoire des sciences et des beaux arts (http://books. google. com/?id=30gDAAAAMAAI& 

pg=PP151&lpg=PP151&dq=grimaldi+diffraction+date:0-1800). Paris: Impr. de S. A. S.; Chez E. Ganeau. pp. 149. . 
[5] Sir David Brewster (1831). A Treatise on Optics (http://books.google.com/?id=opYAAAAAMAAI&pg=RAl-PA95&lpg=RAl-PA95& 

dq=grimaldi+diffraction+date:0-1840). London: Longman, Rees, Orme, Brown & Green and lohn Taylor, pp. 95. . 
[6] Letter from lames Gregory to lohn Collins, dated 13 May 1673. Reprinted in: Correspondence of Scientific Men of the Seventeenth 

Century...., ed. Stephen lordan Rigaud (Oxford, England: Oxford University Press, 1841), vol. 2, pages 251-255; see especially page 254. 

Available on-line at: Books.Google.com (http://books.google.com/books?id=0h45L_66bcYC&pg=PA254&dq=feather+ovals& 



Diffraction 15 

ei=5jlaSsLQKJnkygTilLz8CA&ie=ISO-8859-l&output=html). 
[7] Young, Thomas (1804-01-01). "The Bakerian Lecture: Experiments and calculations relative to physical optics" (http://books.google.com/ 

?id=7AZGAAAAMAAJ&pg=PAl). Philosophical Transactions of the Royal Society of London (Royal Society of London.) 94: 1-16. 

doi:10.1098/rstl.l804.0001. . (Note: This lecture was presented before the Royal Society on 24 November 1803.) 
[8] Augustin-Jean Fresnel (1816) "Memoire sur la diffraction de la lumiere ... ," Annates de la Chemie et de Physique, 2nd series, vol. 1, pages 

239-281. (Presented before VAcademie des sciences on 15 October 1815.) Available on-line at: Bibnum.education.fr (http://www.bibnum. 

education.fr/physique/optique/premier-memoire-sur-la-diffraction-de-la-lumiere) (French) 
[9] Augustin-Jean Fresnel (1826) "Memoire sur la diffraction de la lumiere," Me'moires de VAcademie des Sciences (Paris), vol. 5, pages 33-475. 

(Summitted to VAcademie des sciences of Paris on 20 April 1818.) 
[10] Christiaan Huygens, Traite de la lumiere (Leiden, Netherlands: Pieter van der Aa, 1690), Chapter 1. (Note: Huygens published his Traitem 

1690; however, in the preface to his book, Huygens states that in 1678 he first communicated his book to the French Royal Academy of 

Sciences.) 
[11] Brezger, B.; Hackermuller, L.; Uttenthaler, S.; Petschinka, J.; Arndt, M.; Zeilinger, A. (February 2002). "Matter— Wave Interferometer for 

Large Molecules" (http://homepage.univie.ac.at/Lucia.Hackermueller/unsereArtikel/Brezger2002a.pdf) (reprint). Physical Review 

Letters 88 (10): 100404. doi: 10. 1103/PhysRevLett.88. 100404. PMID 11909334. . Retrieved 2007-04-30. 
[12] John M. Cowley (1975) Diffraction physics (North-Holland, Amsterdam) ISBN 444 10791 6 

External links 

• Diffraction and Crystallography for beginners (http://www.xtal.iqfr.csic.es/Cristalografia/index-en.html) 

• Do Sensors "Outresolve" Lenses? (http://luminous-landscape.com/tutorials/resolution.shtml); on lens and 
sensor resolution interaction. 

• Diffraction and acoustics, (http://www.acoustics.salford.ac.uk/feschools/waves/diffract.htm) 

• Diffraction in photography, (http://www.johnsankey.ca/diffraction.html) 

• On Diffraction (http://www.mathpages.com/home/kmath636/kmath636.htm) at MathPages. 

• Diffraction pattern calculators (http://demonstrations.wolfram.com/search.html?query=diffraction) at The 
Wolfram Demonstrations Project 

• Wave Optics (http://www.lightandmatter.com/html_books/5op/ch05/ch05.html) — A chapter of an online 
textbook. 

• 2-D wave Java applet (http://www.falstad.com/wave2d/) — Displays diffraction patterns of various slit 
configurations. 

• Diffraction Java applet (http://www.falstad.com/diffraction/) — Displays diffraction patterns of various 2-D 
apertures. 

• Diffraction approximations illustrated (http://www.mit.edu/~birge/diffraction/) — MIT site that illustrates the 
various approximations in diffraction and intuitively explains the Fraunhofer regime from the perspective of 
linear system theory. 

• Gap (http://www.phy.hk/wiki/englishhtm/Diffraction.htm) Obstacle (http://www.phy.hk/wiki/ 
englishhtm/Diffraction2.htm) Corner (http://www.phy.hk/wiki/englishhtm/Diffraction3.htm) — Java 
simulation of diffraction of water wave. 

• Google Maps (http://maps. google. com/maps?q=Panama+canal&hl=en&ie=UTF8&om=l&z=16&ll=9. 
385048,-79.918799&spn=0.015539,0.027122&t=k&iwloc=addr) - Satellite image of Panama Canal entry 
ocean wave diffraction. 

• Google Maps (http://maps. google. com/maps?f=q&source=s_q&hl=en&geocode=&q=&sll=52. 788632,1. 
609969&sspn=0.010472,0.016093&ie=UTF8&t=h&ll=52.788217,1.606772&spn=0.010472,0.016093& 
z=16) and Bing Maps (http://www.bing. com/maps/?v=2&cp=52. 788763840321245-1. 
6073888540267944&lvl=16&sty=h&eo=0) - Aerial photo of waves diffracting through sea barriers at Sea 
Palling in Norfolk, UK. 

• Diffraction Effects (http://www.cvimellesgriot.com/products/Documents/TechnicalGuide/ 
Diffraction_Effects .pdf) 

• An Introduction to The Wigner Distribution in Geometric Optics (http://scripts.mit.edu/~raskar/lightfields/ 
index. php?title=An_Introduction_to_The_Wigner_Distribution_in_Geometric_Optics) 



Diffraction 16 

• DoITPoMS Teaching and Learning Package - Diffraction and Imaging (http://www.doitpoms.ac.uk/tlplib/ 
diffraction/index. php) 



Uniform theory of diffraction 



In numerical analysis, the uniform geometrical theory of diffraction (UTD) is a high frequency method for solving 
electromagnetic scattering problems from electrically small discontinuities or discontinuities in more than one 
dimension at the same point. UTD is an extension of Joseph Keller's geometrical theory of diffraction (GTD). 

The uniform theory of diffraction approximates near field electromagnetic fields as quasi optical and uses ray 
diffraction to determine diffraction coefficients for each diffracting object-source combination. These coefficients 
are then used to calculate the field strength and phase for each direction away from the diffracting point. 

These fields are then added to the incident fields and reflected fields to obtain a total solution. 

See also 

• Electromagnetic modeling 

References 

[1] R. G. Kouyoumjian and P. H. Pathak, "A uniform geometrical theory of diffraction for an edge in a perfectly conducting surface," Proc. 

IEEE, vol. 62, pp. 1448-1461, November 1974. 
[2] J. B. Keller, "Geometrical theory of diffraction" (http://www.opticsinfobase.org/abstract.cfm?URI=josa-52-2-116), J. Opt. Soc. Am., vol. 

52, no. 2, pp. 116-130, 1962. 

External links 

• Overview of Asymptotic Expansion Methods in Electromagnetics (http://www.cvel.clemson.edu/modeling/ 
tutorials/techniques/gtd-utd/gtd-utd.html) 



Crystallography 



17 



Crystallography 






A crystalline solid: atomic resolution image of 

strontium titanate. Brighter atoms are Sr and 

darker ones are Ti. 



Crystallography is the experimental science of the arrangement of 
atoms in solids. The word "crystallography" derives from the Greek 
words crystallon = cold drop / frozen drop, with its meaning extending 
to all solids with some degree of transparency, and grapho = write. 

Before the development of X-ray diffraction crystallography (see 

below), the study of crystals was based on their geometry. This 

involves measuring the angles of crystal faces relative to theoretical 

reference axes (crystallographic axes), and establishing the symmetry 

of the crystal in question. The former is carried out using a goniometer. 

The position in 3D space of each crystal face is plotted on a 

stereographic net, e.g. Wulff net or Lambert net. In fact, the pole to each face is plotted on the net. Each point is 

labelled with its Miller index. The final plot allows the symmetry of the crystal to be established. 

Crystallographic methods now depend on the analysis of the diffraction patterns of a sample targeted by a beam of 
some type. Although X-rays are most commonly used, the beam is not always electromagnetic radiation. For some 
purposes electrons or neutrons are used. This is facilitated by the wave properties of the particles. Crystallographers 
often explicitly state the type of illumination used when referring to a method, as with the terms X-ray diffraction, 
neutron diffraction and electron diffraction. 

These three types of radiation interact with the specimen in different ways. X-rays interact with the spatial 
distribution of the valence electrons, while electrons are charged particles and therefore feel the total charge 
distribution of both the atomic nuclei and the surrounding electrons. Neutrons are scattered by the atomic nuclei 
through the strong nuclear forces, but in addition, the magnetic moment of neutrons is non-zero. They are therefore 
also scattered by magnetic fields. When neutrons are scattered from hydrogen-containing materials, they produce 
diffraction patterns with high noise levels. However, the material can sometimes be treated to substitute hydrogen for 
deuterium. Because of these different forms of interaction, the three types of radiation are suitable for different 
crystallographic studies. 



Theory 

Generally, an image of a small object is made using a lens to focus the illuminating radiation, as is done with the 
rays of the visible spectrum in light microscopy. However, the wavelength of visible light (about 4000 to 7000 
angstroms) is three orders of magnitude longer than the length of typical atomic bonds and atoms themselves (about 
1 to 2 angstroms). Therefore, obtaining information about the spatial arrangement of atoms requires the use of 
radiation with shorter wavelengths, such as X-rays. Employing shorter wavelengths implied abandoning microscopy 
and true imaging, however, because there exists no material from which a lens capable of focusing this type of 
radiation can be created. (That said, scientists have had some success focusing X-rays with microscopic Fresnel zone 
plates made from gold, and by critical-angle reflection inside long tapered capillaries.) Diffracted x-ray beams 
cannot be focused to produce images, so the sample structure must be reconstructed from the diffraction pattern. 
Sharp features in the diffraction pattern arise from periodic, repeating structure in the sample, which are often very 
strong due to coherent reflection of many photons from many regularly spaced instances of similar structure, while 
non-periodic components of the structure result in diffuse (and usually weak) diffraction features. 

Because of their highly ordered and repetitive structure, crystals give diffraction patterns of sharp Bragg reflection 
spots, and are ideal for analyzing the structure of solids. 



Crystallography 18 

Notation 

• Coordinates in square brackets such as [100] denote a direction vector (in real space). 

• Coordinates in angle brackets or chevrons such as <100> denote a family of directions which are related by 
symmetry operations. In the cubic crystal system for example, <100> would mean [100], [010], [001] or the 
negative of any of those directions. 

• Miller indices in parentheses such as (100) denote a plane of the crystal structure, and regular repetitions of that 
plane with a particular spacing. In the cubic system, the normal to the (hkl) plane is the direction [hkl], but in 
lower-symmetry cases, the normal to (hkl) is not parallel to [hkl]. 

• Indices in curly brackets or braces such as {100} denote a family of planes and their normals which are equivalent 
in cubic materials due to symmetry operations, much the way angle brackets denote a family of directions. In 
non-cubic materials, <hkl> is not necessarily perpendicular to {hkl}. 

Technique 

Some materials studied using crystallography, proteins for example, do not occur naturally as crystals. Typically, 
such molecules are placed in solution and allowed to crystallize over days, weeks, or months through vapor 
diffusion. A drop of solution containing the molecule, buffer, and precipitants is sealed in a container with a 
reservoir containing a hygroscopic solution. Water in the drop diffuses to the reservoir, slowly increasing the 
concentration and allowing a crystal to form. If the concentration were to rise more quickly, the molecule would 
simply precipitate out of solution, resulting in disorderly granules rather than an orderly and hence usable crystal. 

Once a crystal is obtained, data can be collected using a beam of radiation. Although many universities that engage 
in crystallographic research have their own X-ray producing equipment, synchrotrons are often used as X-ray 
sources, because of the purer and more complete patterns such sources can generate. Synchrotron sources also have a 
much higher intensity of X-ray beams, so data collection takes a fraction of the time normally necessary at weaker 
sources. 

Producing an image from a diffraction pattern requires sophisticated mathematics and often an iterative process of 
modelling and refinement. In this process, the mathematically predicted diffraction patterns of an hypothesized or 
"model" structure are compared to the actual pattern generated by the crystalline sample. Ideally, researchers make 
several initial guesses, which through refinement all converge on the same answer. Models are refined until their 
predicted patterns match to as great a degree as can be achieved without radical revision of the model. This is a 
painstaking process, made much easier today by computers. 

The mathematical methods for the analysis of diffraction data only apply to patterns, which in turn result only when 
waves diffract from orderly arrays. Hence crystallography applies for the most part only to crystals, or to molecules 
which can be coaxed to crystallize for the sake of measurement. In spite of this, a certain amount of molecular 
information can be deduced from the patterns that are generated by fibers and powders, which while not as perfect as 
a solid crystal, may exhibit a degree of order. This level of order can be sufficient to deduce the structure of simple 
molecules, or to determine the coarse features of more complicated molecules. For example, the double-helical 
structure of DNA was deduced from an X-ray diffraction pattern that had been generated by a fibrous sample). 



Crystallography 



19 



Crystallography in materials engineering 

Crystallography is a tool that is often employed by materials 
scientists. In single crystals, the effects of the crystalline 
arrangement of atoms is often easy to see macroscopically, because 
the natural shapes of crystals reflect the atomic structure. In addition, 
physical properties are often controlled by crystalline defects. The 
understanding of crystal structures is an important prerequisite for 
understanding crystallographic defects. Mostly, materials do not 
occur in a single crystalline, but poly-crystalline form, such that the 
powder diffraction method plays a most important role in structural 
determination. 




An example of a cubic lattice 



A number of other physical properties are linked to crystallography. 

For example, the minerals in clay form small, flat, platelike structures. Clay can be easily deformed because the 
platelike particles can slip along each other in the plane of the plates, yet remain strongly connected in the direction 
perpendicular to the plates. Such mechanisms can be studied by crystallographic texture measurements. 

In another example, iron transforms from a body-centered cubic (bcc) structure to a face-centered cubic (fee) 
structure called austenite when it is heated. The fee structure is a close-packed structure, and the bcc structure is not, 
which explains why the volume of the iron decreases when this transformation occurs. 

Crystallography is useful in phase identification. When performing any process on a material, it may be desired to 
find out what compounds and what phases are present in the material. Each phase has a characteristic arrangement of 
atoms. Techniques like X-ray diffraction can be used to identify which patterns are present in the material, and thus 
which compounds are present. 

Crystallography covers the enumeration of the symmetry patterns which can be formed by atoms in a crystal and for 
this reason has a relation to group theory and geometry. See symmetry group. 



Biology 

X-ray crystallography is the primary method for determining the molecular conformations of biological 
macromolecules, particularly protein and nucleic acids such as DNA and RNA. In fact, the double-helical structure 

of DNA was deduced from crystallographic data. The first crystal structure of a macromolecule was solved in 1958 

T21 131 

A three-dimensional model of the myoglobin molecule obtained by X-ray analysis. The Protein Data Bank 

(PDB) is a freely accessible repository for the structures of proteins and other biological macromolecules. Computer 

programs like RasMol or Pymol can be used to visualize biological molecular structures. 

Electron crystallography has been used to determine some protein structures, most notably membrane proteins and 
viral capsids. 



Crystallography 20 

Scientists of note 

William Barlow 

John Desmond Bernal 

William Henry Bragg 

William Lawrence Bragg 

Auguste Bravais 

Martin Julian Buerger 

Francis Crick 

Pierre Curie 

Peter Debye 

Boris Delone 

Paul Peter Ewald 

Evgraf Stepanovich Fedorov 

Rosalind Franklin 

Georges Friedel 

Paul Heinrich von Groth 

Rene Just Haiiy 

Carl Hermann 

Johann Friedrich Christian Hessel 

Dorothy Crowfoot Hodgkin 

Robert Huber 

Aaron Klug 

Max von Laue 

Kathleen Lonsdale 

Ernest-Francois Mallard 

Charles-Victor Mauguin 

William Hallowes Miller 

Friedrich Mohs 

Paul Niggli 

Arthur Lindo Patterson 

Max Perutz 

Hugo Rietveld 

Jean-Baptiste L. Rome de l'lsle 

Paul Scherrer 

Arthur Moritz Schonflies 

Constance Tipper 

Don Craig Wiley 

Ada Yonath 

Rajagopala Chidambaram 

Tej P. Singh 

Christian Samuel Weiss 

Ralph Walter Graystone Wyckoff 



Crystallography 21 

See also 

Atomic packing factor 

Condensed matter physics 

Crystal engineering 

Crystal growth 

Crystal optics 

Crystal system 

Crystal 

Crystallite 

Crystallization processes 

Crystallographic database 

Crystallographic group 

Dynamical theory of diffraction 

Electron crystallography 

Euclidean plane isometry 

Fixed points of isometry groups in Euclidean space 

Fractional coordinates 

Group action 

Laser-heated pedestal growth 

Materials science 

Metallurgy 

Mineralogy 

Neutron crystallography 

Neutron diffraction at OPAL 

Permutation group 

Point group 

Solid state chemistry 

Space group 

Symmetric group 

References 

[1] A. Snigirev et al. (2007). "Two-step hard X-ray focusing combining Fresnel zone plate and single-bounce ellipsoidal capillary". Journal of 

Synchrotron Radiation 14 (Pt4): 326-330. doi:10.1107/S0909049507025174. PMID 17587657. 
[2] Kendrew, J.C. et al. (1958) 
[3] (Nature 181, 662—666). (http://www.nature.com/physics/looking-back/kendrew/kendrew.pdf) 

Further reading 

• Burns, G.; Glazer, A.M. (1990). Space Groups for Scientists and Engineers (2nd ed.). Boston: Academic Press, 
Inc. ISBN 0-12-145761-3. 

• Clegg, W (1998). Crystal Structure Determination (Oxford Chemistry Primer). Oxford: Oxford University Press. 
ISBN 0-19-855-901-1. 

• Drenth, J (1999). Principles of Protein X-Ray Crystallography. New York: Springer- Verlag. 
ISBN 0-387-98587-5. 

• Giacovazzo, C; Monaco HL, Viterbo D, Scordari F, Gilli G, Zanotti G, and Catti M (1992). Fundamentals of 
Crystallography. Oxford: Oxford University Press. ISBN 0-19-855578-4. 

• Glusker, JP; Lewis M, Rossi M (1994). Crystal Structure Analysis for Chemists and Biologists. New York: VCH 
Publishers. ISBN 0-471-18543-4. 



Crystallography 22 

• O'Keeffe, M.; Hyde, B.G. (1996). Crystal Structures; I. Patterns and Symmetry. Washington, DC: Mineralogical 
Society of America, Monograph Series. ISBN 0-939950-40-5. 

Applied Computational Powder Diffraction Data Analysis 

• Edited by R. A. Young (1993). Young, R.A.. ed. The Rietveld Method. Oxford: Oxford University Press & 
International Union of Crystallography. ISBN 0-19-855577-6. 

External links 

• American Crystallographic Association (http://www.AmerCrystalAssn.org/) 

• Learning Crystallography (http://www.xtal.iqfr.csic.es/Cristalografia/index-en.html) 

• Crystal Lattice Structures (http://cst-www.nrl.navy.mil/lattice/spcgrp/) 

• Vega Science Trust Interviews on Crystallography (http://www.vega.org.uk) Freeview video interviews with 
Max Pertuz, Rober Huber and Aaron Klug. 

• Commission on Crystallographic Teaching, Pamphlets (http://www.iucr.org/iucr-top/comm/cteach/ 
pamphlets.html) 

• Ames Laboratory, US DOE Crystallography Research Resources (http://www.mcbmm.ameslab.gov/index. 
html) 



Paracrystalline 



Paracrystalline materials are defined as having short and medium range ordering in their lattice (similar to the 
liquid crystal phases) but lacking long-range ordering at least in one direction. 

Ordering is the regularity in which atoms appear in a predictable lattice, as measured from one point. In a highly 
ordered, perfectly crystalline material, or single crystal, the location of every atom in the structure can be described 
exactly measuring out from a single origin. Conversely, in a disordered structure such as a liquid or amorphous solid, 
the location of the first and perhaps second nearest neighbors can be described from an origin (with some degree of 
uncertainty) and the ability to predict locations decreases rapidly from there out. The distance at which atom 
locations can be predicted is referred to as the correlation length £ . A paracrystalline material exhibits correlation 
somewhere between the fully amorphous and fully crystalline. 

The primary, most accessible source of crystallinity information is X-ray diffraction, although other techniques may 
be needed to observe the complex structure of paracrystalline materials, such as fluctuation electron microscopy 
in combination with Density of states modeling of electronic and vibrational states. 

Paracrystalline Model 

The paracrystalline model is a revision of the Continuous Random Network model first proposed by W. H. 

mi 
Zachariasen in 1932 . The paracrystal model is defined as highly strained, microcrystalline grains surrounded by 

fully amorphous material . This is a higher energy state then the continuous random network model. The 

important distinction between this model and the microcrystalline phases is the lack of defined grain boundaries and 

highly strained lattice parameters, which makes calculations of molecular and lattice dynamics difficult. A general 

theory of paracrystals has been formulated in a basic textbook , and then further developed/refined by various 

authors. An example of a paracrystalline, or partially disordered lattice is showin in the following figure. 



Paracrystalline 



23 









j. 



j 



^j 






-L 



-> 



, 



i 



J 



J 



*-J 



^ 






* Si 



Applications 

The paracrystal model has been useful, for example, in describing the state of partially amorphous semiconductor 
materials after deposition. It has also been successfully applied to synthetic polymers, liquid crystals, 
biopoloymers , and biomembranes . 



See also 

• X-ray scattering 

• Amorphous solid 

• Single Crystal 

• Polycrystalline 

• Crystallography 

• DNA 

• X-ray pattern of a B-DNA Paracrystal 



[10] 



Notes 

[1] Voyles, et al. Structure and physical properties of paracrystalline atomistic models of amorphous silicon. J. Ap. Phys., 90(2001) 4437, doi: 

10.1063/1.1407319 
[2] Biswas, P, et al. J. Phys.:Condens. Matter, 19 (2007) 455202, doi:10.1088/0953-8984/19/45/455202 

[3] Nakhmanson, Voyles, Mousseau, Barkema, and Drabold. Phys. Rev. B 63(2001) 235207. doi: 10.1 103/PhysRevB.63.235207 
[4] Zachariasen, W.H., J. Am. Chem. Soc, 54(1932) 3841. 
[5] J.M. Cowley, Diffraction Studies on Non-Cryst. Substan. 13 (1981) 

[6] Hosemann R., Bagchi R.N., Direct analysis of diffraction by matter, North-Holland Pubis., Amsterdam — New York, 1962 
[7] Bessel functions and diffraction by helical structures http://planetphysics.org/encyclopedia/ 

BesselFunctionsAndTheirApplicationsToDiffractionByHelicalStructures.html 
[8] X-Ray Diffraction Patterns of Double-Helical Deoxyribonucleic Acid (DNA) Crystals and Paracrystalline Fibers http://planetphysics.org/ 

encyclopedia/BesselFunctionsApplicationsToDiffractionByHelicalStructures.html 
[9] Baianu I.C., X-ray scattering by partially disordered membrane systems, Acta Cryst. A, 34 (1978), 751—753. 
[10] http://commons.wikimedia.Org/wiki/File:ABDNAxrgpj.jpg 



Quantum optics 24 



Quantum optics 



Quantum optics is a field of research in physics, dealing with the application of quantum mechanics to phenomena 
involving light and its interactions with matter. 

History of quantum optics 

Light is made up of particles called photons and hence inherently is "grainy" (quantized). Quantum optics is the 
study of the nature and effects of light as quantized photons. The first indication that light might be quantized came 
from Max Planck in 1899 when he correctly modeled blackbody radiation. By assuming that, Bohr showed that the 
atoms were also quantized, in the sense that they could only emit discrete amounts of energy. The understanding of 
the interaction between light and matter following these developments not only formed the basis of quantum optics 
but were also crucial for the development of quantum mechanics as a whole. However, the subfields of quantum 
mechanics dealing with matter-light interaction were principally regarded as research into matter rather than into 
light; hence one rather spoke of atom physics and quantum electronics in 1960. Laser science — i.e., research into 
principles, design and application of these devices — became an important field, and the quantum mechanics 
underlying the laser's principles was studied now with more emphasis on the properties of light, and the name 
quantum optics became customary. 

As laser science needed good theoretical foundations, and also because research into these soon proved very fruitful, 
interest in quantum optics rose. Following the work of Dirac in quantum field theory, George Sudarshan, Roy J. 
Glauber, and Leonard Mandel applied quantum theory to the electromagnetic field in the 1950s and 1960s to gain a 
more detailed understanding of photodetection and the statistics of light (see degree of coherence). This led to the 
introduction of the coherent state as a quantum description of laser light and the realization that some states of light 
could not be described with classical waves. In 1977, Kimble et al. demonstrated the first source of light which 
required a quantum description: a single atom that emitted one photon at a time. This was the first conclusive 
evidence that light was made up of photons. Another quantum state of light with certain advantages over any 
classical state, squeezed light, was soon proposed. At the same time, development of short and ultrashort laser 
pulses — created by Q switching and modelocking techniques — opened the way to the study of unimaginably fast 
("ultrafast") processes. Applications for solid state research (e.g. Raman spectroscopy) were found, and mechanical 
forces of light on matter were studied. The latter led to levitating and positioning clouds of atoms or even small 
biological samples in an optical trap or optical tweezers by laser beam. This, along with Doppler cooling was the 
crucial technology needed to achieve the celebrated Bose-Einstein condensation. 

Other remarkable results are the demonstration of quantum entanglement, quantum teleportation, and (recently, in 
1995) quantum logic gates. The latter are of much interest in quantum information theory, a subject which partly 
emerged from quantum optics, partly from theoretical computer science. 

Today's fields of interest among quantum optics researchers include parametric down-conversion, parametric 
oscillation, even shorter (attosecond) light pulses, use of quantum optics for quantum information, manipulation of 
single atoms, Bose-Einstein condensates, their application, and how to manipulate them (a sub-field often called 
atom optics), coherent perfect absorbers, and much more. 

Research into quantum optics that aims to bring photons into use for information transfer and computation is now 
often called photonics to emphasize the claim that photons and photonics will take the role that electrons and 
electronics now have. 



Quantum optics 25 

Concepts of quantum optics 



Quantum optics operators 

Ladder operators 



Creation and annihilation operators 
Displacement operator 



Rotation operator (quantum mechanics) 
Squeeze operator 



Anti-symmetric operator 

[1] 

According to quantum theory, light may be considered not only as an electro-magnetic wave but also as a "stream" 
of particles called photons which travel with c, the vacuum speed of light. These particles should not be considered 
to be classical billiard balls, but as quantum mechanical particles described by a wavefunction spread over a finite 
region. Each particle carries one quantum of energy equal to hf, where h is Planck's constant and f is the frequency of 
the light. The postulation of the quantization of light by Max Planck in 1899 and the discovery of the general validity 
of this idea in Albert Einstein's 1905 explanation of the photoelectric effect soon led physicists to realize the 
possibility of population inversion and the possibility of the laser. 

This kind of use of statistical mechanics is the fundament of most concepts of quantum optics: Light is described in 
terms of field operators for creation and annihilation of photons — i.e. in the language of quantum electrodynamics. 

A frequently encountered state of the light field is the coherent state as introduced by George Sudarshan in 1963. 
This state, which can be used to approximately describe the output of a single-frequency laser well above the laser 
threshold, exhibits Poissonian photon number statistics. Via certain nonlinear interactions, a coherent state can be 
transformed into a squeezed coherent state, which can exhibit super- or sub- Poissonean photon statistics. Such light 
is called squeezed light. Other important quantum aspects are related to correlations of photon statistics between 
different beams. For example, parametric nonlinear processes can generate so-called twin beams, where ideally each 
photon of one beam is associated with a photon in the other beam. 

Atoms are considered as quantum mechanical oscillators with a discrete energy spectrum with the transitions 
between the energy eigenstates being driven by the absorption or emission of light according to Einstein's theory 
with the oscillator strength depending on the quantum numbers of the states. 

For solid state matter one uses the energy band models of solid state physics. This is important as understanding how 
light is detected (typically by a solid-state device that absorbs it) is crucial for understanding experiments. 

See also 

• Optics 

• Optical phase space 

• Optical physics 

• Nonclassical light 

References 

• L. Mandel, E. Wolf Optical Coherence and Quantum Optics (Cambridge 1995) 

• D. F. Walls and G. J. Milburn Quantum Optics (Springer 1994) 

• C. W. Gardiner and Peter Zoller, Quantum Noise, (Springer 2004). 

• M. O. Scully and M. S. Zubairy Quantum Optics (Cambridge 1997) 



Quantum optics 



26 



W. P. Schleich Quantum Optics in Phase Space (Wiley 2001) 



External links 

• An introduction to quantum optics of the light field 

[3] 



[2] 



Encyclopedia of laser physics and technology , with content on quantum optics (particularly quantum noise in 

lasers), by Riidiger Paschotta. 

Qwiki - A quantum physics wiki devoted to providing technical resources for practicing quantum physicists. 

Quantiki - a free-content WWW resource in quantum information science that anyone can edit. 

Various Quantum Optics Reports 



References 

[1] http://en.wikipedia.org/wiki/Template:Quantum 

[2] http://gerdbreitenbach.de/gallery 

[3] http://www.rp-photonics.com/encyclopedia.html 

[4] http://qwiki. stanford.edu/wiki/Quantum_Optics 

[5] http://www.quantiki.org/ 

[6] http://www.physics.drexel.edu/~tim/Decoherence/index.html 



X-rays 



X-radiation (composed of X-rays) is a form of 
electromagnetic radiation. X-rays have a wavelength in 
the range of 0.01 to 10 nanometers, corresponding to 
frequencies in the range 30 petahertz to 30 exahertz (3 x 
10 Hz to 3 x 10 Hz) and energies in the range 120 eV 
to 120 keV. They are shorter in wavelength than UV rays 
and longer than gamma rays. In many languages, 
X-radiation is called Rontgen radiation, after Wilhelm 
Conrad Rontgen, who is generally credited as its 
discoverer, and who had named it X-radiation to signify 
an unknown type of radiation. Correct spelling of 
X-ray(s) in the English language includes the variants 
x-ray(s) and X ray(s). XRAY is used as the phonetic 
pronunciation for the letter x. 



X-rays from about 0.12 to 12 keV (10 to 0.10 nm 
wavelength) are classified as "soft" X-rays, and from 
about 12 to 120 keV (0.10 to 0.01 nm wavelength) as 
"hard" X-rays, due to their penetrating abilities. 

Hard X-rays can penetrate solid objects, and their most 
common use is to take images of the inside of objects in 
diagnostic radiography and crystallography. As a result, 
the term X-ray is metonymically used to refer to a 
radiographic image produced using this method, in 
addition to the method itself. By contrast, soft X-rays can 



£ 

CD 

C 
" 




Long-waves 
X-rays are part of the electromagnetic spectrum. 



X-rays 27 

hardly be said to penetrate matter at all; for instance, the attenuation length of 600 eV (~ 2 nm) x-rays in water is less 
than 1 micrometer. X-rays are a form of ionizing radiation, and exposure to them can be a health hazard. 

The distinction between X-rays and gamma rays has changed in recent decades. Originally, the electromagnetic 
radiation emitted by X-ray tubes had a longer wavelength than the radiation emitted by radioactive nuclei (gamma 
rays). Older literature distinguished between X- and gamma radiation on the basis of wavelength, with radiation 
shorter than some arbitrary wavelength, such as 10~ m, defined as gamma rays. However, as shorter wavelength 
continuous spectrum "X-ray" sources such as linear accelerators and longer wavelength "gamma ray" emitters were 
discovered, the wavelength bands largely overlapped. The two types of radiation are now usually distinguished by 
their origin: X-rays are emitted by electrons outside the nucleus, while gamma rays are emitted by the nucleus. 

[7] [8] 

Units of measure and exposure 

The measure of X-rays ionizing ability is called the exposure: 

• The coulomb per kilogram (C/kg) is the SI unit of ionizing radiation exposure, and it is the amount of radiation 
required to create one coulomb of charge of each polarity in one kilogram of matter. 

• The roentgen (R) is an obsolete traditional unit of exposure, which represented the amount of radiation required to 
create one electrostatic unit of charge of each polarity in one cubic centimeter of dry air. 1 .00 roentgen = 
2.58xlO" 4 C/kg 

However, the effect of ionizing radiation on matter (especially living tissue) is more closely related to the amount of 
energy deposited into them rather than the charge generated. This measure of energy absorbed is called the absorbed 
dose: 

• The gray (Gy), which has units of (Joules/kilogram), is the SI unit of absorbed dose, and it is the amount of 
radiation required to deposit one joule of energy in one kilogram of any kind of matter. 

• The rad is the (obsolete) corresponding traditional unit, equal to 10 millijoules of energy deposited per kilogram. 
100rad= 1.00 gray. 

The equivalent dose is the measure of the biological effect of radiation on human tissue. For X-rays it is equal to the 
absorbed dose. 

• The sievert (Sv) is the SI unit of equivalent dose, which for X-rays is numerically equal to the gray (Gy). 

• The Roentgen equivalent man (rem) is the traditional unit of equivalent dose. For X-rays it is equal to the rad or 
10 millijoules of energy deposited per kilogram. 1.00 Sv = 100 rem. 

Medical X-rays are a significant source of man-made radiation exposure, accounting for 58% in the United States in 
1987, but since most radiation exposure is natural (82%), medical X-rays only account for 10% of total American 
radiation exposure. 

Reported dosage due to dental X-rays seems to vary significantly. Depending on the source, a typical dental X-ray of 
a human results in an exposure of perhaps, 3, 40, 300, or as many as 900 mrems (30 to 9,000 u,Sv). 

Sources 



X-rays 



28 



X-ray K-series spectral line wavelengths (nm) for some common target materials. [14] 



Target 


Kfr 


Kp 2 


Ka, 


Ka 2 


Fe 


0.17566 


0.17442 


0.193604 


0.193998 


Co 


0.162079 


0.160891 


0.178897 


0.179285 


Ni 


0.15001 


0.14886 


0.165791 


0.166175 


Cu 


0.139222 


0.138109 


0.154056 


0.154439 


Zr 


0.070173 


0.068993 


0.078593 


0.079015 


Mo 


0.063229 


0.062099 


0.070930 


0.071359 




The main source of x-ray radiation is from outer space (e.g. the sun, 
black holes, and supernovae). Man-made X-rays are generated by an 
X-ray tube, a vacuum tube that uses a high voltage to accelerate the 
electrons released by a hot cathode to a high velocity. The high 
velocity electrons collide with a metal target, the anode, creating the 

ri7i 

X-rays. In medical X-ray tubes the target is usually tungsten or a 
more crack-resistant alloy of rhenium (5%) and tungsten (95%), but 
sometimes molybdenum for more specialized applications, such as 
when soft X-rays are needed as in mammography. In crystallography, a 
copper target is most common, with cobalt often being used when 
fluorescence from iron content in the sample might otherwise present a 
problem. 

The maximum energy of the produced X-ray photon is limited by the 
energy of the incident electron, which is equal to the voltage on the 
tube, so an 80 kV tube cannot create X-rays with an energy greater 
than 80 keV. When the electrons hit the target, X-rays are created by 
two different atomic processes: 

1 . X-ray fluorescence: If the electron has enough energy it can knock 
an orbital electron out of the inner electron shell of a metal atom, 
and as a result electrons from higher energy levels then fill up the 
vacancy and X-ray photons are emitted. This process produces an 
emission spectrum of X-rays at a few discrete frequencies, 
sometimes referred to as the spectral lines. The spectral lines 

generated depend on the target (anode) element used and thus are called characteristic lines. Usually these are 
transitions from upper shells into K shell (called K lines), into L shell (called L lines) and so on. 

2. Bremsstrahlung: This is radiation given off by the electrons as they are scattered by the strong electric field near 
the high-Z (proton number) nuclei. These X-rays have a continuous spectrum. The intensity of the X-rays 
increases linearly with decreasing frequency, from zero at the energy of the incident electrons, the voltage on the 
X-ray tube. 

So the resulting output of a tube consists of a continuous bremsstrahlung spectrum falling off to zero at the tube 

voltage, plus several spikes at the characteristic lines. The voltages used in diagnostic X-ray tubes, and thus the 

n 8i 
highest energies of the X-rays, range from roughly 20 to 150 kV. 

Both of these X-ray production processes are very inefficient, with a production efficiency of only about one percent, 
and hence, to produce a usable flux of X-rays, most of the electric power consumed by the tube is released as waste 
heat. The X-ray tube must be designed to dissipate this excess heat. 



Hand mit Ringen (Hand with Rings): print of 

Wilhelm Rontgen's first "medical" X-ray, of his 

wife's hand, taken on 22 December 1895 and 

presented to Professor Ludwig Zehnder of the 

Physik Institut, University of Freiburg, on 1 

January 1896 



X-rays 29 

In medical diagnostic applications, the low energy (soft) X-rays are unwanted, since they are totally absorbed by the 
body, increasing the dose. Hence, a thin metal sheet, often of aluminum, called an X-ray filter, is usually placed over 
the window of the X-ray tube, filtering out the low energy components in the spectrum. This is called hardening the 
beam. 

Radiographs obtained using X-rays can be used to identify a wide spectrum of pathologies. Because the body 
structures being imaged in medical applications are large compared to the wavelength of the X-rays, the X-rays can 
be analyzed as particles rather than waves. (This is in contrast to X-ray crystallography, where their wave-like nature 
is more important because the wavelength is comparable to the sizes of the structures being imaged.) 

To make an X-ray image of human or animal bones, short X-ray pulses illuminate the body or limb, with 
radiographic film placed behind it. Any bones that are present absorb most of the X-ray photons by photoelectric 
processes. This is because bones have a higher electron density than soft tissues. Note that bones contain a high 
percentage of calcium (20 electrons per atom), potassium (19 electrons per atom) magnesium (12 electrons per 
atom), and phosphorus (15 electrons per atom). The X-rays that pass through the flesh leave a latent image in the 
photographic film. When the film is developed, the parts of the image corresponding to higher X-ray exposure are 
dark, leaving a white shadow of bones on the film. 

To generate an image of the cardiovascular system, including the arteries and veins (angiography) an initial image is 
taken of the anatomical region of interest. A second image is then taken of the same region after iodinated contrast 
material has been injected into the blood vessels within this area. These two images are then digitally subtracted, 
leaving an image of only the iodinated contrast outlining the blood vessels. The radiologist or surgeon then compares 
the image obtained to normal anatomical images to determine if there is any damage or blockage of the vessel. 

A specialized source of X-rays which is becoming widely used in research is synchrotron radiation, which is 
generated by particle accelerators. Its unique features are X-ray outputs many orders of magnitude greater than those 

ri9i 

of X-ray tubes, wide X-ray spectra, excellent collimation, and linear polarization. 

Detectors 
Photographic plate 

The detection of X-rays is based on various methods. The most commonly known methods are photographic plates, 
photographic film in cassettes, and rare earth screens. Regardless of what is "catching" the image, they are all 
categorized as "Image Receptors" (IR). 

Before the advent of the digital computer and before the invention of digital imaging, photographic plates were used 
to produce most radiographic images. The images were produced right on the glass plates. Photographic film largely 
replaced these plates, and it was used in X-ray laboratories to produce medical images. In more recent years, 
computerized and digital radiography has been replacing photographic film in medical and dental applications, 
though film technology remains in widespread use in industrial radiography processes (e.g. to inspect welded 
seams). Photographic plates are mostly things of history, and their replacement, the "intensifying screen", is also 
fading into history. The metal silver (formerly necessary to the radiographic & photographic industries) is a 
non-renewable resource. Thus it is beneficial that this is now being replaced by digital (DR) and computed (CR) 
technology. Where photographic films required wet processing facilities, these new technologies do not. The digital 
archiving of images utilizing these new technologies also saves storage space. 

Since photographic plates are sensitive to X-rays, they provide a means of recording the image, but they also 
required much X-ray exposure (to the patient), hence intensifying screens were devised. They allow a lower dose to 
the patient, because the screens take the X-ray information and intensify it so that it can be recorded on film 
positioned next to the intensifying screen. 



X-rays 30 

The part of the patient to be X-rayed is placed between the X-ray source and the image receptor to produce a shadow 
of the internal structure of that particular part of the body. X-rays are partially blocked ("attenuated") by dense 
tissues such as bone, and pass more easily through soft tissues. Areas where the X-rays strike darken when 
developed, causing bones to appear lighter than the surrounding soft tissue. 

Contrast compounds containing barium or iodine, which are radiopaque, can be ingested in the gastrointestinal tract 
(barium) or injected in the artery or veins to highlight these vessels. The contrast compounds have high atomic 
numbered elements in them that (like bone) essentially block the X-rays and hence the once hollow organ or vessel 
can be more readily seen. In the pursuit of a non-toxic contrast material, many types of high atomic number elements 
were evaluated. For example, the first time the forefathers used contrast it was chalk, and was used on a cadaver's 
vessels. Unfortunately, some elements chosen proved to be harmful — for example, thorium was once used as a 
contrast medium (Thorotrast) — which turned out to be toxic in some cases (causing injury and occasionally death 
from the effects of thorium poisoning). Modern contrast material has improved, and while there is no way to 
determine who may have a sensitivity to the contrast, the incidence of "allergic-type reactions" are low. (The risk is 
comparable to that associated with penicillin.) 

Photostimulable phosphors (PSPs) 

An increasingly common method is the use of photostimulated luminescence (PSL), pioneered by Fuji in the 1980s. 
In modern hospitals a photostimulable phosphor plate (PSP plate) is used in place of the photographic plate. After 
the plate is X-rayed, excited electrons in the phosphor material remain "trapped" in "colour centres" in the crystal 
lattice until stimulated by a laser beam passed over the plate surface. The light given off during laser stimulation is 
collected by a photomultiplier tube and the resulting signal is converted into a digital image by computer technology, 
which gives this process its common name, computed radiography (also referred to as digital radiography). The 
PSP plate can be reused, and existing X-ray equipment requires no modification to use them. 

Geiger counter 

Initially, most common detection methods were based on the ionization of gases, as in the Geiger-Muller counter: a 
sealed volume, usually a cylinder, with a mica, polymer or thin metal window contains a gas, a cylindrical cathode 
and a wire anode; a high voltage is applied between the cathode and the anode. When an X-ray photon enters the 
cylinder, it ionizes the gas and forms ions and electrons. Electrons accelerate toward the anode, in the process 
causing further ionization along their trajectory. This process, known as a Townsend avalanche, is detected as a 
sudden current, called a "count" or "event". 

In order to gain energy spectrum information, a diffracting crystal may be used to first separate the different photons. 
The method is called wavelength dispersive X-ray spectroscopy (WDX or WDS). Position-sensitive detectors are 
often used in conjunction with dispersive elements. Other detection equipment that is inherently energy-resolving 
may be used, such as the aforementioned proportional counters. In either case, use of suitable pulse-processing 
(MCA) equipment allows digital spectra to be created for later analysis. 

For many applications, counters are not sealed but are constantly fed with purified gas, thus reducing problems of 
contamination or gas aging. These are called "flow counters". 



X-rays 



31 



Scintillators 

Some materials such as sodium iodide (Nal) can "convert" an X-ray photon to a visible photon; an electronic 
detector can be built by adding a photomultiplier. These detectors are called "scintillators", filmscreens or 
"scintillation counters". The main advantage of using these is that an adequate image can be obtained while 
subjecting the patient to a much lower dose of X-rays. 




X-ray during cholecystectomy 



Image intensification 

X-rays are also used in "real-time" procedures such as angiography or 
contrast studies of the hollow organs (e.g. barium enema of the small 
or large intestine) using fluoroscopy acquired using an X-ray image 
intensifier. Angioplasty, medical interventions of the arterial system, 
rely heavily on X-ray-sensitive contrast to identify potentially treatable 
lesions. 

Direct semiconductor detectors 

Since the 1970s, new semiconductor detectors have been developed 

(silicon or germanium doped with lithium, Si(Li) or Ge(Li)). X-ray 

photons are converted to electron-hole pairs in the semiconductor and 

are collected to detect the X-rays. When the temperature is low enough (the detector is cooled by Peltier effect or 

even cooler liquid nitrogen), it is possible to directly determine the X-ray energy spectrum; this method is called 

energy dispersive X-ray spectroscopy (EDX or EDS); it is often used in small X-ray fluorescence spectrometers. 

These detectors are sometimes called "solid state detectors". Detectors based on cadmium telluride (CdTe) and its 

alloy with zinc, cadmium zinc telluride, have an increased sensitivity, which allows lower doses of X-rays to be 

used. 

Practical application in medical imaging started in the 1990s. Currently amorphous selenium is used in commercial 
large area flat panel X-ray detectors for mammography and chest radiography. Current research and development is 
focused around pixel detectors, such as CERN's energy resolving Medipix detector. 

Note: A standard semiconductor diode, such as a 1N4007, will produce a small amount of current when placed in an 
X-ray beam. A test device once used by Medical Imaging Service personnel was a small project box that contained 
several diodes of this type in series, which could be connected to an oscilloscope as a quick diagnostic. 

Silicon drift detectors (SDDs), produced by conventional semiconductor fabrication, now provide a cost-effective 
and high resolving power radiation measurement. Unlike conventional X-ray detectors, such as Si(Li)s, they do not 
need to be cooled with liquid nitrogen. 

Scintillator plus semiconductor detectors (indirect detection) 

With the advent of large semiconductor array detectors it has become possible to design detector systems using a 
scintillator screen to convert from X-rays to visible light which is then converted to electrical signals in an array 
detector. Indirect Flat Panel Detectors (FPDs) are in widespread use today in medical, dental, veterinary and 
industrial applications. 

The array technology is a variant on the amorphous silicon TFT arrays used in many flat panel displays, like the ones 
in computer laptops. The array consists of a sheet of glass covered with a thin layer of silicon that is in an amorphous 
or disordered state. At a microscopic scale, the silicon has been imprinted with millions of transistors arranged in a 
highly ordered array, like the grid on a sheet of graph paper. Each of these thin film transistors (TFTs) is attached to 
a light-absorbing photodiode making up an individual pixel (picture element). Photons striking the photodiode are 
converted into two carriers of electrical charge, called electron-hole pairs. Since the number of charge carriers 



X-rays 



32 



produced will vary with the intensity of incoming light photons, an electrical pattern is created that can be swiftly 
converted to a voltage and then a digital signal, which is interpreted by a computer to produce a digital image. 
Although silicon has outstanding electronic properties, it is not a particularly good absorber of X-ray photons. For 
this reason, X-rays first impinge upon scintillators made from e.g. gadolinium oxysulfide or caesium iodide. The 
scintillator absorbs the X-rays and converts them into visible light photons that then pass onto the photodiode array. 



Visibility to the human eye 

While generally considered invisible to the human eye, in special circumstances X-rays can be visible. Brandes, 

in an experiment a short time after Rontgen's landmark 1895 paper, reported after dark adaptation and placing his 

r2ii 
eye close to an X-ray tube, seeing a faint "blue-gray" glow which seemed to originate within the eye itself. Upon 

hearing this, Rontgen reviewed his record books and found he too had seen the effect. When placing an X-ray tube 

on the opposite side of a wooden door Rontgen had noted the same blue glow, seeming to emanate from the eye 

itself, but thought his observations to be spurious because he only saw the effect when he used one type of tube. 

Later he realized that the tube which had created the effect was the only one powerful enough to make the glow 

plainly visible and the experiment was thereafter readily repeatable. The knowledge that X-rays are actually faintly 

visible to the dark-adapted naked eye has largely been forgotten today; this is probably due to the desire not to repeat 

what would now be seen as a recklessly dangerous and potentially harmful experiment with ionizing radiation. It is 

not known what exact mechanism in the eye produces the visibility: it could be due to conventional detection 

(excitation of rhodopsin molecules in the retina), direct excitation of retinal nerve cells, or secondary detection via, 

for instance, X-ray induction of phosphorescence in the eyeball with conventional retinal detection of the secondarily 

produced visible light. 

Though X-rays are otherwise invisible it is possible to see the ionization of the air molecules if the intensity of the 

T221 
X-ray beam is high enough. The beamline from the wiggler at the ID 11 at ESRF is one example of such high 

intensity. 



Medical uses 

Since Rontgen's discovery that X-rays can identify bone structures, 

X-rays have been developed for their use in medical imaging, the first 

1241 
use was less than a month after his seminal paper on the subject. 

Radiology is a specialized field of medicine. Radiologists employ 

radiography and other techniques for diagnostic imaging. This is 

probably the most common use of X-ray technology. 

X-rays are especially useful in the detection of pathology of the 
skeletal system, but are also useful for detecting some disease 
processes in soft tissue. Some notable examples are the very common 
chest X-ray, which can be used to identify lung diseases such as 
pneumonia, lung cancer or pulmonary edema, and the abdominal 
X-ray, which can detect intestinal obstruction, free air (from visceral 
perforations) and free fluid (in ascites). X-rays may also be used to 
detect pathology such as gallstones (which are rarely radiopaque) or 
kidney stones which are often (but not always) visible. Traditional 
plain X-rays are less useful in the imaging of soft tissues such as the 
brain or muscle. Imaging alternatives for soft tissues are computed 
axial tomography (CAT or CT scanning), magnetic resonance imaging (MRI) or ultrasound. The latter two do not 




Head CT scan (transverse plane) slice — a modern 
application of X-rays 



X-rays 



33 



subject the individual to ionizing radiation. In addition to plain X-rays and CT scans, physicians use fluoroscopy as 
an X-ray test methodology. This method often uses administration of a medical contrast material (intravenously, 
orally or via enema). Examples include cardiac catheterization (to examine for coronary artery blockages) and 
Barium swallow (to examine for esophageal disorders. 

Since 2005, X-rays are listed as a carcinogen by the U.S. government. The use of X-rays as a treatment is known 
as radiotherapy and is largely used for the management (including palliation) of cancer; it requires higher radiation 
energies than for imaging alone. 



Health risks 

X-rays are a relatively safe method of investigation and the radiation 
exposure is relatively low, depending upon the study. Experimental 
and epidemiological data, currently do not support the proposition that 
there is a threshold dose of radiation below which there is no increased 

[271 [2R1 

risk of cancer. However, this is under increasing doubt. 

Diagnostic X-rays account for 14% of the total annual radiation 

[29] 
exposure from man-made and natural sources worldwide. It is 

estimated that the additional radiation will increase a person's 

cumulative risk of getting cancer by age 75 by 0.6—1.8%. The 

amount of absorbed radiation depends upon the type of X-ray test and 

[311 

the body part involved. CT and fluoroscopy entail higher doses of 
radiation than do plain X-rays. 

To place the increased risk in perspective, a plain chest X-ray or dental 
X-ray will expose a person to the same amount from background 

radiation that we are exposed to (depending upon location) everyday 

T321 
over 10 days. Each such X-ray would add less than 1 per 1,000,000 

to the lifetime cancer risk. An abdominal or chest CT would be the 




equivalent to 2—3 years of background radiation, increasing the lifetime cancer risk between 1 per 10,000 and 1 per 

T321 

1,000. For instance, the effective dose to the torso from a CT scan of the chest of a 14 year old girl is about 

T331 
5mSv. These numbers are very small compared to the roughly 40% chance of developing any cancer during our 



lifetime 



13-1 1 



It should be noted that the accurate estimation of effective doses due to CT is difficult. For instance, the 

[35] 



estimation uncertainty range is about ±19% to ±32% for adult head scans depending upon the method used. 

Fathers exposed to diagnostic x-rays are more likely to have infants who contract leukemia, especially if exposure is 
closer to conception or includes two or more X-rays of the lower gastrointestinal (GI) tract or lower abdomen. 
The risk of radiation is greater to unborn babies, so in pregnant patients, the benefits of the investigation (X-ray) 

[371 [3R1 

should be balanced with the potential hazards to the unborn fetus. In the US, there are an estimated 

[311 

62,000,000 CT scans performed annually, including more than 4,000,000 on children. Avoiding unnecessary 
X-rays (especially CT scans) will reduce radiation dose and any associated cancer risk 



[39] 



Shielding 

3 

Lead is the most common shield against X-rays because of its high density (11340 kg/m ), stopping power, ease of 
installation and low cost. The maximum range of a high-energy photon such as an X-ray in matter is infinite; at 
every point in the matter traversed by the photon, there is a probability of interaction. Thus there is a very small 
probability of no interaction over very large distances. The shielding of photon beam is therefore exponential (with 
an attenuation length being close to the radiation length of the material); doubling the thickness of shielding will 
square the shielding effect. 



X-rays 



34 



The following table shows the recommended thickness of lead shielding in function of X-ray energy, from the 
Recommendations by the Second International Congress of Radiology. 



X-Rays generated by peak 

voltages 

not exceeding 


Minimum 

thickness 
of Lead 


75 kV 


1.0 mm 


100 kV 


1.5 mm 


125 kV 


2.0 mm 


150 kV 


2.5 mm 


175 kV 


3.0 mm 


200 kV 


4.0 mm 


225 kV 


5.0 mm 


300 kV 


9.0 mm 


400 kV 


15.0 mm 


500 kV 


22.0 mm 


600 kV 


34.0 mm 


900 kV 


51.0 mm 



Other uses 




Other notable uses of X-rays include 

• X-ray crystallography in which the pattern produced by the 
diffraction of X-rays through the closely spaced lattice of atoms in a 
crystal is recorded and then analysed to reveal the nature of that 

lattice. A related technique, fiber diffraction, was used by Rosalind 

T411 
Franklin to discover the double helical structure of DNA. 

• X-ray astronomy, which is an observational branch of astronomy, 
which deals with the study of X-ray emission from celestial objects. 

• X-ray microscopic analysis, which uses electromagnetic radiation in 
the soft X-ray band to produce images of very small objects. 

• X-ray fluorescence, a technique in which X-rays are generated 
within a specimen and detected. The outgoing energy of the X-ray 
can be used to identify the composition of the sample. 

• Industrial radiography uses X-rays for inspection of industrial parts, 
particularly welds. 

• Paintings are often X-rayed to reveal the underdrawing and 

pentimenti or alterations in the course of painting, or by later restorers. Many pigments such as lead white show 
well in X-ray photographs. 

• Airport security luggage scanners use X-rays for inspecting the interior of luggage for security threats before 
loading on aircraft. 

• Border security truck scanners use X-rays for inspecting the interior of trucks for at country borders. 

• X-ray fine art photography 

• Roentgen Stereophotogrammetry is used to track movement of bones based on the implantation of markers 



Each dot, called a reflection, in this diffraction 

pattern forms from the constructive interference 

of scattered X-rays passing through a crystal. The 

data can be used to determine the crystalline 

structure. 



X-rays 35 

• X-ray photoelectron spectroscopy is a chemical analysis technique relying on the photoelectric effect, usually 
employed in surface science. 

History 
Discovery 

German physicist Wilhelm Rontgen is usually credited as the 
discoverer of X-rays because he was the first to systematically study 
them, though he is not the first to have observed their effects. He is 
also the one who gave them the name "X-rays", though many referred 
to these as "Rontgen rays" for several decades after their discovery and 

X-ray fine art photography of needlefish by Peter 

to this day in some languages, including Rontgen's native German, and D . 

Swedish. 




X-rays were found emanating from Crookes tubes, experimental discharge tubes invented around 1875, by scientists 
investigating the cathode rays, that is energetic electron beams, that were first created in the tubes. Crookes tubes 
created free electrons by ionization of the residual air in the tube by a high DC voltage of anywhere between a few 
kilovolts and 100 kV. This voltage accelerated the electrons coming from the cathode to a high enough velocity that 
they created X-rays when they struck the anode or the glass wall of the tube. Many of the early Crookes tubes 

undoubtedly radiated X-rays, because early researchers noticed effects that were attributable to them, as detailed 

T421 
below. Wilhelm Rontgen was the first to systematically study them, in 1895. 

The important early researchers in X-rays were Ivan Pulyui, William Crookes, Johann Wilhelm Hittorf, Eugen 
Goldstein, Heinrich Hertz, Philipp Lenard, Hermann von Helmholtz, Nikola Tesla, Thomas Edison, Charles Glover 
Barkla, Max von Laue, and Wilhelm Conrad Rontgen. 

Johann Hittorf 

German physicist Johann Hittorf (1824—1914), a co-inventor and early researcher of the Crookes tube, found when 
he placed unexposed photographic plates near the tube, that some of them were flawed by shadows, though he did 
not investigate this effect. 

Ivan Pulyui 

In 1877 Ukrainian-born Pulyui, a lecturer in experimental physics at the University of Vienna, constructed various 

T431 
designs of vacuum discharge tube to investigate their properties. He continued his investigations when appointed 

professor at the Prague Polytechnic and in 1886 he found that sealed photographic plates became dark when exposed 

to the emanations from the tubes. Early in 1896, just a few weeks after Rontgen published his first X-ray photograph, 

[431 

Pulyui published high-quality X-ray images in journals in Paris and London. Although Pulyui had studied with 

Rontgen at the University of Strasbourg in the years 1873—75, his biographer Gaida (1997) asserts that his 

1431 
subsequent research was conducted independently. 

Nikola Tesla 

In April 1887, Nikola Tesla began to investigate X-rays using high voltages and tubes of his own design, as well as 
Crookes tubes. From his technical publications, it is indicated that he invented and developed a special 

[44] [451 

single-electrode X-ray tube, which differed from other X-ray tubes in having no target electrode. The 

principle behind Tesla's device is called the Bremsstrahlung process, in which a high-energy secondary X-ray 
emission is produced when charged particles (such as electrons) pass through matter. By 1892, Tesla performed 
several such experiments, but he did not categorize the emissions as what were later called X-rays. Tesla generalized 



X-rays 36 

the phenomenon as radiant energy of "invisible" kinds. Tesla stated the facts of his methods concerning 

various experiments in his 1897 X-ray lecture before the New York Academy of Sciences. Also in this lecture, 
Tesla stated the method of construction and safe operation of X-ray equipment. His X-ray experimentation by 

vacuum high field emissions also led him to alert the scientific community to the biological hazards associated with 

v [49] 

X-ray exposure. 

Fernando Sanford 

X-rays were generated and detected by Fernando Sanford (1854—1948), the foundation Professor of Physics at 
Stanford University, in 1891. From 1886 to 1888 he had studied in the Hermann Helmholtz laboratory in Berlin, 
where he became familiar with the cathode rays generated in vacuum tubes when a voltage was applied across 
separate electrodes, as previously studied by Heinrich Hertz and Philipp Lenard. His letter of January 6, 1893 
(describing his discovery as "electric photography") to The Physical Review was duly published and an article 
entitled Without Lens or Light, Photographs Taken With Plate and Object in Darkness appeared in the San Francisco 
Examiner. 

Philipp Lenard 

Philipp Lenard, a student of Heinrich Hertz, wanted to see whether cathode rays could pass out of the Crookes tube 
into the air. He built a Crookes tube (later called a "Lenard tube") with a "window" in the end made of thin 
aluminum, facing the cathode so the cathode rays would strike it. He found that something came through, that 



would expose photographic plates and cause fluorescence. He measured the penetrating power of these rays through 

[521 
various materials. It has been suggested that at least some of these "Lenard rays" were actually X-rays. Hermann 

von Helmholtz formulated mathematical equations for X-rays. He postulated a dispersion theory before Rontgen 

T531 
made his discovery and announcement. It was formed on the basis of the electromagnetic theory of light. 

However, he did not work with actual X-rays. 



Wilhelm Rontgen 

On November 8, 1895, German physics professor Wilhelm Rontgen stumbled on X-rays while experimenting with 

Lenard and Crookes tubes and began studying them. He wrote an initial report "On a new kind of ray: A preliminary 

T541 
communication" and on December 28, 1895 submitted it to the Wiirzburg's Physical-Medical Society journal. 

This was the first paper written on X-rays. Rontgen referred to the radiation as "X", to indicate that it was an 

unknown type of radiation. The name stuck, although (over Rontgen's great objections) many of his colleagues 

suggested calling them Rontgen rays. They are still referred to as such in many languages, including German and 

Russian. Rontgen received the first Nobel Prize in Physics for his discovery. 

There are conflicting accounts of his discovery because Rontgen had his lab notes burned after his death, but this is a 
likely reconstruction by his biographers: Rontgen was investigating cathode rays with a fluorescent screen 
painted with barium platinocyanide and a Crookes tube which he had wrapped in black cardboard so the visible light 
from the tube wouldn't interfere. He noticed a faint green glow from the screen, about 1 meter away. He realized 
some invisible rays coming from the tube were passing through the cardboard to make the screen glow. He found 
they could also pass through books and papers on his desk. Rontgen threw himself into investigating these unknown 
rays systematically. Two months after his initial discovery, he published his paper. 

Rontgen discovered its medical use when he saw a picture of his wife's hand on a photographic plate formed due to 
X-rays. His wife's hand's photograph was the first ever photograph of a human body part using X-rays. When she 
saw the picture, she said "I have seen my own death." 



X-rays 



37 



Thomas Edison 



J/V in 



In 1895, Thomas Edison investigated materials' ability to fluoresce 

when exposed to X-rays, and found that calcium tungstate was the 

most effective substance. Around March 1896, the fluoroscope he 

developed became the standard for medical X-ray examinations. 

Nevertheless, Edison dropped X-ray research around 1903 after the 

death of Clarence Madison Dally, one of his glassblowers. Dally had a 

habit of testing X-ray tubes on his hands, and acquired a cancer in 

them so tenacious that both arms were amputated in a futile attempt to 

save his life. At the 1901 Pan-American Exposition in Buffalo, New 

York, an assassin shot President William McKinley twice at close 

range with a .32 caliber revolver. The first bullet was removed but the second remained lodged somewhere in his 

stomach. McKinley survived for some time and requested that Thomas Edison "rush an X-ray machine to Buffalo to 

find the stray bullet. It arrived, but was not used as McKinley died of septic shock due to bacterial infection." 




Diagram of a water cooled X-ray tube 
(simplified/outdated) 



Frank Austin and the Frost brothers 

The first medical X-ray made in the United States was obtained using a discharge tube of Pulyui's design. In January 
1896, on reading of Rontgen's discovery, Frank Austin of Dartmouth College tested all of the discharge tubes in the 
physics laboratory and found that only the Pulyui tube produced X-rays. This was a result of Pulyui's inclusion of an 
oblique "target" of mica, used for holding samples of fluorescent material, within the tube. On 3 February 1896 
Gilman Frost, professor of medicine at the college, and his brother Edwin Frost, professor of physics, exposed the 
wrist of Eddie McCarthy, whom Edwin had treated some weeks earlier for a fracture, to the X-rays and collected the 
resulting image of the broken bone on gelatin photographic plates obtained from Howard Langill, a local 



photographer also interested in Rontgen's work. 



[24] 




20th century and beyond 

The many applications of X-rays immediately generated enormous 
interest. Workshops began making specialized versions of Crookes 
tubes for generating X-rays, and these first generation cold cathode or 
Crookes X-ray tubes were used until about 1920. 

Crookes tubes were unreliable. They had to contain a small quantity of 

gas (invariably air) as a current will not flow in such a tube if they are 

fully evacuated. However as time passed the X-rays caused the glass to 

absorb the gas, causing the tube to generate "harder" X-rays until it 

soon stopped operating. Larger and more frequently used tubes were 

provided with devices for restoring the air, known as "softeners". 

These often took the form of a small side tube which contained a small 

piece of mica: a substance that traps comparatively large quantities of 

air within its structure. A small electrical heater heated the mica and caused it to release a small amount of air, thus 

restoring the tube's efficiency. However the mica had a limited life and the restore process was consequently difficult 

to control. 

In 1904, John Ambrose Fleming invented the thermionic diode valve (vacuum tube). This used a hot cathode which 
permitted current to flow in a vacuum. This idea was quickly applied to X-ray tubes, and heated cathode X-ray tubes, 
called Coolidge tubes, replaced the troublesome cold cathode tubes by about 1920. 



A male technician taking an x-ray of a female 

patient in 1940. This image was used to argue 

that exposure to radiation during the x-ray 

procedure would be a myth. 



X-rays 



38 



Two years later, physicist Charles Barkla discovered that X-rays could be scattered by gases, and that each element 
had a characteristic X-ray. He won the 1917 Nobel Prize in Physics for this discovery. Max von Laue, Paul Knipping 
and Walter Friedrich observed for the first time the diffraction of X-rays by crystals in 1912. This discovery, along 
with the early works of Paul Peter Ewald, William Henry Bragg and William Lawrence Bragg gave birth to the field 
of X-ray crystallography. The Coolidge tube was invented the following year by William D. Coolidge which 
permitted continuous production of X-rays; this type of tube is still in use today. 

The use of X-rays for medical purposes (to develop into the field of 
radiation therapy) was pioneered by Major John Hall-Edwards in 
Birmingham, England. In 1908, he had to have his left arm amputated 



owing to the spread of X-ray dermatitis 
invented in the 1950s. 



[57] 



The X-ray microscope was 



The Chandra X-ray Observatory, launched on July 23, 1999, has been 
allowing the exploration of the very violent processes in the universe 
which produce X-rays. Unlike visible light, which is a relatively stable 
view of the universe, the X-ray universe is unstable, it features stars 
being torn apart by black holes, galactic collisions, and novas, neutron 
stars that build up layers of plasma that then explode into space. 




ROSAT image of X-ray fluorescence of, and 

occultation of the X-ray background by, the 

Moon 



An X-ray laser device was proposed as part of the Reagan 
Administration's Strategic Defense Initiative in the 1980s, but the first and only test of the device (a sort of laser 
"blaster", or death ray, powered by a thermonuclear explosion) gave inconclusive results. For technical and political 
reasons, the overall project (including the X-ray laser) was de-funded (though was later revived by the second Bush 
Administration as National Missile Defense using different technologies). 



See also 

Backscatter X-ray 

detective quantum efficiency 

High energy X-rays 

Nray 

Neutron radiation 

Radiologic technologist 

Resonant inelastic X-ray scattering (RIXS) 

Small angle X-ray scattering (SAXS) 

X-ray absorption spectroscopy 

X-ray generation 

X-ray marker 

X-ray nanoprobe 

X-ray optics 

X-ray vision 

X-ray welding 

X-ray reflectivity 



X-rays 39 

Notes 

[I] Novelline, Robert. Squire's Fundamentals of Radiology. Harvard University Press. 5th edition. 1997. ISBN 0674833392. 
[2] Oxford English Dictionary http://www.oed.com 

[3] http://physics.nist.gov/cgi-bin/ffast/ffast.pl?Formula=H2O&gtype=5&range=S&lower=0.300&upper=2.00&density=1.00 

[4] Dendy, P. P.; B. Heaton (1999). Physics for Diagnostic Radiology (http://books.google.com/?id=lBTQvsQIs4wC&pg=PA12). USA: 

CRC Press, p. 12. ISBN 0750305916. . 
[5] Charles Hodgman, Ed. (1961). CRC Handbook of Chemistry and Physics, 44th Ed.. USA: Chemical Rubber Co.. p. 2850. 
[6] Feynman, Richard; Robert Leighton, Matthew Sands (1963). The Feynman Lectures on Physics, Vol.1. USA: Addison- Wesley, pp. 2—5. 

ISBN 0201021161. 
[7] LAnnunziata, Michael; Mohammad Baradei (2003). Handbook of Radioactivity Analysis (http://books. google. com/?id=b519el0OPT0C& 

pg=PA58&dq=gamma+x-ray). Academic Press, p. 58. ISBN 0124366031. . 
[8] Grupen, Claus; G. Cowan, S. D. Eidelman, T. Stroh (2005). Astroparticle Physics. Springer, p. 109. ISBN 3540253122. 
[9] US National Research Council (2006). Health Risks from Low Levels of Ionizing Radiation, BEIR 7 phase 2 (http://books.google.com/ 

?id=Uqj40zBKlHwC&pg=PA5). National Academies Press, pp. 5, fig.PS-2. ISBN 030909156X. ., data credited to NCRP (US National 

Committee on Radiation Protection) 1987 
[10] http://www.doctorspiller.com/Dental%20_X-Rays.htm and http://www.dentalgentlecare.com/x-ray_safety.htm 

[II] (http://hss.energy.gov/NuclearSafety/NSEA/fire/trainingdocs/radem3.pdf) 
[12] (http://www.hawkhill.eom/l 14s.html) 

[13] http://www.solarstorms.org/SWChapter8.html and http://www.powerattunements.com/x-ray.html 

[14] David R. Lide, ed (1994). CRC Handbook of Chemistry and Physics 75th edition. CRC Press, pp. 10-227. ISBN 0-8493-0475-X. 

[15] Kevles, Bettyann Holtzmann (1996). Naked to the Bone Medical Imaging in the Twentieth Century. Camden, NJ: Rutgers University Press. 

pp. 19-22. ISBN 0813523583. 
[16] Sample, Sharron (2007-03-27). "X-Rays" (http://science.hq.nasa.gov/kids/imagers/ems/xrays.html). The Electromagnetic Spectrum. 

NASA. . Retrieved 2007-12-03. 
[17] Whaites, Eric; Roderick Cawson (2002). Essentials of Dental Radiography and Radiology (http://books. google. com/?id=x6ThiifBPcsC& 

dq=radiography+kilovolt+x-ray+machine). Elsevier Health Sciences, pp. 15—20. ISBN 044307027X. . 
[18] Bushburg, Jerrold; Anthony Seibert, Edwin Leidholdt, John Boone (2002). The Essential Physics of Medical Imaging (http://books. google. 

com/?id=VZvqqaQ5DvoC&pg=PT33&dq=radiography+kerma+rem+Sievert). USA: Lippincott Williams & Wilkins. p. 1 16. 

ISBN 0683301187.. 
[19] Emilio, Burattini; Antonella Ballerna (1994). "Preface" (http://books.google.com/books?id=VEld4080nekC&pg=PA129& 

dq="synchrotron+radiation"+x-ray+advantages&as_brr=3). . IOS Press, pp. xv. ISBN 9051992483. . Retrieved 2008-11-11. 
[20] Martin, Dylan (2005). "X-Ray Detection" (http://www.u.arizona.edu/~dwmartin/). University of Arizona Optical Sciences Center. . 

Retrieved 2008-05-19. 
[21] Frame, Paul. "Wilhelm Rontgen and the Invisible Light" (http://www.orau.org/ptp/articlesstories/invisiblelight.htm). Tales from the 

Atomic Age. Oak Ridge Associated Universities. . Retrieved 2008-05-19. 
[22] http://www.esrf.eu/UsersAndScience/Experiments/MaterialsScience/faisceau 

[23] Easements of Modern X-Ray Physics. John Wiley & Sons Ltd,. 2001. pp. 40-41. ISBN 0-471-49858-0. 
[24] Spiegel, Peter K (1995). "The first clinical X-ray made in America — 100 years" (http://www.ajronline.Org/cgi/reprint/164/l/241.pdf). 

American Journal of Roentgenology (Leesburg, VA: American Roentgen Ray Society) 164 (1): 241-243. ISSN: 1546-3141. PMID 7998549. . 
[25] Herman, Gabor T. (2009). Fundamentals of Computerized Tomography: Image Reconstruction from Projections (2nd ed.). Springer. 

ISBN 978-1-85233-617-2 
[26] "11th Report on Carcinogens" (http://ntp.niehs.nih.gov/ntp/roc/tocll.html). Ntp.niehs.nih.gov. . Retrieved 2010-11-08. 
[27] Upton, AC (2003). "The state of the art in the 1990s: NCRP report No. 136 on the scientific bases for linearity in the dose-response 

relationship for ionizing radiation". Health Physics 85: 15—22. 
[28] Calabrese and Baldwin, Toxicology rethinks its central belief, Nature, 421, pp. 691-2, 13 February 2003. 
[29] United Nations Scientific Committee on the Effects of Atomic Radiation. New York. United Nations, 2000 
[30] Berrington; de Gonzalez, A; Darby, S (2004). "Risk of cancer from diagnostic X-rays: estimates for the UK and 14 other countries". Lancet 

363: 345-351. 
[31] Brenner DJ and Hall EJ (2007). "Computed tomography- an increasing source of radiation exposure." (http://www.nejm.org/doi/full/10. 

1056/NEJMra072149). New England Journal of Medicine 357: 2277-2284. . 
[32] (http://www.radiologyinfo.org/en/safety/index.cfm?pg=sfty_xray)IRadiological Society of North America and American College of 

Radiology 
[33] Caon, M., Bibbo, G. & Pattison, J. (2000) Monte Carlo calculated effective dose to teenage girls from computed tomography examinations, 

Radiation Protection Dosimetry 90(4):445-448. 
[34] (http://seer.cancer.gov/csr/1975_2006/browse_csr.php?section=2&page=sect_02_table. ll.html#tablel)INational Cancer Institute: 

Surveillance Epidemiology and End Results (SEER) data 
[35] Gregory KG, Bibbo G and Pattison JE (2008) On the uncertanties in effective dose estimates of adult CT head scans, Medical Physics 

35(8):3501-3510. 



X-rays 40 

[36] Xiao-Ou, Shu; et al (December 1994). "Association of paternal diagnostic X-ray exposure with risk of infant leukemia" (http://www.ncbi. 

nlm.nih.gov/pubmed/7881337). Cancer Epidemiology, Biomarkers & Prevention (American Association for Cancer Research) 3 (8): 645. 

ISSN 1538-7755. PMID 7881337. . 
[37] Stewart, Alice M; Webb, J.W.; Giles, B.D.; Hewitt, D. (1956). "Preliminary Communication: Malignant Disease in Childhood and 

Diagnostic Irradiation In-Utero". Lancet 271 (6940): 447. PMID 13358242. 
[38] "Pregnant Women and Radiation Exposure" (http://emedicinelive.com/index.php/Women-s-Health/ 

pregnant-women-and-radiation-exposure.html). eMedicine Live online medical consultation. Medscape. 28 December 2008. . Retrieved 

2009-01-16. 
[39] Donnelly, CF (2005). "Reducing radiation dose associated with pediatric CT by decreasing unnecessary examinations". American Journal 

Roentgenology 32: 242-244. 
[40] Alchemy Art Lead Products — Lead Shielding Sheet Lead For Shielding Applications (http://www.alchemycastings.com/pdf/SheetLead. 

pdf). Retrieved 2008-12-07. 
[41] Kasai, Nobutami; Masao Kakudo (2005). X-ray diffraction by macromolecules. Tokyo: Kodansha. pp. 291—2. ISBN 3540253173. 
[42] The history, development, and impact of computed imaging in neurological diagnosis and neurosurgery: CT, MRI, DTI: Nature Precedings 

DOI: 10.1038/npre.2009.3267.5 (http://precedings.nature.eom/documents/3267/version/5). 
[43] Gaida, Roman; et al. (1997). "Ukrainian Physicist Contributes to the Discovery of X-Rays" (http://web.archive.org/web/ 

20080528172938/http://www. meduniv.lviv.ua/oldsite/puluj. html). Mayo Foundation for Medical Education and Research. Archived 

from the original (http://www.meduniv.lviv.ua/oldsite/puluj.html) on 2008-05-28. . Retrieved 2008-04-06. 
[44] Morton, William James, and Edwin W. Hammer, American Technical Book Co., 1896. Page 68. 
[45] U.S. Patent 514170 (http://www.google.com/patents?vid=514170), Incandescent Electric Light, and U.S. Patent 454622 (http://www. 

google.com/patents ?vid=454622), System of Electric Lighting. 
[46] Cheney, Margaret, " Tesla: Man Out of Time (http://books. google.com/books ?vid=ISBN0743215362)". Simon and Schuster, 2001. Page 

77. 
[47] Thomas Commerford Martin (ed.), " The Inventions, Researches and Writings of Nikola Tesla (http://books.google.com/ 

books ?vid=OCLC04049568)". Page 252 "When it forms a drop, it will emit visible and invisible waves. [...]". (ed., this material originally 

appeared in an article by Nikola Tesla in The Electrical Engineer of 1894.) 
[48] Nikola Tesla, "The stream of Lenard and Roentgen and novel apparatus for their production", Apr. 6, 1897. 
[49] Cheney, Margaret, Robert Uth, and Jim Glenn, " Tesla, master of lightning (http://books. google. com/books?vid=ISBN0760710058)". 

Barnes & Noble Publishing, 1999. Page 76. ISBN 0760710058 
[50] Wyman, Thomas (Spring 2005). "Fernando Sanford and the Discovery of X-rays". "Imprint", from the Associates of the Stanford University 

Libraries: 5—15. 
[51] Thomson, Joseph J. (1903). The Discharge of Electricity through Gasses (http://books. google. com/?id=Ryw4AAAAMAAJ& 

pg=PA138). USA: Charles Scribner's Sons. pp. 182-186. . 
[52] Thomson, 1903,p.l85 
[53] Wiedmann's Annalen, Vol. XLVIII 
[54] Stanton, Arthur (1896-01-23). "Wilhelm Conrad Rontgen On a New Kind of Rays: translation of a paper read before the Wurzburg Physical 

and Medical Society, 1895" (http://www.nature.com/nature/journal/v53/nl369/pdf/053274b0.pdf) (Subscription-only access — 

(http://scholar.google.co.uk/scholar?hl=en&lr=&q=author:Stanton+intitle:Wilhelm+Conrad+RAfntgen+On+a+New+Kind+ 

of+Rays:+translation+of+a+paper+read+before+the+WA 1 /4rzburg+Physical+and+Medical+Society,+ 1895& 

as_publication=[[Nature+(journal)INature]]&as_ylo=1896&as_yhi=1896&btnG=Search)). Nature 53 (1369): 274-6. 

doi: 10.1038/053274b0. see also pp. 268 and 276 of the same issue. 
[55] Peters, Peter (1995). "W. C. Roentgen and the discovery of x-rays" (http://www.medcyclopaedia.com/library/radiology/chapter01. 

aspx). Ch.l Textbook of Radiology. Medcyclopedia.com, GE Healthcare. . Retrieved 2008-05-05. 
[56] National Library of Medicine. " Could X-rays Have Saved President William McKinley? (http://www.nlm.nih.gov/visibleproofs/ 

galleries/cases/mckinley.html)" Visible Proofs: Forensic Views of the Body. 
[57] (http://www.birmingham.gov.uk/xraylBirmingham.gov.uk) 



X-rays 41 

References 

• NASA (http://imagers.gsfc.nasa.gov/ems/xrays.html) Goddard Space Flight centre introduction to X-rays. 

External links 

• Example Radiograph: Fractured Humerus (http://www.rtstudents.com/x-rays/broken-humerus-xray.htm) 

• A Photograph of an X-ray Machine (http://www.iuk.edu/~koalhe/img/Equipment/xray.jpg) 

• X-ray Safety (http://www.x-raysafety.com/) 

• An X-ray tube demonstration (Animation) (http://www.ionactive.co.uk/multi-media_video.html?m=4) 

• 1896 Article: "On a New Kind of Rays" (http://web.archive.Org/web/20070710033139/http://deutsche. 
nature . com/phy sics/7 . pdf) 

• "Digital X-Ray Technologies Project" (http://docs.google.com/ 
fileview?id=OB89CZuXbiY7mNmQxYmVlNDktNjBiZSOONjcwLTgOODgtZjc3NWUwOWUxZDg5&hl=tr) 

• A video of a medical X-ray procedure example (http://nursing-resource.com/?p=198#videolink) 

• What is Radiology? (http://rad.usuhs.mil/rad/home/whatis.html) a simple tutorial 

• 50,000 X-ray, MRI, and CT pictures (http://rad.usuhs.edu/medpix) MedPix medical image database 

• Index of Early Bremsstrahlung Articles (http://www.datasync.com/~rsfl/bremindx.htm) 

• Extraordinary X-Rays (http://www.life.com/image/first/in-gallery/44881/extraordinary-x-rays) — slideshow 
by Life magazine 



Electron 



42 



Electron 



Electron 










& US 1 




Experiments with a Crookes tube first demonstrated the particle nature of electrons. In this illustration, the profile of the 
cross-shaped target is projected against the tube face at right by a beam of electrons. 


Composition: 


[21 
Elementary particle 


Particle statistics: 


Fermionic 


Group: 


Lepton 


Generation: 


First 


Interaction: 


Gravity, Electromagnetic, Weak 


Symbol(s): 


e-,p- 


Antiparticle: 


Positron (also called antielectron) 


Theorized: 


Richard Laming (1838-1 851), [3] 

G. Johnstone Stoney (1874) and others. [4] [5] 


Discovered: 


J. J. Thomson (1897) [6] 


Mass: 


9.10938215(45) x 10" 31 kg [7] 5.4857990943(23) x 10" 4 u [7] 
[1822.88850204(77)]"' u [ ] 
0.510998910(13) MeV/c 2[7] 


Electric charge: 


-le [9] 

-1.602176487(40) x 10" 19 C [7] -4.803 x 10" 10 esu [10] 


Magnetic moment: 


-1.00115965218111 u [7] 

B 


Spin: 


\ 



The electron is a subatomic particle carrying a negative electric charge. It has no known components or substructure. 
Therefore, the electron is generally believed to be an elementary particle. An electron has a mass that is 
approximately 1/1836 that of the proton. The intrinsic angular momentum (spin) of the electron is a half-integer 
value in units of h, which means that it is a fermion. The antiparticle of the electron is called the positron. The 
positron is identical to the electron except that it carries electrical and other charges of the opposite sign. When an 
electron collides with a positron, both particles may either scatter off each other or be totally annihilated, producing a 
pair (or more) of gamma ray photons. Electrons, which belong to the first generation of the lepton particle family, 



Electron 43 

ri3i 

participate in gravitational, electromagnetic and weak interactions. Electrons, like all matter, have quantum 
mechanical properties of both particles and waves, so they can collide with other particles and be diffracted like 
light. However, this duality is best demonstrated in experiments with electrons, due to their tiny mass. Since an 
electron is a fermion, no two electrons can occupy the same quantum state, in accordance with the Pauli exclusion 
principle. 

The concept of an indivisible amount of electric charge was theorized to explain the chemical properties of atoms, 

mi 
beginning in 1838 by British natural philosopher Richard Laming; the name electron was introduced for this 

charge in 1894 by Irish physicist George Johnstone Stoney. The electron was identified as a particle in 1897 by J. J. 

Thomson and his team of British physicists. 

In many physical phenomena, such as electricity, magnetism, and thermal conductivity, electrons play an essential 
role. An electron in motion relative to an observer generates a magnetic field, and will be deflected by external 
magnetic fields. When an electron is accelerated, it can absorb or radiate energy in the form of photons. Electrons, 
together with atomic nuclei made of protons and neutrons, make up atoms. However, electrons contribute less than 
0.06% to an atom's total mass. The attractive Coulomb force between an electron and a proton causes electrons to be 
bound into atoms. The exchange or sharing of the electrons between two or more atoms is the main cause of 
chemical bonding. 

According to theory, most electrons in the universe were created in the big bang, but they may also be created 
through beta decay of radioactive isotopes and in high-energy collisions, for instance when cosmic rays enter the 
atmosphere. Electrons may be destroyed through annihilation with positrons, and may be absorbed during 
nucleosynthesis in stars. Laboratory instruments are capable of containing and observing individual electrons as well 
as electron plasma, whereas dedicated telescopes can detect electron plasma in outer space. Electrons have many 
applications, including welding, cathode ray tubes, electron microscopes, radiation therapy, lasers and particle 
accelerators. 

History 

The ancient Greeks noticed that amber attracted small objects when rubbed with fur. Apart from lightning, this 

ri7i 
phenomenon is humanity's earliest recorded experience with electricity. In his 1600 treatise De Magnete, the 

English scientist William Gilbert coined the New Latin term electricus, to refer to this property of attracting small 

objects after being rubbed. Both electric and electricity are derived from the Latin electrum (also the root of the 

alloy of the same name), which came from the Greek word rj^eKxpov (elektron) for amber. 

In 1737 C. F. du Fay and Hawksbee independently discovered what they believed to be two kinds of frictional 
electricity; one generated from rubbing glass, the other from rubbing resin. From this, Du Fay theorized that 
electricity consists of two electrical fluids, "vitreous" and "resinous", that are separated by friction and that neutralize 

ri9i 

each other when combined. A decade later Benjamin Franklin proposed that electricity was not from different 
types of electrical fluid, but the same electrical fluid under different pressures. He gave them the modern charge 

[201 r211 T221 

nomenclature of positive and negative respectively. Franklin thought that the charge carrier was positive. 

Between 1838 and 1851, British natural philosopher Richard Laming developed the idea that an atom is composed of 
a core of matter surrounded by subatomic particles that had unit electric charges. Beginning in 1846, German 
physicist William Weber theorized that electricity was composed of positively and negatively charged fluids, and 
their interaction was governed by the inverse square law. After studying the phenomenon of electrolysis in 1874, 
Irish physicist George Johnstone Stoney suggested that there existed a "single definite quantity of electricity", the 

charge of a monovalent ion. He was able to estimate the value of this elementary charge e by means of Faraday's 

T231 
laws of electrolysis. However, Stoney believed these charges were permanently attached to atoms and could not 

be removed. In 1881, German physicist Hermann von Helmholtz argued that both positive and negative charges 

were divided into elementary parts, each of which "behaves like atoms of electricity". 




Electron 44 

In 1894, Stoney coined the term electron to describe these elementary charges, saying, "... an estimate was made of 

the actual amount of this most remarkable fundamental unit of electricity, for which I have since ventured to suggest 

T241 
the name electron" . The word electron is a combination of the word electric and the suffix -on, with the latter 

now used to designate a subatomic particle, such as a proton or neutron. 

Discovery 

The German physicist Johann Wilhelm Hittorf undertook the study of 
electrical conductivity in rarefied gases. In 1869, he discovered a glow 
emitted from the cathode that increased in size with decrease in gas 
pressure. In 1876, the German physicist Eugen Goldstein showed that 
the rays from this glow cast a shadow, and he dubbed them cathode 

[98] 

rays. During the 1870s, the English chemist and physicist Sir 

William Crookes developed the first cathode ray tube to have a high 

[291 
vacuum inside. He then showed that the luminescence rays 

A beam of electrons deflected in a circle by a 

appearing within the tube carried energy and moved from the cathode . „ , ,[27] 

rr ° OJ magnetic field 

to the anode. Furthermore, by applying a magnetic field, he was able to 

deflect the rays, thereby demonstrating that the beam behaved as though it were negatively charged. In 1879, 

he proposed that these properties could be explained by what he termed 'radiant matter'. He suggested that this was a 
fourth state of matter, consisting of negatively charged molecules that were being projected with high velocity from 
the cathode. 

The German-born British physicist Arthur Schuster expanded upon Crookes' experiments by placing metal plates in 
parallel to the cathode rays and applying an electric potential between the plates. The field deflected the rays toward 
the positively charged plate, providing further evidence that the rays carried negative charge. By measuring the 
amount of deflection for a given level of current, in 1890 Schuster was able to estimate the charge-to-mass ratio of 
the ray components. However, this produced a value that was more than a thousand times greater than what was 

[30] [33] 

expected, so little credence was given to his calculations at the time. 

1141 

In 1896, the British physicist J. J. Thomson, with his colleagues John S. Townsend and H. A. Wilson, performed 
experiments indicating that cathode rays really were unique particles, rather than waves, atoms or molecules as was 
believed earlier. Thomson made good estimates of both the charge e and the mass m, finding that cathode ray 
particles, which he called "corpuscles," had perhaps one thousandth of the mass of the least massive ion known: 
hydrogen. He showed that their charge to mass ratio, elm, was independent of cathode material. He further 

showed that the negatively charged particles produced by radioactive materials, by heated materials and by 
illuminated materials were universal. The name electron was again proposed for these particles by the Irish 

physicist George F. Fitzgerald, and the name has since gained universal acceptance. 

While studying naturally fluorescing minerals in 1896, the French physicist Henri Becquerel discovered that they 
emitted radiation without any exposure to an external energy source. These radioactive materials became the subject 
of much interest by scientists, including the New Zealand physicist Ernest Rutherford who discovered they emitted 
particles. He designated these particles alpha and beta, on the basis of their ability to penetrate matter. In 1900, 
Becquerel showed that the beta rays emitted by radium could be deflected by an electric field, and that their 
mass-to-charge ratio was the same as for cathode rays. This evidence strengthened the view that electrons existed 

. , . [37] [38] 

as components of atoms. 

The electron's charge was more carefully measured by the American physicist Robert Millikan in his oil-drop 
experiment of 1909, the results of which he published in 1911. This experiment used an electric field to prevent a 
charged droplet of oil from falling as a result of gravity. This device could measure the electric charge from as few as 
1—150 ions with an error margin of less than 0.3%. Comparable experiments had been done earlier by Thomson's 
team, using clouds of charged water droplets generated by electrolysis, and in 1911 by Abram Ioffe, who 



Electron 



45 



independently obtained the same result as Millikan using charged microparticles of metals, then published his results 

[39] 
in 1913. However, oil drops were more stable than water drops because of their slower evaporation rate, and thus 

more suited to precise experimentation over longer periods of time. 

Around the beginning of the twentieth century, it was found that under certain conditions a fast moving charged 
particle caused a condensation of supersaturated water vapor along its path. In 1911, Charles Wilson used this 
principle to devise his cloud chamber, allowing the tracks of charged particles, such as fast-moving electrons, to be 
photographed. 



Atomic theory 

By 1914, experiments by physicists Ernest Rutherford, Henry Moseley, 
James Franck and Gustav Hertz had largely established the structure of 
an atom as a dense nucleus of positive charge surrounded by 

[421 

lower-mass electrons. In 1913, Danish physicist Niels Bohr 

postulated that electrons resided in quantized energy states, with the 

energy determined by the angular momentum of the electron's orbits 

about the nucleus. The electrons could move between these states, or 

orbits, by the emission or absorption of photons at specific frequencies. 

By means of these quantized orbits, he accurately explained the 

[43] 
spectral lines of the hydrogen atom. However, Bohr's model failed 

to account for the relative intensities of the spectral lines and it was 

unsuccessful in explaining the spectra of more complex atoms 



[42] 



Increasing energy 
of orbits 




A photon is emitted 
with energy E =hf 



The Bohr model of the atom, showing states of 
electron with energy quantized by the number n. 

An electron dropping to a lower orbit emits a 

photon equal to the energy difference between the 

orbits. 



Chemical bonds between atoms were explained by Gilbert Newton 

Lewis, who in 1916 proposed that a covalent bond between two atoms 

is maintained by a pair of electrons shared between them. Later, in 

1923, Walter Heitler and Fritz London gave the full explanation of the 

electron-pair formation and chemical bonding in terms of quantum mechanics. In 1919, the American chemist 

Irving Langmuir elaborated on the Lewis' static model of the atom and suggested that all electrons were distributed 

in successive "concentric (nearly) spherical shells, all of equal thickness". The shells were, in turn, divided by 

him in a number of cells each containing one pair of electrons. With this model Langmuir was able to qualitatively 

explain the chemical properties of all elements in the periodic table, which were known to largely repeat 

themselves according to the periodic law 



[47] 



In 1924, Austrian physicist Wolfgang Pauli observed that the shell-like structure of the atom could be explained by a 
set of four parameters that defined every quantum energy state, as long as each state was inhabited by no more than a 
single electron. (This prohibition against more than one electron occupying the same quantum energy state became 
known as the Pauli exclusion principle.) The physical mechanism to explain the fourth parameter, which had two 
distinct possible values, was provided by the Dutch physicists Abraham Goudsmith and George Uhlenbeck when 
they suggested that an electron, in addition to the angular momentum of its orbit, could possess an intrinsic angular 
momentum. This property became known as spin, and explained the previously mysterious splitting of 



spectral lines observed with a high-resolution spectrograph; this phenomenon is known as fine structure splitting 



[50] 



Electron 



46 



Quantum mechanics 

In his 1924 dissertation Recherche s sur la theorie des quanta (Research on Quantum Theory), French physicist 
Louis de Broglie hypothesized that all matter possesses a De Broglie wave similar to light. That is, under the 
appropriate conditions, electrons and other matter would show properties of either particles or waves. The 
corpuscular properties of a particle are demonstrated when it is shown to have a localized position in space along its 
trajectory at any given moment. Wave-like nature is observed, for example, when a beam of light is passed 
through parallel slits and creates interference patterns. In 1927, the interference effect was demonstrated with a beam 
of electrons by English physicist George Paget Thomson with a thin metal film and by American physicists Clinton 
Davisson and Lester Germer using a crystal of nickel. 

The success of de Broglie's prediction led to the publication, by Erwin 
Schrodinger in 1926, of the Schrodinger equation that successfully 
describes how electron waves propagated. Rather than yielding a 
solution that determines the location of an electron over time, this 
wave equation can be used to predict the probability of finding an 
electron near a position. This approach was later called quantum 
mechanics, which provided an extremely close derivation to the energy 
states of an electron in a hydrogen atom. Once spin and the 
interaction between multiple electrons were considered, quantum 
mechanics allowed the configuration of electrons in atoms with higher 
atomic numbers than hydrogen to be successfully predicted 




[56] 



In 1928, building on Wolfgang Pauli's work, Paul Dirac produced a 

model of the electron - the Dirac equation, consistent with relativity 

theory, by applying relativistic and symmetry considerations to the 

hamiltonian formulation of the quantum mechanics of the 

electro-magnetic field. In order to resolve some problems within his 

relativistic equation, in 1930 Dirac developed a model of the vacuum 

as an infinite sea of particles having negative energy, which was 

dubbed the Dirac sea. This led him to predict the existence of a 

positron, the antimatter counterpart of the electron. This particle 

was discovered in 1932 by Carl D. Anderson, who proposed calling standard electrons negatrons, and using electron 

as a generic term to describe both the positively and negatively charged variants. This usage of the term 'negatron' is 



Orbital s ((= 0, m^=0) 

In quantum mechanics, the behavior of an 

electron in an atom is described by an orbital, 

which is a probability distribution rather than an 

orbit. In the figure, the shading indicates the 

relative probability to "find" the electron, having 

the energy corresponding to the given quantum 

numbers, at that point. 



still occasionally encountered today, and it may be shortened to 'negaton' 



[59] [60] 



In 1947 Willis Lamb, working in collaboration with graduate student Robert Rutherford, found that certain quantum 
states of hydrogen atom, which should have the same energy, were shifted in relation to each other, the difference 
being the Lamb shift. About the same time, Polykarp Kusch, working with Henry M. Foley, discovered the magnetic 
moment of the electron is slightly larger than predicted by Dirac's theory. This small difference was later called 
anomalous magnetic dipole moment of the electron. To resolve these issues, a refined theory called quantum 
electrodynamics was developed by Sin-Itiro Tomonaga, Julian Schwinger and Richard P. Feynman in the late 



1940s 



[61] 



Electron 



47 



Particle accelerators 

With the development of the particle accelerator during the first half of the twentieth century, physicists began to 
delve deeper into the properties of subatomic particles. The first successful attempt to accelerate electrons using 
Electromagnetic induction was made in 1942 by Donald Kerst. His initial betatron reached energies of 2.3 MeV, 
while subsequent betatrons achieved 300 MeV. In 1947, synchrotron radiation was discovered with a 70 MeV 
electron synchrotron at General Electric. This radiation was caused by the acceleration of electrons, moving near the 
speed of light, through a magnetic field 



[63] 



With a beam energy of 1.5 GeV, the first high-energy particle collider was ADONE, which began operations in 
1968. This device accelerated electrons and positrons in opposite directions, effectively doubling the energy of 
their collision when compared to striking a static target with an electron. The Large Electron-Positron Collider 
(LEP) at CERN, which was operational from 1989 to 2000, achieved collision energies of 209 GeV and made 
important measurements for the Standard Model of particle physics. 



Characteristics 



Classification 



Three Generations 
of Matter (Fermions) 



In the Standard Model of particle physics, 
electrons belong to the group of subatomic 
particles called leptons, which are believed 
to be fundamental or elementary particles. 
Electrons have the lowest mass of any 
charged lepton (or electrically charged 
particle of any type) and belong to the 
first-generation of fundamental particles. 
The second and third generation contain 
charged leptons, the muon and the tau, 
which are identical to the electron in charge, 
spin and interactions, but are more massive. 
Leptons differ from the other basic 
constituent of matter, the quarks, by their 
lack of strong interaction. All members of 
the lepton group are fermions, because they 

all have half-odd integer spin; the electron 

u ■ 1/ [69] 

has spin / . 

Fundamental properties 

The invariant mass of an electron is 

approximately 9.109 x 10~ kilogram, or 5.489 x 10~ atomic mass unit. On the basis of Einstein's principle of 

mass— energy equivalence, this mass corresponds to a rest energy of 0.511 MeV. The ratio between the mass of a 

proton and that of an electron is about 1836. Astronomical measurements show that the proton-to-electron 

T711 
mass ratio has held the same value for at least half the age of the universe, as is predicted by the Standard Model. 

Electrons have an electric charge of -1.602 x 10" coulomb, which is used as a standard unit of charge for 
subatomic particles. Within the limits of experimental accuracy, the electron charge is identical to the charge of a 

1721 

proton, but with the opposite sign. As the symbol e is used for the elementary charge, the electron is commonly 
symbolized by e~, where the minus sign indicates the negative charge. The positron is symbolized by e because it 





i 


II 


III 




mass^* 


2.4 MeV 


1.27 GeV 


171.2 GeV 





charge-* 
spin-* 


lU 




% t 

v 2 I 


:y 


name-* 


up 


charm 


top 


photon 




4.8 MeV 


104 MeV 


4.2 GeV 







rd 


V2 O 


:b 


: g 


O 


down 


strange 


bottom 


gluon 




<2.2 eV 


<0.17 MeV 


<15.5 MeV 


91.2 GeV n 




V 


V 


V 


: z 




electron 
neutrino 


muon 
neutrino 


tau 
neutrino 


weak 
force 




0.511 MeV 


105.7 MeV 


1.777 GeV 


80.4 GeV 

+ 


to 

c 
o 

4-> 


1 p 


V, li 


It 


fW 


Q. 

5 


electron 


1 
muon 


tau 


weak 
force 



U 



c 
o 
in 

o 

CO. 



Standard Model of elementary particles. The electron is at lower left. 



Electron 



48 



has the same properties as the electron but with a positive rather than negative charge. 

1 T71 
The electron has an intrinsic angular momentum or spin of / . This property is usually stated by referring to the 

electron as a spin-/ particle. For such particles the spin magnitude is / h. while the result of the 

measurement of a projection of the spin on any axis can only be ± V . In addition to spin, the electron has an intrinsic 

T71 T741 T751 

magnetic moment along its spin axis. It is approximately equal to one Bohr magneton, which is a physical 

—24 T71 

constant equal to 9.274 009 15(23) x 10 joules per tesla. The orientation of the spin with respect to the 

momentum of the electron defines the property of elementary particles known as helicity. 

T21 T771 
The electron has no known substructure. Hence, it is defined or assumed to be a point particle with a point 

ri2i 

charge and no spatial extent. Observation of a single electron in a Penning trap shows the upper limit of the 

—22 r"7Qi 

particle's radius is 10 meters. There is a physical constant called the "classical electron radius", with the much 
larger value of 2.8179 x 10~ m. However, the terminology comes from a simplistic calculation that ignores the 
effects of quantum mechanics; in reality, the so-called classical electron radius has little to do with the true 



fundamental structure of the electron 



[79] [80] 



There are elementary particles that spontaneously decay into less massive particles. An example is the muon, which 
decays into an electron, a neutrino and an antineutrino, with a mean lifetime of 2.2 x 10" seconds. However, the 
electron is thought to be stable on theoretical grounds: the electron is the least massive particle with non-zero electric 

roil 

charge, so its decay would violate charge conservation. The experimental lower bound for the electron's mean 
lifetime is 4.6 x 10 years, at a 90% confidence level. 



[83] 



Quantum properties 

As with all particles, electrons can act as waves. This is called the wave— particle duality and can be demonstrated 
using the double-slit experiment. The wave-like nature of the electron allows it to pass through two parallel slits 
simultaneously, rather than just one slit as would be the case for a classical particle. In quantum mechanics, the 
wave-like property of one particle can be described mathematically as a complex-valued function, the wave function, 
commonly denoted by the Greek letter psi (tp). When the absolute value of this function is squared, it gives the 
probability that a particle will be observed near a location — a probability density. 

Electrons are identical particles because they cannot be distinguished 
from each other by their intrinsic physical properties. In quantum 
mechanics, this means that a pair of interacting electrons must be able 
to swap positions without an observable change to the state of the 
system. The wave function of fermions, including electrons, is 
antisymmetric, meaning that it changes sign when two electrons are 
swapped; that is, ip{r , r ) = -ip(r , r ), where the variables r and r 
correspond to the first and second electrons, respectively. Since the 
absolute value is not changed by a sign swap, this corresponds to equal 
probabilities. Bosons, such as the photon, have symmetric wave 




functions instead 



[83] 



Example of an antisymmetric wave function for a 

quantum state of two identical fermions in a 

2-dimensional box. If the particles swap position, 

the wave function inverts its sign. 



In the case of antisymmetry, solutions of the wave equation for 

interacting electrons result in a zero probability that each pair will 

occupy the same location or state. This is responsible for the Pauli exclusion principle, which precludes any two 

electrons from occupying the same quantum state. This principle explains many of the properties of electrons. For 

example, it causes groups of bound electrons to occupy different orbitals in an atom, rather than all overlapping each 



other in the same orbit 



[83] 



Electron 49 

Virtual particles 

Physicists believe that empty space may be continually creating pairs of virtual particles, such as a positron and 
electron, which rapidly annihilate each other shortly thereafter. The combination of the energy variation needed to 
create these particles, and the time during which they exist, fall under the threshold of detectability expressed by the 
Heisenberg uncertainty relation, AE'At > h. In effect, the energy needed to create these virtual particles, AE, can be 
"borrowed" from the vacuum for a period of time, At, so that their product is no more than the reduced Planck 
constant, h ~ 6.6 x 10 eV«s. Thus, for a virtual electron, At is at most 1.3 x 10 s. 





s 

e 




electron 


A schematic depiction of virtual 
-positron pairs appearing at random near 
an electron (at lower left) 



While an electron— positron virtual pair is in existence, the coulomb 
force from the ambient electric field surrounding an electron causes a 
created positron to be attracted to the original electron, while a created 
electron experiences a repulsion. This causes what is called vacuum 
polarization. In effect, the vacuum behaves like a medium having a 
dielectric permittivity more than unity. Thus the effective charge of an 
electron is actually smaller than its true value, and the charge decreases 
with increasing distance from the electron. This polarization 

was confirmed experimentally in 1997 using the Japanese TRISTAN 

rooi 

particle accelerator. Virtual particles cause a comparable shielding 
effect for the mass of the electron. 

The interaction with virtual particles also explains the small (about 

0.1%) deviation of the intrinsic magnetic moment of the electron from the Bohr magneton (the anomalous magnetic 

moment). The extraordinarily precise agreement of this predicted difference with the experimentally 

1911 
determined value is viewed as one of the great achievements of quantum electrodynamics. 

In classical physics, the angular momentum and magnetic moment of an object depend upon its physical dimensions. 

Hence, the concept of a dimensionless electron possessing these properties might seem inconsistent. The apparent 

paradox can be explained by the formation of virtual photons in the electric field generated by the electron. These 

[92] 
photons cause the electron to shift about in a jittery fashion (known as zitterbewegung), which results in a net 

rj21 [93] 

circular motion with precession. This motion produces both the spin and the magnetic moment of the electron. 
In atoms, this creation of virtual photons explains the Lamb shift observed in spectral lines. 

Interaction 

An electron generates an electric field that exerts an attractive force on a particle with a positive charge, such as the 
proton, and a repulsive force on a particle with a negative charge. The strength of this force is determined by 
Coulomb's inverse square law. When an electron is in motion, it generates a magnetic field. The 
Ampere-Maxwell law relates the magnetic field to the mass motion of electrons (the current) with respect to an 
observer. It is this property of induction which supplies the magnetic field that drives an electric motor. The 
electromagnetic field of an arbitrary moving charged particle is expressed by the Lienard— Wiechert potentials, which 
are valid even when the particle's speed is close to that of light (relativistic). 



Electron 



50 



When an electron is moving through a magnetic field, it is subject to 

the Lorentz force that exerts an influence in a direction perpendicular 

to the plane defined by the magnetic field and the electron velocity. 

This centripetal force causes the electron to follow a helical trajectory 

through the field at a radius called the gyroradius. The acceleration 

from this curving motion induces the electron to radiate energy in the 

[97] [98] [99] 
form of synchrotron radiation. The energy emission in turn 

causes a recoil of the electron, known as the Abraham-Lorentz-Dirac 

force, which creates a friction that slows the electron. This force is 

caused by a back-reaction of the electron's own field upon itself. 

In quantum electrodynamics the electromagnetic interaction between 

particles is mediated by photons. An isolated electron that is not 

undergoing acceleration is unable to emit or absorb a real photon; 

doing so would violate conservation of energy and momentum. 

Instead, virtual photons can transfer momentum between two charged particles. It is this exchange of virtual photons 

that, for example, generates the Coulomb force. Energy emission can occur when a moving electron is deflected 

by a charged particle, such as a proton. The acceleration of the electron results in the emission of Bremsstrahlung 



q < 0] 




v y^ 


B 


q ^\ q = 


kL) 


q > o\ 




A particle with charge q (at left) is 


moving with 


velocity v through a magnetic field B that is 


oriented toward the viewer. For an 


electron, q is 


negative so it follows a curved trajectory toward 


the top. 





radiation 



[102] 




h-f=E 2 -E 1 




i 



Here, Bremsstrahlung is produced by an electron 

e deflected by the electric field of an atomic 

nucleus. The energy change E - E determines 

the frequency /of the emitted photon. 



An inelastic collision between a photon (light) and a solitary (free) electron is called Compton scattering. This 
collision results in a transfer of momentum and energy between the particles, which modifies the wavelength of the 
photon by an amount called the Compton shift. The maximum magnitude of this wavelength shift is him c, 
which is known as the Compton wavelength. For an electron, it has a value of 2.43 x 10~ m. When the 
wavelength of the light is long (for instance, the wavelength of the visible light is 0.4—0.7 |xm) the wavelength shift 
becomes negligible. Such interaction between the light and free electrons is called Thomson scattering or Linear 
Thomson scattering. 

The relative strength of the electromagnetic interaction between two charged particles, such as an electron and a 
proton, is given by the fine-structure constant. This value is a dimensionless quantity formed by the ratio of two 
energies: the electrostatic energy of attraction (or repulsion) at a separation of one Compton wavelength, and the rest 



Electron 



51 



energy of the charge. It is given by a ~ 7.297353 x 10 , which is approximately equal to /.._. 

When electrons and positrons collide, they annihilate each other, giving rise to two or more gamma ray photons. If 
the electron and positron have negligible momentum, a positronium atom can form before annihilation results in two 
or three gamma ray photons totalling 1.022 MeV. On the other hand, high-energy photons may transform 

into an electron and a positron by a process called pair production, but only in the presence of a nearby charged 
particle, such as a nucleus. 

In the theory of electroweak interaction, the left-handed component of electron's wavefunction forms a weak isospin 
doublet with the electron neutrino. This means that during weak interactions, electron neutrinos behave like 
electrons. Either member of this doublet can undergo a charged current interaction by emitting or absorbing a W and 
be converted into the other member. Charge is conserved during this reaction because the W boson also carries a 
charge, canceling out any net change during the transmutation. Charged current interactions are responsible for the 
phenomenon of beta decay in a radioactive atom. Both the electron and electron neutrino can undergo a neutral 
current interaction via a Z exchange, and this is responsible for neutrino-electron elastic scattering. 



Atoms and molecules 

An electron can be bound to the nucleus of an 
atom by the attractive Coulomb force. A system 
of several electrons bound to a nucleus is called 
an atom. If the number of electrons is different 
from the nucleus' electrical charge, such an atom 
is called an ion. The wave-like behavior of a 
bound electron is described by a function called 
an atomic orbital. Each orbital has its own set of 
quantum numbers such as energy, angular 
momentum and projection of angular 
momentum, and only a discrete set of these 
orbitals exist around the nucleus. According to 
the Pauli exclusion principal each orbital can be 
occupied by up to two electrons, which must 
differ in their spin quantum number. 

Electrons can transfer between different orbitals 
by the emission or absorption of photons with an 
energy that matches the difference in 
potential. Other methods of orbital transfer 




Probability densities for the first few hydrogen atom orbitals, seen in 
cross-section. The energy level of a bound electron determines the orbital it 
occupies, and the color reflects the probability to find the electron at a given 

position. 



include collisions with particles, such as 

electrons, and the Auger effect. In order to escape the atom, the energy of the electron must be increased above 
its binding energy to the atom. This occurs, for example, with the photoelectric effect, where an incident photon 
exceeding the atom's ionization energy is absorbed by the electron 



[113] 



The orbital angular momentum of electrons is quantized. Because the electron is charged, it produces an orbital 
magnetic moment that is proportional to the angular momentum. The net magnetic moment of an atom is equal to the 
vector sum of orbital and spin magnetic moments of all electrons and the nucleus. The nuclear magnetic moment is, 
however, negligible in comparison to the effect from the electrons. The magnetic moments of the electrons that 
occupy the same orbital (so called, paired electrons) cancel each other out 



[114] 



The chemical bond between atoms occurs as a result of electromagnetic interactions, as described by the laws of 
quantum mechanics. The strongest bonds are formed by the sharing or transfer of electrons between atoms, 



Electron 



52 



allowing the formation of molecules. Within a molecule, electrons move under the influence of several nuclei, 
and occupy molecular orbitals; much as they can occupy atomic orbitals in isolated atoms. A fundamental factor 
in these molecular structures is the existence of electron pairs. These are electrons with opposed spins, allowing them 
to occupy the same molecular orbital without violating the Pauli exclusion principle (much like in atoms). Different 
molecular orbitals have different spatial distribution of the electron density. For instance, in bonded pairs (i.e. in the 
pairs that actually bind atoms together) electrons can be found with the maximal probability in a relatively small 
volume between the nuclei. On the contrary, in non-bonded pairs electrons are distributed in a large volume around 



nuclei 



[117] 



Conductivity 

If a body has more or fewer electrons than are required to balance the 
positive charge of the nuclei, then that object has a net electric charge. 
When there is an excess of electrons, the object is said to be negatively 
charged. When there are fewer electrons than the number of protons in 
nuclei, the object is said to be positively charged. When the number of 
electrons and the number of protons are equal, their charges cancel 
each other and the object is said to be electrically neutral. A 
macroscopic body can develop an electric charge through rubbing, by 
the triboelectric effect. 

Independent electrons moving in vacuum are termed free electrons. 
Electrons in metals also behave as if they were free. In reality the 
particles that are commonly termed electrons in metals and other solids 
are quasi-electrons — quasi-particles, which have the same electrical 
charge, spin and magnetic moment as real electrons but may have a 

M221 

different mass. When free electrons — both in vacuum and 

metals — move, they produce a net flow of charge called an electric 
current, which generates a magnetic field. Likewise a current can be created by a changing magnetic field. These 




A lightning discharge consists primarily of a flow 
of electrons. The electric potential needed 

for lightning may be generated by a triboelectric 
' effecJ 119][12 ° f 



interactions are described mathematically by Maxwell's equations 



[123] 



At a given temperature, each material has an electrical conductivity that determines the value of electric current 
when an electric potential is applied. Examples of good conductors include metals such as copper and gold, whereas 
glass and Teflon are poor conductors. In any dielectric material, the electrons remain bound to their respective atoms 
and the material behaves as an insulator. Most semiconductors have a variable level of conductivity that lies between 
the extremes of conduction and insulation. On the other hand, metals have an electronic band structure 

containing partially filled electronic bands. The presence of such bands allows electrons in metals to behave as if 
they were free or delocalized electrons. These electrons are not associated with specific atoms, so when an electric 
field is applied, they are free to move like a gas (called Fermi gas) through the material much like free electrons. 

Because of collisions between electrons and atoms, the drift velocity of electrons in a conductor is on the order of 
millimeters per second. However, the speed at which a change of current at one point in the material causes changes 
in currents in other parts of the material, the velocity of propagation, is typically about 75% of light speed. This 
occurs because electrical signals propagate as a wave, with the velocity dependent on the dielectric constant of the 
material. 

Metals make relatively good conductors of heat, primarily because the delocalized electrons are free to transport 
thermal energy between atoms. However, unlike electrical conductivity, the thermal conductivity of a metal is nearly 
independent of temperature. This is expressed mathematically by the Wiedemann-Franz law, which states that 
the ratio of thermal conductivity to the electrical conductivity is proportional to the temperature. The thermal 



Electron 



53 



disorder in the metallic lattice increases the electrical resistivity of the material, producing a temperature dependence 



for electrical current 



[128] 



When cooled below a point called the critical temperature, materials can undergo a phase transition in which they 
lose all resistivity to electrical current, in a process known as superconductivity. In BCS theory, this behavior is 
modeled by pairs of electrons entering a quantum state known as a Bose— Einstein condensate. These Cooper pairs 
have their motion coupled to nearby matter via lattice vibrations called phonons, thereby avoiding the collisions with 

M291 

atoms that normally create electrical resistance. (Cooper pairs have a radius of roughly 100 nm, so they can 



overlap each other.) 
uncertain. 



[130] 



However, the mechanism by which higher temperature superconductors operate remains 



Electrons inside conducting solids, which are quasi-particles themselves, when tightly confined at temperatures close 

[1311 [1321 

to absolute zero, behave as though they had split into two other quasiparticles: spinons and holons. The 

former carries spin and magnetic moment, while the latter electrical charge. 



Motion and energy 

According to Einstein's theory of special relativity, as an electron's speed approaches the speed of light, from an 
observer's point of view its relativistic mass increases, thereby making it more and more difficult to accelerate it 
from within the observer's frame of reference. The speed of an electron can approach, but never reach, the speed of 
light in a vacuum, c. However, when relativistic electrons — that is, electrons moving at a speed close to c — are 
injected into a dielectric medium such as water, where the local speed of light is significantly less than c, the 

electrons temporarily travel faster than light in the medium. As they interact with the medium, they generate a faint 

[1331 
light called Cherenkov radiation. 



V, 
10 
9 
8 
7 
6 
5 
4 
3 
2 
1 


i 
























J 




1 




J 




__^-~^' 








Lore 

va 


c V 

ntz factor as a function of velocity. It starts at 
lue 1 and goes to infinity as v approaches c. 



The effects of special relativity are based on a quantity known as the Lorentz factor, defined as 7=1/^/1— i^/c 2 
where v is the speed of the particle. The kinetic energy K of an electron moving with velocity v is: 



K, 



(7-i; 



m e <r, 



where m is the mass of electron. For example, the Stanford linear accelerator can accelerate an electron to roughly 

II 341 2 

51 GeV. This gives a value of nearly 100,000 for y, since the mass of an electron is 0.51 MeV/c . The 

relativistic momentum of this electron is 100,000 times the momentum that classical mechanics would predict for an 
electron at the same speed 



[135] 



Since an electron behaves as a wave, at a given velocity it has a characteristic de Broglie wavelength. This is given 
by 2 = hip where h is the Planck constant and p is the momentum. For the 51 GeV electron above, the 
wavelength is about 2.4 x 10~ m, small enough to explore structures well below the size of an atomic nucleus. 



Electron 



54 



Formation 

The Big Bang theory is the most widely accepted scientific theory to 
explain the early stages in the evolution of the Universe. For the 
first millisecond of the Big Bang, the temperatures were over 
10 billion kelvins and photons had mean energies over a million 
electronvolts. These photons were sufficiently energetic that they could 
react with each other to form pairs of electrons and positrons, 







P? a 


Nucleus 


y 


Electron (e~) 


"*• 


G4-- 






Photon (y) 


\ 


Positron (e + ) 


Pair production caused by 


the collision of a 


photon with 


an atomic nucleus 



7 + 7 ^ e + + e , 
where y is a photon, e is a positron and e~ is an electron. Likewise, positron-electron pairs annihilated each other 
and emitted energetic photons. An equilibrium between electrons, positrons and photons was maintained during this 
phase of the evolution of the Universe. After 15 seconds had passed, however, the temperature of the universe 

dropped below the threshold where electron-positron formation could occur. Most of the surviving electrons and 

n 3Ri 
positrons annihilated each other, releasing gamma radiation that briefly reheated the universe. 

For reasons that remain uncertain, during the process of leptogenesis there was an excess in the number of electrons 

11391 
over positrons. Hence, about one electron in every billion survived the annihilation process. This excess matched 

the excess of protons over anti-protons, in a condition known as baryon asymmetry, resulting in a net charge of zero 

for the universe. The surviving protons and neutrons began to participate in reactions with each other — in 

the process known as nucleosynthesis, forming isotopes of hydrogen and helium, with trace amounts of lithium. This 

process peaked after about five minutes. Any leftover neutrons underwent negative beta decay with a half-life of 

about a thousand seconds, releasing a proton and electron in the process, 

n => p + e~ + v e , 
where n is a neutron, p is a proton and v is an electron antineutrino. For about the next 300,000—400,000 years, the 

excess electrons remained too energetic to bind with atomic nuclei. What followed is a period known as 

11441 
recombination, when neutral atoms were formed and the expanding universe became transparent to radiation. 

11441 

Roughly one million years after the big bang, the first generation of stars began to form. Within a star, stellar 
nucleosynthesis results in the production of positrons from the fusion of atomic nuclei. These antimatter particles 
immediately annihilate with electrons, releasing gamma rays. The net result is a steady reduction in the number of 
electrons, and a matching increase in the number of neutrons. However, the process of stellar evolution can result in 
the synthesis of radioactive isotopes. Selected isotopes can subsequently undergo negative beta decay, emitting an 
electron and antineutrino from the nucleus. An example is the cobalt-60 ( Co) isotope, which decays to form 
nickel-60 ( 60 Ni). [146] 



Electron 



55 



- 35 km 




N = 10 



At the end of its lifetime, a star with more than 
about 20 solar masses can undergo gravitational 
collapse to form a black hole. According to 
classical physics, these massive stellar objects 
exert a gravitational attraction that is strong 
enough to prevent anything, even 
electromagnetic radiation, from escaping past the 
Schwarzschild radius. However, it is believed 
that quantum mechanical effects may allow 
Hawking radiation to be emitted at this distance. 
Electrons (and positrons) are thought to be 
created at the event horizon of these stellar 
remnants. 

When pairs of virtual particles (such as an 
electron and positron) are created in the vicinity 
of the event horizon, the random spatial 
distribution of these particles may permit one of them to appear on the exterior; this process is called quantum 
tunneling. The gravitational potential of the black hole can then supply the energy that transforms this virtual particle 
into a real particle, allowing it to radiate away into space. In exchange, the other member of the pair is given 
negative energy, which results in a net loss of mass-energy by the black hole. The rate of Hawking radiation 
increases with decreasing mass, eventually causing the black hole to evaporate away until, finally, it explodes. 

20 

Cosmic rays are particles traveling through space with high energies. Energy events as high as 3.0 x 10 eV have 
been recorded. When these particles collide with nucleons in the Earth's atmosphere, a shower of particles is 
generated, including pions. More than half of the cosmic radiation observed from the Earth's surface consists of 
muons. The particle called a muon is a lepton which is produced in the upper atmosphere by the decay of a pion. A 
muon, in turn, can decay to form an electron or positron. Thus, for the negatively charged pion n 



M(e) = 18% 
N(y)=18% 



An extended air shower generated by an energetic cosmic ray striking the 
Earth's atmosphere 



[152] 



TV 



/'■ 



fl +v. 



/J-' 



+ V e + V. 



fit 



where u is a muon and v is a muon neutrino. 



Observation 

Remote observation of electrons requires detection of their radiated 
energy. For example, in high-energy environments such as the corona 
of a star, free electrons form a plasma that radiates energy due to 
Bremsstrahlung. Electron gas can undergo plasma oscillation, which is 
waves caused by synchronized variations in electron density, and these 
produce energy emissions that can be detected by using radio 
telescopes. 

The frequency of a photon is proportional to its energy. As a bound 
electron transitions between different energy levels of an atom, it will 
absorb or emit photons at characteristic frequencies. For instance, 
when atoms are irradiated by a source with a broad spectrum, distinct absorption lines will appear in the spectrum of 

transmitted radiation. Each element or molecule displays a characteristic set of spectral lines, such as the hydrogen 
spectral series. Spectroscopic measurements of the strength and width of these lines allow the composition and 




Aurorae are mostly caused by energetic electrons 

..... , [153] 

precipitating into the atmosphere. 



Electron 



56 



physical properties of a substance to be determined. 

In laboratory conditions, the interactions of individual electrons can be observed by means of particle detectors, 
which allow measurement of specific properties such as energy, spin and charge. The development of the Paul 
trap and Penning trap allows charged particles to be contained within a small region for long durations. This enables 
precise measurements of the particle properties. For example, in one instance a Penning trap was used to contain a 
single electron for a period of 10 months. The magnetic moment of the electron was measured to a precision of 
eleven digits, which, in 1980, was a greater accuracy than for any other physical constant. 

The first video images of an electron's energy distribution were captured by a team at Lund University in Sweden, 
February 2008. The scientists used extremely short flashes of light, called attosecond pulses, which allowed an 
electron's motion to be observed for the first time. 

The distribution of the electrons in solid materials can be visualized by angle resolved photoemission spectroscopy 
(ARPES). This technique employs the photoelectric effect to measure the reciprocal space — a mathematical 
representation of periodic structures that is used to infer the original structure. ARPES can be used to determine the 
direction, speed and scattering of electrons within the material. 

Plasma applications 



Particle beams 



[163], 



Electron beams are used in welding, which allows energy densities 

7 —2 

up to 10 W'cm across a narrow focus diameter of 0.1—1.3 mm and 
usually does not require a filler material. This welding technique must 
be performed in a vacuum, so that the electron beam does not interact 
with the gas prior to reaching the target, and it can be used to join 
conductive materials that would otherwise be considered unsuitable for 
welding. [164] [165] 

Electron beam lithography (EBL) is a method of etching 
semiconductors at resolutions smaller than a micron. This 

technique is limited by high costs, slow performance, the need to 
operate the beam in the vacuum and the tendency of the electrons to 
scatter in solids. The last problem limits the resolution to about 10 nm. 
For this reason, EBL is primarily used for the production of small 



numbers of specialized integrated circuits 



[167] 




During a NASA wind tunnel test, a model of the 

Space Shuttle is targeted by a beam of electrons, 

simulating the effect of ionizing gases during 
, [162] 
re-entry. 



Electron beam processing is used to irradiate materials in order to change their physical properties or sterilize 
medical and food products. In radiation therapy, electron beams are generated by linear accelerators for 

treatment of superficial tumors. Because an electron beam only penetrates to a limited depth before being absorbed, 
typically up to 5 cm for electron energies in the range 5—20 MeV, electron therapy is useful for treating skin lesions 
such as basal cell carcinomas. An electron beam can be used to supplement the treatment of areas that have been 
irradiated by X-rays. 

Particle accelerators use electric fields to propel electrons and their antiparticles to high energies. As these particles 
pass through magnetic fields, they emit synchrotron radiation. The intensity of this radiation is spin dependent, 
which causes polarization of the electron beam — a process known as the Sokolov— Ternov effect. The polarized 
electron beams can be useful for various experiments. Synchrotron radiation can also be used for cooling the electron 
beams, which reduces the momentum spread of the particles. Once the particles have accelerated to the required 
energies, separate electron and positron beams are brought into collision. The resulting energy emissions are 

[1721 

observed with particle detectors and are studied in particle physics. 



Electron 57 

Imaging 

Low-energy electron diffraction (LEED) is a method of bombarding a crystalline material with a collimated beam of 

electrons, then observing the resulting diffraction patterns to determine the structure of the material. The required 

ri73i 
energy of the electrons is typically in the range 20—200 eV. The reflection high energy electron diffraction 



(RHEED) technique uses the reflection of a beam of electrons fired at various low angles to characterize the surface 

of cr 

[175]' 



of crystalline materials. The beam energy is typically in the range 8—20 keV and the angle of incidence is 1—4°. 



The electron microscope directs a focused beam of electrons at a specimen. As the beam interacts with the material, 

some electrons change their properties, such as movement direction, angle, relative phase and energy. By recording 

these changes in the electron beam, microscopists can produce atomically resolved image of the material. In blue 

[1771 
light, conventional optical microscopes have a diffraction-limited resolution of about 200 nm. By comparison, 

electron microscopes are limited by the de Broglie wavelength of the electron. This wavelength, for example, is 

n 7ri 
equal to 0.0037 nm for electrons accelerated across a 100,000-volt potential. The Transmission Electron 

Aberration-corrected Microscope is capable of sub-0.05 nm resolution, which is more than enough to resolve 

individual atoms. This capability makes the electron microscope a useful laboratory instrument for high 

resolution imaging. However, electron microscopes are expensive instruments that are costly to maintain. 

There are two main types of electron microscopes: transmission and scanning. Transmission electron microscopes 
function in a manner similar to overhead projector, with a beam of electrons passing through a slice of material then 
being projected by lenses on a photographic slide or a charge-coupled device. In scanning electron microscopes, the 
image is produced by rastering a finely focused electron beam, as in a TV set, across the studied sample. The 
magnifications range from lOOx to l,000,000x or higher for both microscope types. The scanning tunneling 
microscope uses quantum tunneling of electrons from a sharp metal tip into the studied material and can produce 
atomically resolved images of its surface. 

Other 

In the free electron laser (FEL), a relativistic electron beam is passed through a pair of undulators containing arrays 
of dipole magnets, whose fields are oriented in alternating directions. The electrons emit synchrotron radiation, 
which, in turn, coherently interacts with the same electrons. This leads to the strong amplification of the radiation 
field at the resonance frequency. FEL can emit a coherent high-brilliance electromagnetic radiation with a wide 

range of frequencies, from microwaves to soft X-rays. These devices can be used in the future for manufacturing, 

n R^i 
communication and various medical applications, such as soft tissue surgery. 

Electrons are at the heart of cathode ray tubes, which are used extensively as display devices in laboratory 
instruments, computer monitors and television sets. In a photomultiplier tube, every photon striking the 

photocathode initiates an avalanche of electrons that produces a detectable current pulse. Vacuum tubes use the 
flow of electrons to manipulate electrical signals, and they played a critical role in the development of electronics 
technology. However, they have been largely supplanted by solid-state devices such as the transistor. 

See also 

• Anyon • g-factor 

• Covalent bonding • Spintronics 

• Electride • Stern— Gerlach experiment 

• Electron bubble • Zeeman effect 

• Exoelectron emission 



Electron 



58 



Notes 

[1] Dahl, Per F. (1997). Flash of the Cathode Rays: A History of J J Thomson's Electron (http://books.google.com/?id=xUzaWGocMdMC& 

printsec=frontcover). CRC Press, p. 72. ISBN 0750304537. . 
[2] Eichten, Estia J.; Peskin, Michael E.; Peskin, Michael (1983). "New Tests for Quark and Lepton Substructure". Physical Review Letters 50 

(11): 811-814. doi:10.1103/PhysRevLett.50.811. 
[3] Farrar, Wilfred V. (1969). "Richard Laming and the Coal-Gas Industry, with His Views on the Structure of Matter". Annals of Science 25: 

243-254. doi:10.1080/00033796900200141. 
[4] Arabatzis, Theodore (2006). Representing Electrons: A Biographical Approach to Theoretical Entities (http://books.google.com/ 

?id=rZHT-chpLmAC&pg=PA70). University of Chicago Press, pp. 70-74. ISBN 0226024210. . 
[5] Buchwald, Jed Z.; Warwick, Andrew (2001). Histories of the Electron: The Birth of Microphysics (http://books.google.com/ 

?id=lyqqhlIdCOoC&pg=PA195). MIT Press, pp. 195-203. ISBN 0262524244. . 
[6] Thomson, Joseph John (1897). "Cathode Rays" (http://web.lemoyne.edu/~GIUNTA/thomsonl897.html). Philosophical Magazine 44: 

293.. 
[7] The original source for COD ATA is: 

Mohr, Peter J.; Taylor, Barry N.; Newell, David B. (2006-06-06). "CODATA recommended values of the 
fundamental physical constants". Reviews of Modern Physics 80: 633—730. doi:10. 1103/RevModPhys.80.633. 

Individual physical constants from the CODATA are available at: 

"The NIST Reference on Constants, Units and Uncertainty" (http:/ / physics, nist. gov/ cuu/ ). National 
Institute of Standards and Technology. . Retrieved 2009-01-15. 

[8] The fractional version's denominator is the inverse of the decimal value (along with its relative standard uncertainty of 4.2 x 10 u). 

[9] The electron's charge is the negative of elementary charge, which has a positive value for the proton. 

[10 

[11 



[12 
[13 

[14 
[15 

[16 

[17 
[18 

[19 

[20 

[21 

[22 

[23 

[24 
[25 
[26 
[27 

[28 
[29 

[30 

[31 



Purcell, Edward M. (1985). Electricity and Magnetism. Berkeley Physics Course Volume 2. McGraw-Hill. ISBN 0-07-004908-4. 

"CODATA value: proton-electron mass ratio" (http://physics.nist.gov/cgi-bin/cuu/Value7mpsme). National Institute of Standards and 
Technology. . Retrieved 2009-07-18. 

Curtis, Lorenzo J. (2003). Atomic Structure and Lifetimes: A Conceptual Approach (http://books. google. com/?id=KmwCsuvxClAC& 
pg=PA74). Cambridge University Press, p. 74. ISBN 0521536359. . 

Anastopoulos, Charis (2008). Particle Or Wave: The Evolution of the Concept of Matter in Modern Physics (http://books.google.com/ 
?id=rDEvQZhpltEC&pg=PA236). Princeton University Press, pp. 236-237. ISBN 0691 135 126. . 

Dahl (1997:122-185). 

Wilson, Robert (1997). Astronomy Through the Ages: The Story of the Human Attempt to Understand the Universe (http://books. google. 
com/?id=AoiJ3hA8bQ8C&pg=PA138). CRC Press, p. 138. ISBN 0748407480. . 

Pauling, Linus C. (1960). The Nature of the Chemical Bond and the Structure of Molecules and Crystals: an introduction to modern 
structural chemistry (http://books.google.com/?id=L-lK9HmKmUUC) (3rd ed.). Cornell University Press, pp. 4-10. ISBN 0801403332. . 

Shipley, Joseph T. (1945). Dictionary of Word Origins. The Philosophical Library, p. 133. 

Baigrie, Brian (2006). Electricity and Magnetism: A Historical Perspective (http://books. google. com/?id=3XEc5xkWxi4C&pg=PA7). 
Greenwood Press, pp. 7-8. ISBN 0-3133-3358-0. . 

Keithley, Joseph F. (1999). The Story of Electrical and Magnetic Measurements: From 500 B.C. to the 1940s (http://books.google.com/ 
?id=uwgNAtqSHuQC&pg=PA207). Wiley. ISBN 0-780-31193-0. . 

Benjamin Franklin (1706—1790). (http://scienceworld.wolfram.com/biography/FranklinBenjamin.html) Science World, from Eric 
Weisstein's World of Scientific Biography. 

The Encyclopedia Americana; a library of universal knowledge. (1918). New York: Encyclopedia Americana Corp. 

Myers, Rusty L. (2006). The basics of physics (http://books. google. com/books?id=KnynjL44pI4C). Greenwood Publishing Group, 
p. 242. ISBN 0-313-32857-9. ., Chapter 13, page 242 (http://books.google.com/books?id=KnynjL44pI4C&pg=PA242) 

Barrow, John D. (1983). "Natural Units Before Planck". Royal Astronomical Society Quarterly Journal 24: 24—26. 
Bibcode: 1983QJRAS..24...24B. 

Stoney, George Johnstone (1894). "Of the "Electron," or Atom of Electricity". Philosophical Magazine 38 (5): 418^-20. 

Soukhanov, Anne H. ed. (1986). Word Mysteries & Histories. Houghton Mifflin Company, p. 73. ISBN 0-395-40265-4. 

Guralnik, David B. ed. (1970). Webster's New World Dictionary. Prentice-Hall. p. 450. 

Born, Max; Blin-Stoyle, Roger John; Radcliffe, J. M. (1989). Atomic Physics (http://books.google.com/?id=NmM-KujxMtoC& 
pg=PA26). Courier Dover, p. 26. ISBN 0486659844. . 

Dahl (1997:55-58). 

DeKosky, Robert (1983). "William Crookes and the quest for absolute vacuum in the 1870s". Annals of Science 40 (1): 1—18. 
doi: 10.1080/00033798300200101. 

Leicester, Henry M. (1971). The Historical Background of Chemistry (http://books. google. com/?id=aJZVQnqcwv4C&pg=PA221). 
Courier Dover Publications, pp. 221-222. ISBN 0486610535. . 

Dahl (1997:64-78). 



Electron 59 

[32] Zeeman, Pieter (1907). "Sir William Crookes, F.R.S." (http://books.google.com/?id=UtYRAAAAYAAJ). Nature 11 (1984): 1-3. 

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[33] Dahl (1997:99). 
[34] Thomson, J. J. (1906). "Nobel Lecture: Carriers of Negative Electricity" (http://nobelprize.org/nobel_prizes/physics/laureates/1906/ 

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h 

2^ 



S=y/s(s+l) 

for quantum number s = / 

See: Gupta, M. C. (2001). Atomic and Molecular Spectroscopy (http://books.google.com/?id=0tIAlM6DiQIC&pg=PA81). New Age 

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,, eh 

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2 
electron's rest energy, defined by special relativity (E=mc ). 

From electrostatics theory, the potential energy of a sphere with radius r and charge e is given by: 



p 8neor 



where £ is the vacuum permittivity. For an electron with rest mass m the rest energy is equal to: 

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[182] Bozzola, John J.; Russell, Lonnie Dee (1999). Electron Microscopy: Principles and Techniques for Biologists (http://books.google.com/ 

?id=RqSMzR-IXk0C&pg=PA9) (2nd ed.). Jones & Bartlett Publishers, p. 9. ISBN 0763701920. . 
[183] Freund, Henry P.; Antonsen, Thomas (1996). Principles of Free-Electron Lasers (http://books. google. com/?id=73w9tqTgbiIC& 

pg=PAl). Springer, pp. 1-30. ISBN 0412725401. . 
[184] Kitzmiller, John W. (1995). Television Picture Tubes and Other Cathode-Ray Tubes: Industry and Trade Summary. DIANE Publishing. 

pp. 3-5. ISBN 0788121006. 
[185] Sclater, Neil (1999). Electronic Technology Handbook. McGraw-Hill Professional, pp. 227-228. ISBN 0070580480. 
[186] Staff (2008). "The History of the Integrated Circuit" (http://nobelprize.org/educational_games/physics/integrated_circuit/history/). The 

Nobel Foundation. . Retrieved 2008-10-18. 

References 
External links 

• "The Discovery of the Electron" (http://www.aip.org/history/electron/). American Institute of Physics, Center 
for History of Physics. Retrieved 2006-08-10. 

• "Particle Data Group" (http://pdg.lbl.gov/). University of California. Retrieved 2008-1 1-17. 

• Bock, Rudolf K.; Vasilescu, Angela (1998). The Particle Detector BriefBook (http://physics.web.cern.ch/ 
Physics/ParticleDetector/BriefBook/) (14th ed.). Springer. ISBN 3540641203. Retrieved 2008-10-02. 



Neutron 



65 



Neutron 



Neutron 




The quark structure of the neutron. (The color assignment of individual quarks is not important, only that all three colors are 

present.) 


Classification: 


Baryon 


Composition: 


1 up quark, 2 down quarks 


Particle statistics: 


Fermionic 


Group: 


Hadron 


Interaction: 


Gravity, Weak, Strong 


Symbol(s): 


n, n , N 


Antiparticle: 


Antineutron 


Theorized: 


Ernest Rutherford [1] (1920) 


Discovered: 


James Chadwick [1] (1932) 


Mass: 


1.67492729(28) x 10" 27 kg 
939.565560(81) MeV/c 2 
1.0086649156(6) u [2] 


Mean lifetime: 


885.7(8) s (free) 


Electric charge: 


Oe 
0C 


Electric dipole moment: 


<2.9x 10" 26 ecm 


Electric polarizability: 


1.16(15) x 10" 3 fm 3 


Magnetic moment: 


-1.9130427(5) \i 


Magnetic polarizability: 


3.7(20) x 10" 4 fm 3 


Spin: 


\ 


Isospin: 


\ 


Parity: 


+ 1 


Condensed: 


/(/) = \( l / 2 + ) 



The neutron is a subatomic particle with no net electric charge and a mass slightly larger than that of a proton. With 
the exception of hydrogen, nuclei of atoms consist of protons and neutrons, which are therefore collectively referred 



Neutron 66 

to as nucleons. The number of protons in a nucleus is the atomic number and defines the type of element the atom 
forms. The number of neutrons is the neutron number and determines the isotope of an element. For example, the 
abundant carbon-12 isotope has 6 protons and 6 neutrons, while the very rare radioactive carbon-14 isotope has 6 
protons and 8 neutrons. 

While bound neutrons in stable nuclei are stable, free neutrons are unstable; they undergo beta decay with a mean 
lifetime of just under 15 minutes (885.7 ± 0.8 s). Free neutrons are produced in nuclear fission and fusion. 
Dedicated neutron sources like research reactors and spallation sources produce free neutrons for use in irradiation 
and in neutron scattering experiments. Even though it is not a chemical element, the free neutron is sometimes 
included in tables of nuclides. It is then considered to have an atomic number of zero and a mass number of one, 
and is sometimes referred to as neutronium. 

The neutron has been the key to nuclear power production. After the neutron was discovered in 1932, it was realized 
in 1933 that it might mediate a nuclear chain reaction. In the 1930's, neutrons were used to produce many different 
types of nuclear transmutations. When nuclear fission was discovered in 1938, it was soon realized that this might be 
the mechanism to produce the neutrons for the chain reaction, if the process also produced neutrons, and this was 
proven in 1939, making the path to nuclear power production evident. These events and findings led directly to the 
first nuclear chain reaction which was self-sustaining (1942) and to the first nuclear weapons in 1945. 

Discovery 

In 1920, Ernest Rutherford conceptualised the possible existence of the neutron. In particular, Rutherford considered 
that the disparity found between the atomic number of an atom and its atomic mass could be explained by the 
existence of a neutrally charged particle within the atomic nucleus. 

In 1931 Walther Bothe and Herbert Becker in Germany found that if the very energetic alpha particles emitted from 
polonium fell on certain light elements, specifically beryllium, boron, or lithium, an unusually penetrating radiation 
was produced. At first this radiation was thought to be gamma radiation, although it was more penetrating than any 
gamma rays known, and the details of experimental results were very difficult to interpret on this basis. The next 
important contribution was reported in 1932 by Irene Joliot-Curie and Frederic Joliot in Paris. They showed that if 
this unknown radiation fell on paraffin, or any other hydrogen-containing compound, it ejected protons of very high 
energy. This was not in itself inconsistent with the assumed gamma ray nature of the new radiation, but detailed 
quantitative analysis of the data became increasingly difficult to reconcile with such a hypothesis. 

In 1930 Viktor Ambartsumian and Dmitri Ivanenko in USSR found that contrary to the prevailing opinion of the 

time nucleus can not consist of protons and electrons. They proved that some neutral particles must be present 

mi 
besides the protons. 

In 1932, James Chadwick performed a series of experiments at the University of Cambridge, showing that the 
gamma ray hypothesis was untenable. He suggested that the new radiation consisted of uncharged particles of 
approximately the mass of the proton, and he performed a series of experiments verifying his suggestion. These 
uncharged particles were called neutrons, apparently from the Latin root for neutral and the Greek ending -on (by 
imitation of electron and proton). 

The discovery of the neutron explained a puzzle involving the spin of the nitrogen- 14 nucleus, which had been 
experimentally measured to be 1 h. It was known that atomic nuclei usually had about half as many positive charges 
as if they were composed completely of protons, and in existing models this was often explained by proposing that 
nuclei also contained some "nuclear electrons" to neutralize the excess charge. Thus, nitrogen- 14 would be 
composed of 14 protons and 7 electrons to give it a charge of +7 but a mass of 14 atomic mass units. However, it 
was also known that both protons and electrons carried an intrinsic spin of / fi, and there was no way to arrange an 
odd number (21) of spins ± / ti to give a spin of 1 ti. Instead, when nitrogen- 14 was proposed to consist of 3 pairs of 
protons and neutrons, with an additional unpaired neutron and proton each contributing a spin of / ti in the same 
direction for a total spin of 1 fi, the model became viable. Soon, nuclear neutrons were used to naturally explain spin 



Neutron 



67 



differences in many different nuclides in the same way, and the neutron as a basic structural unit of atomic nuclei 
was accepted. 

Intrinsic properties 



Stability and beta decay 

Under the Standard Model of particle physics, because the neutron 
consists of three quarks, the only possible decay mode without a 
change of baryon number is for one of the quarks to change 
flavour via the weak interaction. The neutron consists of two down 

1 2 

quarks with charge - / e and one up quark with charge + / e, and 
the decay of one of the down quarks into a lighter up quark can be 
achieved by the emission of a W boson. By this means the neutron 
decays into a proton (which contains one down and two up 
quarks), an electron, and an electron antineutrino. 

Outside the nucleus, free neutrons are unstable and have a mean 
lifetime of 885.7 ± 0.8 s (about 14 minutes, 46 seconds); therefore 
the half-life for this process (which differs from the mean lifetime 
by a factor of ln(2) = 0.693) is 613.9 ± 0.8 s (about 10 minutes, 14 
seconds). Free neutrons decay by emission of an electron and an 
electron antineutrino to become a proton, a process known as beta 




The Feynman diagram for beta decay of a neutron into 

a proton, electron, and electron antineutrino via an 

intermediate heavy W boson 



decay 



.[6] 







n 



p + e + v 



Neutrons in unstable nuclei can also decay in this manner. However, inside a nucleus, protons can also transform 

into a neutron via inverse beta decay. This transformation occurs by emission of a antielectron (also called positron) 

and a neutrino: 

+ , + , 
p — > n + e + v 

e 

The transformation of a proton to a neutron inside of a nucleus is also possible through electron capture: 



p + e 



o , 
n + v 



Positron capture by neutrons in nuclei that contain an excess of neutrons is also possible, but is hindered because 
positrons are repelled by the nucleus, and quickly annihilate when they encounter electrons. 

When bound inside of a nucleus, the instability of a single neutron to beta decay is balanced against the instability 
that would be acquired by the nucleus as a whole if an additional proton were to participate in repulsive interactions 
with the other protons that are already present in the nucleus. As such, although free neutrons are unstable, bound 
neutrons are not necessarily so. The same reasoning explains why protons, which are stable in empty space, may 
transform into neutrons when bound inside of a nucleus. 



Electric dipole moment 

The Standard Model of particle physics predicts a tiny separation of positive and negative charge within the neutron 

171 

leading to a permanent electric dipole moment. The predicted value is, however, well below the current sensitivity 
of experiments. From several unsolved puzzles in particle physics, it is clear that the Standard Model is not the final 
and full description of all particles and their interactions. New theories going beyond the Standard Model generally 
lead to much larger predictions for the electric dipole moment of the neutron. Currently, there are at least four 
experiments trying to measure for the first time a finite neutron electric dipole moment, including: 



Neutron 68 

• [8] Cryogenic neutron EDM experiment being set up at the Institut Laue-Langevin 

• [9] nEDM experiment under construction at the new UCN source at the Paul Scherrer Institute 

• [10] nEDM experiment being envisaged at the Spallation Neutron Source 

• [11] nEDM experiment being built at the Institut Laue-Langevin 

Magnetic moment 

Magnetic moment of a neutron is nonzero, unexpected from an electrically neutral particle. This indicates that 
neutron is a composite particle. 

Anti-neutron 

The antineutron is the antiparticle of the neutron. It was discovered by Bruce Cork in the year 1956, a year after the 
antiproton was discovered. CPT-symmetry puts strong constraints on the relative properties of particles and 
antiparticles, so studying antineutrons yields provide stringent tests on CPT-symmetry. The fractional difference in 
the masses of the neutron and antineutron is 9 ± 5 x 10~ . Since the difference is only about 2 standard deviations 
away from zero, this does not give any convincing evidence of CPT-violation. 

Structure and geometry of charge distribution within the neutron 

An article published in 2007 featuring a model-independent analysis concluded that the neutron has a negatively 
charged exterior, a positively charged middle, and a negative core. In a simplified classical view, the negative 
"skin" of the neutron assists it to be attracted to the protons with which it interacts in the nucleus. However, the main 
attraction between neutrons and protons is via the nuclear force, which does not involve charge. 

Neutron compounds 
Dineutrons and tetraneutrons 

The existence of stable clusters of 4 neutrons, or tetraneutrons, has been hypothesised by a team led by 
Francisco-Miguel Marques at the CNRS Laboratory for Nuclear Physics based on observations of the disintegration 
of beryllium- 14 nuclei. This is particularly interesting because current theory suggests that these clusters should not 
be stable. 

The dineutron is another hypothetical particle. 

Neutronium and neutron stars 

At extremely high pressures and temperatures, nucleons and electrons are believed to collapse into bulk neutronic 
matter, called neutronium. This is presumed to happen in neutron stars. 

Detection 

The common means of detecting a charged particle by looking for a track of ionization (such as in a cloud chamber) 
does not work for neutrons directly. Neutrons that elastically scatter off atoms can create an ionization track that is 
detectable, but the experiments are not as simple to carry out; other means for detecting neutrons, consisting of 
allowing them to interact with atomic nuclei, are more commonly used. The commonly used methods to detect 
neutrons can therefore be categorized according to the nuclear processes relied upon, mainly neutron capture or 
elastic scattering. A good discussion on neutron detection is found in chapter 14 of the book Radiation Detection and 
Measurement by Glenn F. Knoll (John Wiley & Sons, 1979). 



Neutron 69 

Neutron detection by neutron capture 

A common method for detecting neutrons involves converting the energy released from such reactions into electrical 
signals. Certain nuclides have neutron deficit and therefore a high probability to absorb a neutron. Upon neutron 
capture, the compound nucleus emits more easily detectable radiation, for example an alpha particle, which is then 

o /: -i f\ OQQ ^qc i^o^ OQO 

detected. The nuclides He, Li, B, U, U, Np and Pu are useful for this purpose. These nuclides are 
rarely found in nature, but can be accumulated through processes such as isotopic enrichment. 

The cross section for the process of neutron capture is much lower at high energies than at low energies. Therefore, 
the detection of neutrons by neutron capture requires a preceding slowing down of neutrons. For this purpose, a 
so-called moderator is used, typically a thick slab of polyethylene. Neutron detection according to the 
moderate-and-capture approach is not capable of measuring neutron energy, precise time of arrival, or direction of 
incidence, since this information is lost during moderation. 

Neutron detection by elastic scattering 

Neutrons can elastically scatter off nuclei, causing the struck nucleus to recoil. Kinematically, a neutron can transfer 
more energy to light nuclei such as hydrogen or helium than to heavier nuclei. Detectors relying on elastic scattering 
are called fast neutron detectors. Recoiling nuclei can ionize and excite further atoms through collisions. Charge 
and/or scintillation light produced in this way can be collected to produce a detected signal. A major challenge in fast 
neutron detection is discerning such signals from erroneous signals produced by gamma radiation in the same 
detector. 

Fast neutron detectors have the advantage of not requiring a moderator, and therefore being capable of measuring the 
neutron's energy, time of arrival, and in certain cases direction of incidence. 

Uses 

The neutron plays an important role in many nuclear reactions. For example, neutron capture often results in neutron 
activation, inducing radioactivity. In particular, knowledge of neutrons and their behavior has been important in the 
development of nuclear reactors and nuclear weapons. The fissioning of elements like uranium-235 and 
plutonium-239 is caused by their absorption of neutrons. 

Cold, thermal and hot neutron radiation is commonly employed in neutron scattering facilities, where the radiation is 
used in a similar way one uses X-rays for the analysis of condensed matter. Neutrons are complementary to the latter 
in terms of atomic contrasts by different scattering cross sections; sensitivity to magnetism; energy range for 
inelastic neutron spectroscopy; and deep penetration into matter. 

The development of "neutron lenses" based on total internal reflection within hollow glass capillary tubes or by 
reflection from dimpled aluminum plates has driven ongoing research into neutron microscopy and neutron/gamma 

u [13] [14] [15] 

ray tomography. 

A major use of neutrons is to excite delayed and prompt gamma rays from elements in materials. This forms the 
basis of neutron activation analysis (NAA) and prompt gamma neutron activation analysis (PGNAA). NAA is most 
often used to analyze small samples of materials in a nuclear reactor whilst PGNAA is most often used to analyze 
subterranean rocks around bore holes and industrial bulk materials on conveyor belts. 

Another use of neutron emitters is the detection of light nuclei, particularly the hydrogen found in water molecules. 
When a fast neutron collides with a light nucleus, it loses a large fraction of its energy. By measuring the rate at 
which slow neutrons return to the probe after reflecting off of hydrogen nuclei, a neutron probe may determine the 
water content in soil. 



Neutron 70 

Sources 

Because free neutrons are unstable, they can be obtained only from nuclear disintegrations, nuclear reactions, and 
high-energy reactions (such as in cosmic radiation showers or accelerator collisions). Free neutron beams are 
obtained from neutron sources by neutron transport. For access to intense neutron sources, researchers must go to 
specialist facilities, such as the ISIS facility in the United Kingdom, which is currently the world's most intense 
pulsed neutron and muon source. 

The neutron's lack of total electric charge makes it difficult to steer or accelerate them. Charged particles can be 
accelerated, decelerated, or deflected by electric or magnetic fields. These methods have little effect on neutrons 
beyond a small effect of an inhomogeneous magnetic field because of the neutron's magnetic moment. Neutrons can 
be controlled by methods that include moderation, reflection and velocity selection. 

Protection 

Exposure to free neutrons can be hazardous, since the interaction of neutrons with molecules in the body can cause 
disruption to molecules and atoms, and can also cause reactions which give rise to other forms of radiation (such as 
protons). The normal precautions of radiation protection apply: avoid exposure, stay as far from the source as 
possible, and keep exposure time to a minimum. Some particular thought must be given to how to protect from 
neutron exposure, however. For other types of radiation, e.g. alpha particles, beta particles, or gamma rays, material 
of a high atomic number and with high density make for good shielding; frequently lead is used. However, this 
approach will not work with neutrons, since the absorption of neutrons does not increase straightforwardly with 
atomic number, as it does with alpha, beta, and gamma radiation. Instead one needs to look at the particular 
interactions neutrons have with matter (see the section on detection above). For example, hydrogen rich materials are 
often used to shield against neutrons, since ordinary hydrogen both scatters and slows neutrons. This often means 
that simple concrete blocks or even paraffin-loaded plastic blocks afford better protection from neutrons than do far 
more dense materials. After slowing, neutrons may then be absorbed with an isotope which has high affinity for slow 
neutrons without causing secondary capture-radiation, such as lithium-6. 

Hydrogen-rich ordinary water affects neutron absorption in nuclear fission reactors: usually neutrons are so strongly 
absorbed by normal water that fuel-enrichment with fissionable isotope is required. The deuterium in heavy water 
has a very much lower absorption affinity for neutrons than does protium (normal light hydrogen). Deuterium is 
therefore used in CANDU-type reactors, in order to slow (moderate) neutron velocity, to increase the probability of 
nuclear fission compared to neutron capture. 

Production 

Various nuclides become more stable by expelling neutrons as a decay 
mode; this is known as neutron emission, and happens commonly 
during spontaneous fission. 

Cosmic radiation interacting with the Earth's atmosphere continuously 
generates neutrons that can be detected at the surface. Even stronger 
neutron radiation is produced at the surface of Mars where the 
atmosphere is thick enough to generate neutrons from cosmic ray 
spallation, but not thick enough to provide significant protection from 

Institut Laue— Langevin (ILL) in Grenoble, 

the neutrons produced. These neutrons not only produce a Martian c tt , t . , , 

r J e France - one or the most important neutron 

surface neutron radiation hazard from direct downward-going neutron research facilities worldwide 

radiation, but also a significant hazard from reflection of neutrons from 

the Martian surface, which will produce reflected neutron radiation penetrating upward into a Martian craft or habitat 
from the floor. 




Neutron 7 1 

Nuclear fission reactors naturally produce free neutrons; their role is to sustain the energy-producing chain reaction. 
The intense neutron radiation can also be used to produce various radioisotopes through the process of neutron 
activation, which is a type of neutron capture. 

Experimental nuclear fusion reactors produce free neutrons as a waste product. However, it is these neutrons that 
possess most of the energy, and converting that energy to a useful form has proved a difficult engineering challenge. 
Fusion reactors which generate neutrons are likely to create around twice the amount of radioactive waste of a 
fission reactor, but the waste is composed of neutron-activated lighter isotopes, which have relatively short (50—100 

years) decay periods as compared to typical half lives of 10,000 years for fission waste, which is long primarily due 

ri7i 
to the long half life of alpha-emitting transuranic actinides. Nuclear power#Solid waste 

Neutron temperature 
Thermal neutron 

—21 

A thermal neutron is a free neutron that is Boltzmann distributed with kT = 0.0253 eV (4.0x10 J) at room 
temperature. This gives characteristic (not average, or median) speed of 2.2 km/s. The name 'thermal' comes from 
their energy being that of the room temperature gas or material they are permeating, (see kinetic theory for energies 
and speeds of molecules). After a number of collisions (often in the range of 10—20) with nuclei, neutrons arrive at 
this energy level, provided that they are not absorbed. 

In many substances, thermal neutrons have a much larger effective cross-section than faster neutrons, and can 
therefore be absorbed more easily by any atomic nuclei that they collide with, creating a heavier — and often 
unstable — isotope of the chemical element as a result. 

Most fission reactors use a neutron moderator to slow down, or thermalize the neutrons that are emitted by nuclear 
fission so that they are more easily captured, causing further fission. Others, called fast breeder reactors, use fission 
energy neutrons directly. 

Cold neutrons 

These neutrons are thermal neutrons that have been equilibrated in a very cold substance such as liquid deuterium. 
These are produced in neutron scattering research facilities. 

Ultracold neutrons 

Ultracold neutrons are produced by inelastically scattering cold neutrons in substances with a temperature of a few 
kelvins, such as solid deuterium or superfluid helium. An alternative production method is the mechanical 
deceleration of cold neutrons. 

Fission energy neutron 

A fast neutron is a free neutron with a kinetic energy level close to 2 MeV (20 TJ/kg), hence a speed of 28,000 
km/s. They are named fission energy or fast neutrons to distinguish them from lower-energy thermal neutrons, and 
high-energy neutrons produced in cosmic showers or accelerators. Fast neutrons are produced by nuclear processes 
such as nuclear fission. 

Fast neutrons can be made into thermal neutrons via a process called moderation. This is done with a neutron 
moderator. In reactors, typically heavy water, light water, or graphite are used to moderate neutrons. 



Neutron 



72 



Fusion neutron 

D-T (deuterium-tritium) fusion is the fusion 
reaction that produces the most energetic 
neutrons, with 14.1 MeV of kinetic energy 
and traveling at 17% of the speed of light. 
D-T fusion is also the easiest fusion reaction 
to ignite, reaching near-peak rates even 
when the deuterium and tritium nuclei have 
only a thousandth as much kinetic energy as 
the 14.1 MeV that will be produced. 

14.1 MeV neutrons have about 10 times as 
much energy as fission neutrons, and are 
very effective at fissioning even non-fissile 
heavy nuclei, and these high-energy fissions 
produce more neutrons on average than 
fissions by lower-energy neutrons. This 
makes D-T fusion neutron sources such as 
proposed tokamak power reactors useful for 
transmutation of transuranic waste. 14.1 
MeV neutrons can also produce neutrons by knocking them loose from nuclei. 

On the other hand, these very high energy neutrons are less likely to simply be captured without causing fission or 
spallation. For these reasons, nuclear weapon design extensively utilizes D-T fusion 14.1 MeV neutrons to cause 
more fission. Fusion neutrons are able to cause fission in ordinarily non-fissile materials, such as depleted uranium 
(uranium-238), and these materials have used in the jackets of thermonuclear weapons. Fusion neutrons also can 
cause fission in substances that are unsuitable or difficult to make into primary fission bombs, such as reactor grade 
plutonium. This physical fact thus causes ordinary non-weapons grade materials to become of concern in certain 
nuclear proliferation discussions and treaties. 

Other fusion reactions produce much less energetic neutrons. D-D fusion produces a 2.45 MeV neutron and helium-3 

3 

half of the time, and produces tritium and a proton but no neutron the other half of the time. D- He fusion produces 
no neutron. 



temperature [keV] 


77 io° 10 1 io 2 l 


f 


% IO" 21 




=? 10" 22 

" IO" 23 






fl IO" 24 
ro 

c io" 25 
o 

u 

<U IO" 27 

1C 










D-T 

D-D 

- D-He3 


/ / 


, 


~ 2 10" 1 10° 10 


i 


temperature [billion Kelvin] 


The fusion reaction rate increases rapidly with temperature until it maximizes and 


then gradually drops off. The DT rate peaks at a lower temperature (about 70 keV, 


or 800 million kelvins) and at a higher value than other reactions commonly 


considered for fusion energy. 



Neutron 



73 



Intermediate-energy neutrons 

A fission energy neutron that has slowed 
down but not yet reached thermal energies is 
called an epithermal neutron. 

Cross sections for both capture and fission 
reactions often have multiple resonance 
peaks at specific energies in the epithermal 
energy range. These are of less significance 
in a fast neutron reactor where most 
neutrons are absorbed before slowing down 
to this range, or in a well-moderated thermal 
reactor where epithermal neutrons mostly 
interact with moderator nuclei, not with 
either fissile or fertile actinide nuclides. 
However, in a partially moderated reactor 
with more interactions of epithermal 
neutrons with heavy metal nuclei, there are 
greater possibilities for transient changes in 
reactivity which might make reactor control 
more difficult. 



.239 36% v ?4f ) fj S sile fission% 




1% 3% Cm 13% V/oCm S l% ' Cm 



Transmutation flow in LWR which is a thermal-spectrum reactor 



Ratios of capture reactions to fission 
reactions are also worse (more captures without 
epithermal-spectrum reactors using these fuels less 
also usually result in a nuclide which is not fissile 
fast neutrons. The exception is uranium-233 of the 
energies. 



fission) in most nuclear fuels such as plutonium-239, making 
desirable, as captures not only waste the one neutron captured but 
with thermal or epithermal neutrons, though still fissionable with 
thorium cycle which has good capture-fission ratios at all neutron 



High-energy neutrons 

These neutrons have more energy than fission energy neutrons and are generated as secondary particles by particle 
accelerators or in the atmosphere from cosmic rays. They can have energies as high as tens of joules per neutron. 



See also 

Neutron radiation 

List of particles 

Nuclear reaction 

Thermal reactor 

Fast neutron 

Ionizing radiation 

Isotope 

Neutron flux 

Neutron generator 

Neutron magnetic moment 

Neutron capture nucleosynthesis 

• R-process 

• S-process 



Neutron 74 

• Neutron radiation and the Sievert radiation scale 

Neutron sources 

• Astronomical neutron sources 

• Neutron sources 

• Neutron generator 

Processes involving neutrons 

• Neutron bomb 

• Neutron diffraction 

• Neutron flux 

• Neutron transport 

References 

[I] 1935 Nobel Prize in Physics (http://nobelprize.org/nobel_prizes/physics/laureates/1935/) 

[2] Particle Data Group's Review of Particle Physics 2006 (http://pdg.lbl.gov/2006/tables/bxxx.pdf) 

[3] http://www.nndc.bnl.gov/nudat2 

[4] http://www.springerlink.com/content/ek2ql56624661848/fulltext.pdf 

[5] Chadwick, James (1932). "Possible Existence of a Neutron". Nature 129: 312. doi:10.1038/129312a0. 

[6] Particle Data Group Summary Data Table on Baryons (http://pdg.lbl.gov/2007/tables/bxxx.pdf) 

[7] University of Sussex (20 February 2006). "Pear-shaped particles probe big-bang mystery" (http://www.sussex.ac.uk/press_office/media/ 

media537.shtml). Press release. . Retrieved 2009-12-14. 
[8] http://hepwww.rl.ac.uk/EDM/index_files/CryoEDM.htm 
[9] http://nedm.web.psi.ch/ 
[10] http://p25ext.lanl.gov/edm/edm.html 

[II] http://nrd.pnpi.spb.ru/LabSereb/neutronedm.htm 

[12] G.A. Miller (2007). "Charge Densities of the Neutron and Proton". Physical Review Letters 99: 1 12001. 

doi: 10.1 103/PhysRevLett.99.1 12001. 

[13] Kumakhov, M. A.; Sharov, V. A. (1992). "A neutron lens". Nature 357: 390-391. doi:10.1038/357390a0. 

[14] Physorg.com, "New Way of 'Seeing': A 'Neutron Microscope'" (http://www.physorg.com/news599.html) 

[15] NASA.gov: "NASA Develops a Nugget to Search for Life in Space" (http://www.nasa.gov/vision/earth/technologies/nuggets.html) 

[16] http://www.physicamedica.com/VOLXVII_Sl/20-CLOWDSLEY%20et%20alii.pdf 

[17] http://news.bbc.co.Uk/l/hi/sci/tech/4627237.stm 

• Annotated bibliography for neutrons from the Alsos Digital Library for Nuclear Issues (http://alsos.wlu.edu/ 
qsearch.aspx?browse=science/Neutrons) 

Further reading 

• Knoll, G. F. (2000) Radiation Detection and Measurement 

• Krane, K. S. (1998) Introductory Nuclear Physics 

• Squires, G. L. (1997) Introduction to the Theory of Thermal Neutron Scattering 

• Dewey, M. S., Gilliam, D. M., Nico, J. S., Snow, M. S., Wietfeldt, F. E. NIST Neutron Lifetime Experiment 



Muon 



75 



Muon 



Muon 




The Moon's cosmic ray shadow, as seen in secondary muons generated by cosmic rays in the atmosphere, and detected 700 meters 

below ground, at the Soudan II detector. 



Composition: 
Particle statistics: 



Elementary particle 
Fermionic 



Group: 
Generation: 



Lepton 
Second 



Interaction: 



Symbol(s): 



Gravity, Electromagnetic, 
Weak 



Antiparticle: 



Antimuon (u. ) 



Theorized: 
Discovered: 



Carl D.Anderson (1936) 



Mass: 



105.65836668(38) MeV/c 



Mean lifetime: 
Electric charge: 



2.197034(21) x 10 6 s [1] 



-le 



Color charge: 
Spin: 



None 



'/„ 



The muon (from the Greek letter mu (|i) used to represent it) is an elementary particle similar to the electron, with a 
negative electric charge and a spin of Vi. Together with the electron, the tau, and the three neutrinos, it is classified as 
a lepton. It is an unstable subatomic particle with the second longest mean lifetime (2.2 ps), exceeded only by that of 
the free neutron (-15 min). Like all elementary particles, the muon has a corresponding antiparticle of opposite 
charge but equal mass and spin: the antimuon (also called a positive muon). Muons are denoted by |i~ and 
antimuons by u. . Muons were previously called mu mesons, but are not classified as mesons by modern particle 

physicists (see History). 

2 

Muons have a mass of 105.7 MeV/c , which is about 200 times the mass of an electron. Since the muon's 
interactions are very similar to those of the electron, a muon can be thought of as a much heavier version of the 
electron. Due to their greater mass, muons are not as sharply accelerated when they encounter electromagnetic fields, 
and do not emit as much bremsstrahlung radiation. Thus muons of a given energy penetrate matter far more deeply 
than electrons, since the deceleration of electrons and muons is primarily due to energy loss by this mechanism. 
So-called "secondary muons", generated by cosmic rays hitting the atmosphere, can penetrate to the Earth's surface 



Muon 76 

and into deep mines. 

As with the case of the other charged leptons, the muon has an associated muon neutrino. Muon neutrinos are 
denoted by v . 

History 

Muons were discovered by Carl D. Anderson and Seth Neddermeyer at Caltech in 1936 while studying cosmic 
radiation. Anderson had noticed particles that curved differently from electrons and other known particles when 
passed through a magnetic field. They were negatively charged but curved less sharply than electrons, but more 
sharply than protons, for particles of the same velocity. It was assumed that the magnitude of their negative electric 
charge was equal to that of the electron, and so to account for the difference in curvature, it was supposed that their 
mass was greater than an electron but smaller than a proton. Thus Anderson initially called the new particle a 
mesotron, adopting the prefix meso- from the Greek word for "mid-". Shortly thereafter, additional particles of 
intermediate mass were discovered, and the more general term meson was adopted to refer to any such particle. To 
differentiate between different types of mesons, the mesotron was in 1947 renamed the mu meson (the Greek letter ,u 
(mu) corresponds to m). 

It was soon found that the mu meson significantly differed from other mesons: for example, its decay products 
included a neutrino and an antineutrino, rather than just one or the other, as was observed with other mesons. Other 
mesons were eventually understood to be hadrons — that is, particles made of quarks — and thus subject to the 
residual strong force. In the quark model, a meson is composed of exactly two quarks (a quark and antiquark) unlike 
baryons, which are composed of three quarks. Mu mesons, however, were found to be fundamental particles 
(leptons) like electrons, with no quark structure. Thus, mu mesons were not mesons at all (in the new sense and use 
of the term meson), and so the term mu meson was abandoned, and replaced with the modern term muon. 

Another particle (the pion, with which the muon was initially confused) had been predicted by theorist Hideki 
Yukawa: 

"It seems natural to modify the theory of Heisenberg and Fermi in the following way. The transition of a 
heavy particle from neutron state to proton state is not always accompanied by the mission of light 
particles. The transition is sometimes taken up by another heavy particle." 

The existence of the muon was confirmed in 1937 by J. C. Street and E. C. Stevenson's cloud chamber experiment. 
The discovery of the muon seemed so incongruous and surprising at the time that Nobel laureate I. I. Rabi famously 
quipped, "Who ordered that?" 

In a 1941 experiment on Mount Washington in New Hampshire, muons were used to observe the time dilation 

mi 
predicted by special relativity for the first time. 

Muon sources 

Since the production of muons requires an available center of momentum frame energy of 105.7 MeV, neither 
ordinary radioactive decay events nor nuclear fission and fusion events (such as those occurring in nuclear reactors 
and nuclear weapons) are energetic enough to produce muons. Only nuclear fission produces single-nuclear-event 
energies in this range, but do not produce muons as the production of a single muon would violate the conservation 
of quantum numbers (see under "muon decay" below). 

On Earth, most naturally occurring muons are created by cosmic rays, which consist mostly of protons, many 
arriving from deep space at very high energy 

About 10,000 muons reach every square meter of the earth's surface a minute; these charged particles form as 
by-products of cosmic rays colliding with molecules in the upper atmosphere. Travelling at relativistic speeds, 
muons can penetrate tens of meters into rocks and other matter before attenuating as a result of absorption or 
deflection by other atoms. 



Muon 



77 



— Mark Wolverton (September 2007). "Muons for Peace: New Way to Spot Hidden Nukes Gets Ready to 
Debut" . Scientific American 297 (3): 26—28. 

When a cosmic ray proton impacts atomic nuclei of air atoms in the upper atmosphere, pions are created. These 
decay within a relatively short distance (meters) into muons (the pion's preferred decay product), and neutrinos. The 
muons from these high energy cosmic rays generally continue in about the same direction as the original proton, at a 
very high velocity. Although their lifetime without relativistic effects would allow a half-survival distance of only 
about 0.66 km (660 meters) at most (as seen from Earth) the time dilation effect of special relativity (from the 
viewpoint of the Earth) allows cosmic ray secondary muons to survive the flight to the Earth's surface, since in the 
Earth frame, the muons have a longer half-life due to their velocity. From the viewpoint (inertial frame) of the muon, 
on the other hand, it is the length contraction effect of special relativity which allows this penetration, since in the 
muon frame, its life time is unaffected, but the distance through the atmosphere and earth appears far shorter than 
these distances in the Earth rest-frame. Both are equally valid ways of explaining the fast muon's unusual survival 
over distances. 

Since muons are unusually penetrative of ordinary matter, like neutrinos, they are also detectable deep underground 
(700 meters at the Soudan II detector, pictured above) and underwater, where they form a major part of the natural 
background ionizing radiation. Like cosmic rays, as noted, this secondary muon radiation is also directional. 

The same nuclear reaction described above (i.e. hadron-hadron impacts to produce pion beams, which then quickly 
decay to muon beams over short distances) is used by particle physicists to produce muon beams, such as the beam 
used for the muon g - 2 experiment 



[7] 



Muon decay 

Muons are unstable elementary particles and are 
heavier than electrons and neutrinos but lighter than all 
other matter particles. They decay via the weak 
interaction. Because lepton numbers must be 
conserved, one of the product neutrinos of muon decay 
must be a muon-type neutrino and the other an 
electron-type antineutrino (antimuon decay produces 
the corresponding antiparticles, as detailed below). 
Because charge must be conserved, one of the products 
of muon decay is always an electron of the same charge 
as the muon (a positron if it is a positive muon). Thus 
all muons decay to at least an electron, and two 
neutrinos. Sometimes, besides these necessary 
products, additional other particles that have a net 
charge and spin of zero (i.e. a pair of photons, or an 
electron-positron pair), are produced. 



T 

A 





w 



The most common decay of the muon 



The dominant muon decay mode (sometimes called the 

Michel decay after Louis Michel) is the simplest 

possible: the muon decays to an electron, an electron-antineutrino, and a muon-neutrino. Antimuons, in mirror 

fashion, most often decay to the corresponding antiparticles: a positron, an electron-neutrino, and a 

muon-antineutrino. In formulaic terms, these two decays are: 



/'■ 



+ u e + Vp, [V 



+ v e + i/ l 



V ' 



The mean lifetime of the (positive) muon is 2.197 019 ± 0.000 021 u,s 
lifetimes has been established to better than one part in 10 . 



[8] 



The equality of the muon and anti-muon 



Muon 78 

The tree-level muon decay width is 
G 2 F ml ^ 2 ' 

r= m j 

1927T 3 

where J{x) = 1 — 8x — 12x 2 lnx + 8x 3 — x 4 ; G F ™ the Fermi coupling constant. 

The decay distributions of the electron in muon decays have been parameterised using the so-called Michel 
parameters. The values of these four parameters are predicted unambiguously in the Standard Model of particle 
physics, thus muon decays represent a good test of the space-time structure of the weak interaction. No deviation 
from the Standard Model predictions has yet been found. 

Certain neutrino-less decay modes are kinematically allowed but forbidden in the Standard Model. Examples 
forbidden by lepton flavour conservation are 

fi~ — > e~ + 7 and ^ — > e ~ -\- e + -|- e ~ . 

Observation of such decay modes would constitute clear evidence for physics beyond the Standard Model (BSM). 

—11 —12 

Current experimental upper limits for the branching fractions of such decay modes are in the range 10 to 10 

Muonic atoms 

The muon was the first elementary particle discovered that does not appear in ordinary atoms. Negative muons can, 
however, form muonic atoms (also called mu-mesic atoms), by replacing an electron in ordinary atoms. Muonic 
hydrogen atoms are much smaller than typical hydrogen atoms because the much larger mass of the muon gives it a 
much smaller ground-state wavefunction than is observed for the electron. In multi-electron atoms, when only one of 
the electrons is replaced by a muon, the size of the atom continues to be determined by the other electrons, and the 
atomic size is nearly unchanged. However, in such cases the orbital of the muon continues to be smaller and far 
closer to the nucleus than the atomic orbitals of the electrons. 

A positive muon, when stopped in ordinary matter, can also bind an electron and form an exotic atom known as 
muonium (Mu) atom, in which the muon acts as the nucleus. The positive muon, in this context, can be considered a 
pseudo-isotope of hydrogen with one ninth of the mass of the proton. Because the reduced mass of muonium, and 
hence its Bohr radius, is very close to that of hydrogen, this short-lived "atom" behaves chemically — to a first 
approximation — like hydrogen, deuterium and tritium. 

Use in measurement of the proton charge radius 

The recent culmination of a twelve year experiment investigating the proton's charge radius involved the use of 
muonic hydrogen. This form of hydrogen is composed of a muon orbiting a proton . The Lamb shift in muonic 
hydrogen was measured by driving the muon from the from its 2s state up to an excited 2p state using a laser 
The frequency of the photon required to induce this transition was revealed to be 50 terahertz which, according to 
present theories of quantum electrodynamics, yields a value of 0.84184 ± 0.00067 femtometres for the charge radius 
of the proton. 

Anomalous magnetic dipole moment 

The anomalous magnetic dipole moment is the difference between the experimentally observed value of the 
magnetic dipole moment and the theoretical value predicted by the Dirac equation. The measurement and prediction 

ri3i 

of this value is very important in the precision tests of QED (quantum electrodynamics). The E821 experiment at 
Brookhaven National Laboratory (BNL) studied the precession of muon and anti-muon in a constant external 
magnetic field as they circulated in a confining storage ring. The E821 Experiment reported the following average 

ri4i 

value (from the July 2007 review by Particle Data Group) 



Muon 79 

a = ^— = 0.00116592080(54)(33) 
where the first errors are statistical and the second systematic. 

The difference between the g-factors of the muon and the electron is due to their difference in mass. Because of the 
muon's larger mass, contributions to the theoretical calculation of its anomalous magnetic dipole moment from 
Standard Model weak interactions and from contributions involving hadrons are important at the current level of 
precision, whereas these effects are not important for the electron. The muon's anomalous magnetic dipole moment 
is also sensitive to contributions from new physics beyond the Standard Model, such as supersymmetry. For this 
reason, the muon's anomalous magnetic moment is normally used as a probe for new physics beyond the Standard 
Model rather than as a test of QED (Phys.Lett. B649, 173 (2007) [15] ). 

See also 

• Mumesic atom 

• Muonium 

• Muon spin spectroscopy 

• Muon-catalyzed fusion 

• List of particles 

References 

[I] K. Nakamura et al. (Particle Data Group), J. Phys. G 37, 075021 (2010), URL: http://pdg.lbl.gov 

[2] Yukaya Hideka, On the Interaction of Elementary Particles 1, Proceedings of the Physico-Mathematical Society of Japan (3) 17, 48, pp 

139-148 (1935). (Read 17 November 1934) 
[3] New Evidence for the Existence of a Particle Intermediate Between the Proton and Electron", Phys. Rev. 52, 1003 (1937). 
[4] David H. Frisch and James A. Smith, "Measurement of the Relativistic Time Dilation Using Muons", American Journal of Physics, 31, 342, 

1963, cited by Michael Fowler, " Special Relativity: What Time is it? (http://galileoandeinstein.physics.virginia.edu/lectures/srelwhat. 

html)" 
[5] S. Carroll (2004). Spacetime and Geometry: An Introduction to General Relativity. Addison Wesly. p. 204 
[6] http://www. sciam.com/ article. cfm?id=muons-for-peace 
[7] Brookhaven National Laboratory (30 July 2002). "Physicists Announce Latest Muon g-2 Measurement" (http://www.bnl.gov/bnlweb/ 

pubaf/pr/2002/bnlpr073002.htm). Press release. . Retrieved 2009-11-14. 
[8] (http://arxiv.org/abs/0704. 1981vl) 
[9] https://muhy.web.psi.ch/wiki/ 
[10] TRIUMF Muonic Hydrogen collaboration. "A brief description of Muonic Hydrogen research". Retrieved 2010-11-7 

[II] https://muhy.web.psi.ch/wiki/index.php/Main/Gallery 

[12] Pohl, Randolf et al. "The Size of the Proton" Nature 466, 213-216 (8 July 2010) 

[13] http://www.g-2.bnl.gov/ 

[14] http://pdg.lbl.gov/2007/reviews/g-2_s004219.pdf 

[15] http://arxiv.org/abs/hep-ph/0611102 

• S.H. Neddermeyer, CD. Anderson (1937). "Note on the Nature of Cosmic-Ray Particles". Physical Review 51: 
884-886. doi: 10. 1 103/PhysRev.5 1 .884. 

• J.C. Street, E.C. Stevenson (1937). "New Evidence for the Existence of a Particle of Mass Intermediate Between 
the Proton and Electron". Physical Review 52: 1003-1004. doi: 10. 1 103/PhysRev.52. 1003. 

• G. Feinberg, S. Weinberg (1961). "Law of Conservation of Muons.". Physical Review Letters 6: 381—383. 
doi: 10. 1 103/PhysRevLett.6.38 1 . 

• Serway & Faughn (1995). College Physics (4th ed.). Saunders, p. 841. 

• M. Knecht (2003). "The Anomalous Magnetic Moments of the Electron and the Muon" (http://books. google. 
com/?id=me6ftonVM_EC&pg=PA265&lpg=PA265&dq="The+Anomalous+Magnetic+Moments+of+the+ 
Electron+and+the+Muon"&q="The Anomalous Magnetic Moments of the Electron and the Muon"). In B. 
Duplantier, V. Rivasseau. Poincare Seminar 2002: Vacuum Energy — Renormalization. Progress in Mathematical 
Physics. 30. Birkhauser. p. 265. ISBN 3-7643-0579-7. 



Muon 



80 



• E. Derman (2004). My Life As A Quant. Wiley, pp. 58-62. 

External links 

• Muon anomalous magnetic moment and supersymmetry (http://antwrp.gsfc.nasa.gov/apod/ap050828.html) 

• g-2 (muon anomalous magnetic moment) experiment (http://www.g-2.bnl.gov/) 

• muLan (Measurement of the Positive Muon Lifetime) experiment (http://www.npl.uiuc.edu/exp/mulan/) 

• The Review of Particle Physics (http://pdg.lbl.gov/) 

• The TRIUMF Weak Interaction Symmetry Test (http://twist.triumf.ca/) 



X-ray diffraction 



X-ray scattering techniques are a family of 
non-destructive analytical techniques which reveal 
information about the crystallographic structure, 
chemical composition, and physical properties of 
materials and thin films. These techniques are based on 
observing the scattered intensity of an X-ray beam 
hitting a sample as a function of incident and scattered 
angle, polarization, and wavelength or energy. 

X-ray diffraction techniques 

X-ray diffraction yields the atomic structure of 
materials and is based on the elastic scattering of 
X-rays from the electron clouds of the individual atoms 
in the system. The most comprehensive description of 
scattering from crystals is given by the dynamical 
theory of diffraction 




[l] 



This is an X-ray diffraction pattern formed when X-rays are focused 

on a crystalline material, in this case a protein. Each dot, called a 

reflection, forms from the coherent interference of scattered X-rays 

passing through the crystal. 



Single-crystal X-ray diffraction is a technique used 
to solve the complete structure of crystalline 
materials, ranging from simple inorganic solids to 
complex macromolecules, such as proteins. 

Powder diffraction (XRD) is a technique used to characterise the crystallographic structure, crystallite size (grain 
size), and preferred orientation in polycrystalline or powdered solid samples. Powder diffraction is commonly 
used to identify unknown substances, by comparing diffraction data against a database maintained by the 
International Centre for Diffraction Data. It may also be used to characterize heterogeneous solid mixtures to 
determine relative abundance of crystalline compounds and, when coupled with lattice refinement techniques, 
such as Rietveld refinement, can provide structural information on unknown materials. Powder diffraction is also 
a common method for determining strains in crystalline materials. An effect of the finite crystallite sizes is seen as 
a broadening of the peaks in an X-ray diffraction as is explained by the Scherrer Equation. 

Thin film diffraction and grazing incidence X-ray diffraction may be used to characterize the crystallographic 
structure and preferred orientation of substrate-anchored thin films. 

High-resolution X-ray diffraction is used to characterize thickness, crystallographic structure, and strain in thin 
epitaxial films. It employs parallel-beam optics. 



X-ray diffraction 8 1 

• X-ray pole figure analysis enables one to analyze and determine the distribution of crystalline orientations within 
a crystalline thin-film sample. 

• X-ray rocking curve analysis is used to quantify grain size and mosaic spread in crystalline materials. 

Scattering techniques 
Elastic scattering 

Materials that do not have long range order may also be studied by scattering methods that rely on elastic scattering 
of monochromatic X-rays. 

• Small angle X-ray scattering (SAXS) probes structure in the nanometer to micrometer range by measuring 

121 
scattering intensity at scattering angles 26 close to 0°. 

• X-ray reflectivity is an analytical technique for determining thickness, roughness, and density of single layer and 
multilayer thin films. 

• Wide angle X-ray scattering (WAXS), a technique concentrating on scattering angles 26 larger than 5°. 

Inelastic scattering 

When the energy and angle of the inelastically scattered X-rays are monitored scattering techniques can be used to 
probe the electronic band structure of materials. 

• Compton scattering 

• Resonant inelastic X-ray scattering (RIXS) 

• X-ray Raman scattering 

• X-ray diffraction pattern 

See also 

Structure determination 

Materials Science 

Metallurgy 

Mineralogy 

X-ray crystallography 

X-ray generator 

References 

[1] Azaroff, L. V.; R. Kaplow, N. Kato, R. J. Weiss, A. J. C. Wilson, R. A. Young (1974). X-ray diffraction. McGraw-Hill. 

[2] Glatter, O.; O. Kratky (1982). Small Angle X-ray Scattering (http://physchem.kfunigraz.ac.at/sm/Software.htm). Academic Press. . 

External links 

• International Union of Crystallography (http://www.iucr.ac.uk/) 

• IUCr Crystallography Online (http://www.iucr.org/cww-top/crystal.index.html) 

• The International Centre for Diffraction Data (ICDD) (http://www.icdd.com/) 

• The British Crystallographic Association (http://crystallography.org.uk/) 

• Introduction to X-ray Diffraction (http://www.mrl.ucsb.edu/mrl/centralfacilities/xray/xray-basics/index. 
html) at University of California, Santa Barbara 



Electron diffraction 82 



Electron diffraction 



Electron diffraction refers to the wave nature of electrons. However, from a technical or practical point of view, it 
may be regarded as a technique used to study matter by firing electrons at a sample and observing the resulting 
interference pattern. This phenomenon is commonly known as the wave-particle duality, which states that the 
behavior of a particle of matter (in this case the incident electron) can be described by a wave. For this reason, an 
electron can be regarded as a wave much like sound or water waves. This technique is similar to X-ray and neutron 
diffraction. 

Electron diffraction is most frequently used in solid state physics and chemistry to study the crystal structure of 
solids. Experiments are usually performed in a transmission electron microscope (TEM), or a scanning electron 
microscope (SEM) as electron backscatter diffraction. In these instruments, electrons are accelerated by an 
electrostatic potential in order to gain the desired energy and determine their wavelength before they interact with the 
sample to be studied. 

The periodic structure of a crystalline solid acts as a diffraction grating, scattering the electrons in a predictable 
manner. Working back from the observed diffraction pattern, it may be possible to deduce the structure of the crystal 
producing the diffraction pattern. However, the technique is limited by the phase problem. 

Apart from the study of crystals i.e. electron crystallography, electron diffraction is also a useful technique to study 
the short range order of amorphous solids, and the geometry of gaseous molecules. 

History 

The de Broglie hypothesis, formulated in 1926, predicts that particles should also behave as waves. De Broglie's 
formula was confirmed three years later for electrons (which have a rest-mass) with the observation of electron 
diffraction in two independent experiments. At the University of Aberdeen George Paget Thomson passed a beam of 
electrons through a thin metal film and observed the predicted interference patterns. At Bell Labs Clinton Joseph 
Davisson and Lester Halbert Germer guided their beam through a crystalline grid. Thomson and Davisson shared the 
Nobel Prize for Physics in 1937 for their work. 



Theory 



Electron interaction with matter 

Unlike other types of radiation used in diffraction studies of materials, such as X-rays and neutrons, electrons are 
charged particles and interact with matter through the Coulomb forces. This means that the incident electrons feel the 
influence of both the positively charged atomic nuclei and the surrounding electrons. In comparison, X-rays interact 
with the spatial distribution of the valence electrons, while neutrons are scattered by the atomic nuclei through the 
strong nuclear forces. In addition, the magnetic moment of neutrons is non-zero, and they are therefore also scattered 
by magnetic fields. Because of these different forms of interaction, the three types of radiation are suitable for 
different studies. 



Electron diffraction 83 

Intensity of diffracted beams 

In the kinematical approximation for electron diffraction, the intensity of a diffracted beam is given by: 

J g I V'gl ^ \ ± g| 

Here ^gis the wavefunction of the diffracted beam and -Fgis the so called structure factor which is given by: 
F s = E f &-**■" 

i 

where gis the scattering vector of the diffracted beam, r { is the position of an atom { in the unit cell, and f i is the 

scattering power of the atom, also called the atomic form factor. The sum is over all atoms in the unit cell. 

The structure factor describes the way in which an incident beam of electrons is scattered by the atoms of a crystal 

unit cell, taking into account the different scattering power of the elements through the term f± . Since the atoms are 

spatially distributed in the unit cell, there will be a difference in phase when considering the scattered amplitude 

from two atoms. This phase shift is taken into account by the exponential term in the equation. 

The atomic form factor, or scattering power, of an element depends on the type of radiation considered. Because 

electrons interact with matter though different processes than for example X-rays, the atomic form factors for the 

two cases are not the same. 

Wavelength of electrons 

The wavelength of an electron is given by the de Broglie equation 

V 
Here fi is Planck's constant and pthe relativistic momentum of the electron. \ is called the de Broglie 

wavelength. The electrons are accelerated in an electric potential JJ to the desired velocity: 



\2eU 

V m o 
TTlois the mass of the electron, and e is the elementary charge. The electron wavelength is then given by: 

h h h 



p rriQV \/2rn () eU 

However, in an electron microscope, the accelerating potential is usually several thousand volts causing the electron 
to travel at an appreciable fraction of the speed of light. An SEM may typically operate at an accelerating potential of 
10,000 volts (10 kV) giving an electron velocity approximately 20% of the speed of light, while a typical TEM can 
operate at 200 kV raising the electron velocity to 70% the speed of light. We therefore need to take relativistic 
effects into account. It can be shown that the electron wavelength is then modified according to: 

1 



A 



\Z2moeU /T + 



ell 



2m-oc 2 

C is the speed of light. We recognize the first term in this final expression as the non-relativistic expression derived 
above, while the last term is a relativistic correction factor. The wavelength of the electrons in a 10 kV SEM is then 

— 12 

12.3 x 10 m (12.3 pm) while in a 200 kV TEM the wavelength is 2.5 pm. In comparison the wavelength of X-rays 
usually used in X-ray diffraction is in the order of 100 pm (Cu ka: X=154 pm). 



Electron diffraction 



84 



Electron diffraction in a TEM 

Electron diffraction of solids is usually performed in a Transmission Electron Microscope (TEM) where the 
electrons pass through a thin film of the material to be studied. The resulting diffraction pattern is then observed on a 
fluorescent screen, recorded on photographic film, on imaging plates or using a CCD camera. 

Benefits 

As mentioned above, the wavelength of electron accelerated in a TEM is much smaller than that of the radiation 
usually used for X-ray diffraction experiments. A consequence of this is that the radius of the Ewald sphere is much 
larger in electron diffraction experiments than in X-ray diffraction. This allows the diffraction experiment to reveal 
more of the two dimensional distribution of reciprocal lattice points. 

Furthermore, electron lenses allows the geometry of the diffraction experiment to be varied. The conceptually 
simplest geometry is that of a parallel beam of electrons incident on the specimen. However, by converging the 
electrons in a cone onto the specimen, one can in effect perform a diffraction experiment over several incident angles 
simultaneously. This technique is called Convergent Beam Electron Diffraction (CBED) and can reveal the full three 
dimensional symmetry of the crystal. 

In a TEM, a single crystal grain or particle may be selected for the diffraction experiments. This means that the 
diffraction experiments can be performed on single crystals of nanometer size, whereas other diffraction techniques 
would be limited to studying the diffraction from a multicrystalline or powder sample. Furthermore, electron 
diffraction in TEM can be combined with direct imaging of the sample, including high resolution imaging of the 
crystal lattice, and a range of other techniques. These include solving and refining crystal structures by electron 
crystallography, chemical analysis of the sample composition through energy-dispersive X-ray spectroscopy, 
investigations of electronic structure and bonding through electron energy loss spectroscopy, and studies of the mean 
inner potential through electron holography. 



Diffraction pattern 



Practical aspects 

Figure 1 to the right is a simple sketch of the path of a parallel beam of 

electrons in a TEM from just above the sample and down the column 

to the fluorescent screen. As the electrons pass through the sample, 

they are scattered by the electrostatic potential set up by the constituent 

elements. After the electrons have left the sample they pass through the 

electromagnetic objective lens. This lens acts to collect all electrons 

scattered from one point of the sample in one point on the fluorescent 

screen, causing an image of the sample to be formed. We note that at 

the dashed line in the figure, electrons scattered in the same direction 

by the sample are collected into a single point. This is the back focal 

plane of the microscope, and is where the diffraction pattern is formed. 

By manipulating the magnetic lenses of the microscope, the diffraction 

pattern may be observed by projecting it onto the screen instead of the image. An example of what a diffraction 

pattern obtained in this way may look like is shown in figure 2. 

If the sample is tilted with respect to the incident electron beam, one can obtain diffraction patterns from several 
crystal orientations. In this way, the reciprocal lattice of the crystal can be mapped in three 




1: Sketch of the electron beam-path in a TEM. 



Electron diffraction 



85 



dimensions. By studying the systematic absence of diffraction spots the 
Bravais lattice and any screw axes and glide planes present in the 
crystal structure may be determined. 

Limitations 

Electron diffraction in TEM is subject to several important limitations. 
First, the sample to be studied must be electron transparent, meaning 
the sample thickness must be of the order of 100 nm or less. Careful 
and time consuming sample preparation may therefore be needed. 
Furthermore, many samples are vulnerable to radiation damage caused 
by the incident electrons. 




2: Typical electron diffraction pattern obtained in 
a TEM with a parallel electron beam 



The study of magnetic materials is complicated by the fact that electrons are deflected in magnetic fields by the 
Lorentz force. Although this phenomenon may be exploited to study the magnetic domains of materials by Lorentz 
force microscopy, it may make crystal structure determination virtually impossible. 

Furthermore, electron diffraction is often regarded as a qualitative technique suitable for symmetry determination, 
but too inaccurate for determination of lattice parameters and atomic positions. But there are also several examples 
where unknown crystal structures (both inorganic, organic and biological) have been solved by electron 
crystallography. Lattice parameters of high accuracy can in fact be obtained from electron diffraction, relative errors 
less than 0.1% have been demonstrated. However, the right experimental conditions may be difficult to obtain, and 
these procedures are often viewed as too time consuming and the data too difficult to interpret. X-ray or neutron 
diffraction are therefore often the preferred methods for determining lattice parameters and atomic positions. 

However, the main limitation of electron diffraction in TEM remains the comparatively high level of user interaction 
needed. Whereas both the execution of powder X-ray (and neutron) diffraction experiments and the data analysis are 
highly automated and routinely performed, electron diffraction requires a much higher level of user input. 



See also 

Electron microscope 

Transmission electron microscopy 

Selected area diffraction 

Gas electron diffraction 

RHEED 

Low-energy electron diffraction 

Stereographic projection 

Kikuchi line 

Electron backscatter diffraction 



Electron diffraction 86 

References 

• Leonid A. Bendersky and Frank W. Gayle, "Electron Diffraction Using Transmission Electron Microscopy , 
Journal of Research of the National Institute of Standards and Technology, 106 (2001) pp. 997—1012. 

• Gareth Thomas and Michael J. Goringe (1979). Transmission Electron Microscopy of Materials. John Wiley. 
ISBN 0-471-12244-0. 

External links 

• Remote experiment on electron diffraction (choose English and then "Labs") 

T31 

• Jmol-mediated image/diffraction analysis of an unknown 

References 

[1] http://nvl.nist.gov/pub/nistpubs/jres/106/6/j66ben.pdf 

[2] http://rcl.physik.uni-kl.de 

[3] http://newton.umsl.edU/run//nano/unknownl73.html 



Neutron diffraction 



Neutron diffraction is a method by which neutrons are used to determine the atomic and/or magnetic structure of a 
material. It can be applied to study the structure of crystalline solids (see crystallography), gasses, liquids or 
amorphous materials. Neutron diffraction is a form of elastic scattering where the neutrons scattered from the sample 
have comparable energy to the incident neutrons. The technique is similar to X-ray diffraction but due to the 
different scattering properties of neutrons versus x-rays complementary information can be obtained. A sample to be 
examined is placed in a beam of thermal or cold neutrons and the diffraction pattern intensity from the sample 
provides information of the structure of the material. 

Description 
Principle 

Neutrons are particles found in the atomic nucleus of almost all atoms, but they are bound. The technique requires 
free neutrons and these normally do not occur in nature, because they have limited life-time. In a nuclear reactor, 
however, neutrons can be set free through nuclei decay particularly when fission occurs. All quantum particles can 
exhibit wave phenomena we typically associate with light or sound. Diffraction is one of these phenomena; it occurs 
when waves encounter obstacles whose size is comparable with the wavelength. If the wavelength of a quantum 
particle is short enough, atoms or their nuclei can serve as diffraction obstacles. When a beam of neutrons emanating 
from a reactor is slowed down and selected properly by their speed, their wavelength lies near one Angstrom (0.1 
nanometer), the typical separation between atoms in a solid material. Such a beam can then be used to perform a 
diffraction experiment. Impinging on a crystalline sample it will scatter under a limited number of well-defined 
angles according to the same Bragg's law that describes X-ray diffraction. 

Instrumental requirements 

A neutron diffraction measurement requires a neutron source (e.g. a nuclear reactor or spallation source), a sample 
(the material to be studied), and a detector. Samples sizes are large compared to those used in X-ray diffraction. The 
technique is therefore mostly performed as powder diffraction. At a research reactor other components such as 
crystal monochromators or filters may be needed to select the desired neutron wavelength. Some parts of the setup 
may also be movable. At a spallation source the time of flight technique is used to sort the energies of the incident 



Neutron diffraction 87 

neutrons (Higher energy neutrons are faster), so no monochromator is needed, but rather a series of aperture 
elements synchronized to filter neutron pulses with the desired wavelength. 

Nuclear scattering 

Neutrons interact with matter differently than x-rays. X-rays interact primarily with the electron cloud surrounding 
each atom. The contribution to the diffracted x-ray intensity is therefore larger for atoms with a large atomic number 
(Z) than it is for atoms with a small Z. On the other hand, neutrons interact directly with the nucleus of the atom, and 
the contribution to the diffracted intensity is different for each isotope; for example, regular hydrogen and deuterium 
contribute differently. It is also often the case that light (low Z) atoms contribute strongly to the diffracted intensity 
even in the presence of large Z atoms. The scattering length varies from isotope to isotope rather than linearly with 
the atomic number. An element like Vanadium is a strong scatterer of X-rays, but its nuclei hardly scatter neutrons, 
which is why it often used as a container material. Non-magnetic neutron diffraction is directly sensitive to the 
positions of the nuclei of the atoms. 

A major difference with X-rays is that the scattering is mostly due to the tiny nuclei of the atoms. That means that 
there is no need for an atomic form factor to describe the shape of the electron cloud of the atom and the scattering 
power of an atom does not fall off with the scattering angle as it does for X-rays. Diffractograms therefore can show 
strong well defined diffraction peaks even at high angles, particularly if the experiment is done at low temperatures. 
Many neutron sources are equipped with liquid helium cooling systems that allow to collect data at temperatures 
down to 4.2 K. The superb high angle (i.e. high resolution) information means that the data can give very precise 
values for the atomic positions in the structure. On the other hand, Fourier maps (and to a lesser extent difference 
Fourier maps) derived from neutron data suffer from series termination errors, sometimes so much that the results 
are meaningless. 

Magnetic scattering 

Although neutrons are uncharged, they carry a spin, and therefore interact with magnetic moments, including those 
arising from the electron cloud around an atom. Neutron diffraction can therefore reveal the microscopic magnetic 
structure of a material . 

Magnetic scattering does require an atomic form factor as it is caused by the much larger electron cloud around the 
tiny nucleus. The intensity of the magnetic contribution to the diffraction peaks will therefore dwindle towards 
higher angles. 

History 

The first neutron diffraction experiments were carried out in 1945 by Ernest O. Wollan using the Graphite Reactor at 
Oak Ridge. He was joined shortly thereafter (June 1946) by Clifford Shull, and together they established the basic 
principles of the technique, and applied it successfully to many different materials, addressing problems like the 
structure of ice and the microscopic arrangements of magnetic moments in materials. For this achievement Shull was 
awarded one half of the 1994 Nobel Prize in Physics. Wollan had died in the 1990s. (The other half of the 1994 
Nobel Prize for Physics went to Bert Brockhouse for development of the inelastic scattering technique at the Chalk 
River facility of AECL. This also involved the invention of the triple axis spectrometer). Brockhouse and Shull 
jointly take the somewhat dubious distinction of the longest gap between the work being done (1946) and the Nobel 
Prize being awarded (1994). 



Neutron diffraction 

Uses 

Neutron diffraction is closely related to X-ray powder diffraction . In fact the single crystal version of the 
technique is less commonly used because currently available neutron sources require relatively large samples and 
large single crystals are hard or impossible to come by for most materials. Future developments, however, may well 
change this picture. Because the data is typically a ID powder diffractogram they are usually processed using 
Rietveld refinement. In fact the latter found its origin in neutron diffraction (at Petten in the Netherlands) and was 
later extended for use in X-ray diffraction. 

One practical application of elastic neutron scattering/diffraction is that the lattice constant of metals and other 
crystalline materials can be very accurately measured. Together with an accurately aligned micropositioner a map of 
the lattice constant through the metal can be derived. This can easily be converted to the stress field experienced by 
the material. This has been used to analyse stresses in aerospace and automotive components to give just two 
examples. This technique has led to the development of dedicated stress diffractometers, such as the ENGIN-X 
instrument at the ISIS neutron source. 

Hydrogen, null-scattering and contrast variation 

Neutron diffraction can be used to establish the structure of low atomic number materials like proteins and 
surfactants much more easily with lower flux than at a synchrotron radiation source. This is because some low 
atomic number materials have a higher cross section for neutron interaction than higher atomic weight materials. 

One major advantage of neutron diffraction over X-ray diffraction is that the latter is rather insensitive to the 

1 2 

presence of hydrogen (H) in a structure, whereas the nuclei H and H (i.e. Deuterium, D) are strong scatterers for 
neutrons. This means that the position of hydrogen in a crystal structure and its thermal motions can be determined 
far more precisely with neutrons. In addition the neutron scattering lengths b = -3.7406(1 1) fm and b = 6.671(4) 

Ml H D 

fm , for H and D respectively, have opposite sign allowing for contrast variation. In fact there is a particular 
isotope ratio for which the contribution of the element would cancel, this is called null-scattering. In practice 
however it is not desirable to work with the relatively high concentration of H in such a sample. The scattering 
intensity by H-nuclei has a large ineleastic component and this creates a large continuous background that is more or 
less independent of scattering angle. The elastic pattern typically consists of sharp Bragg reflections if the sample is 
crystalline. They tend to drown in the inelastic background. This is even more serious when the technique is used for 
the study of liquid structure. Nevertheless, by preparing samples with different isotope ratios it is possible to vary the 
scattering contrast enough to highlight one element in an otherwise complicated structure. The variation of other 
elements is possible but usually rather expensive. Hydrogen is inexpensive and particularly interesting because it 
plays an exceptionally large role in biochemical structures and is difficult to study structurally in other ways. 



Further reading 



Lovesey, S. W. (1984). Theory of Neutron Scattering from Condensed Matter; Volume 1: Neutron Scattering. 

Oxford: Clarendon Press. ISBN 0-19-852015-8. 

Lovesey, S. W. (1984). Theory of Neutron Scattering from Condensed Matter; Volume 2: Condensed Matter. 

Oxford: Clarendon Press. ISBN 0-19-852017-4. 

Squires, G.L. (1996). Introduction to the Theory of Thermal Neutron Scattering (2nd ed.). Mineola, New York: 

Dover Publications Inc. ISBN 0-486-69447-X. 



Neutron diffraction 

Applied Computational Powder Diffraction Data Analysis 

• Young, R.A., ed (1993). The Rietveld Method. Oxford: Oxford University Press & International Union of 
Crystallography. ISBN 0-19-855577-6. 

See also 

Bragg diffraction 

Crystallography 

Crystallographic database 

Diffraction 

Electron crystallography 

Electron diffraction 

Neutron crystallography 

Neutron Diffraction at OPAL 

X-ray crystallography 

X-ray diffraction 

X-ray scattering techniques 

References 

[1] Neutron diffraction of magnetic materials / Yu. A. Izyumov, V.E. Naish, and R.P. Ozerov ; translated from Russian by Joachim Buchner. 

New York : Consultants Bureau, cl991.ISBN 0-306-1 1030-X 
[2] Clifford Shull: Early development of neutron scattering. Rev. Mod. Phys. 67 (1995) 753—757 
[3] Neutron powder diffraction by Richard M. Ibberson and William I.F. David, Chapter 5 of Structure determination form powder diffraction 

data IUCr monographphs on crystallography, Oxford scientific publications 2002, ISBN 0-19-850091-2 
[4] Sears, V. F. (1992), "Neutron scattering lengths and cross sections", Neutron News 3: 26—37 

External links 

• National Institute of Standards and Technology Center for Neutron Research (http://www.ncnr.nist.gov/) 



Neutron scattering 90 



Neutron scattering 



Neutron scattering encompasses all scientific techniques whereby the deflection of neutron radiation is used as a 
scientific probe. Neutrons readily interact with atomic nuclei and magnetic fields from unpaired electrons, making a 
useful probe of both structure and magnetic order. Neutron Scattering falls into two basic categories, elastic and 
inelastic. Elastic scattering is when a neutron interacts with a nucleus or electronic magnetic field but does not leave 
it in an excited state, meaning the emitted neutron has the same energy as the injected neutron. Scattering processes 
that involve an energetic excitation or relaxation by the neutron are inelastic: the injected neutron's energy is used or 
increased to create an excitation or by absorbing the excess energy from a relaxation, and consequently the emitted 
neutron's energy is reduced or increased respectively. 

For several good reasons, moderated neutrons provide an ideal tool for the study of almost all forms of condensed 
matter. Firstly, they are readily produced at a nuclear research reactor or a spallation source. Normally in such 
processes neutrons are however produced with much higher energies than are needed. Therefore moderators are 
generally used which slow the neutrons down and therefore produce wavelengths that are comparable to the atomic 
spacing in solids and liquids, and kinetic energies that are comparable to those of dynamic processes in materials. 
Moderators can be made from aluminium and filled with liquid hydrogen (for very long wavelength neutrons) or 

7 8 

liquid methane (for shorter wavelength neutrons). Fluxes of 10 /s - 10 /s are not atypical in most neutron sources 
from any given moderator. 

The neutrons cause pronounced interference and energy transfer effects in scattering experiments. Unlike an x-ray 
photon with a similar wavelength, which interacts with the electron cloud surrounding the nucleus, neutrons interact 
with the nucleus itself. Because the neutron is an electrically neutral particle, it is deeply penetrating, and is therefore 
more able to probe the bulk material. Consequently, it enables the use of a wide range of sample environments that 
are difficult to use with synchrotron x-ray sources. It also has the advantage that the cross sections for interaction do 
not increase with atomic number as they do with radiation from a synchrotron x-ray source. Thus neutrons can be 
used to analyse materials with low atomic numbers like proteins and surfactants. This can be done at synchrotron 
sources but very high intensities are needed which may cause the structures to change. Moreover, the nucleus 
provides a very short range, isotropic potential varying randomly from isotope to isotope, making it possible to tune 
the nuclear scattering contrast to suit the experiment. 

The neutron has an additional advantage over the x-ray photon in the study of condensed matter. It readily interacts 
with internal magnetic fields in the sample. In fact, the strength of the magnetic scattering signal is often very similar 
to that of the nuclear scattering signal in many materials, which allows the simultaneous exploration of both nuclear 
and magnetic structure. Because the neutron scattering amplitude can be measured in absolute units, both the 
structural and magnetic properties as measured by neutrons can be compared quantitatively with the results of other 
characterisation techniques. 

See also 

• Neutron diffraction 

• Small angle neutron scattering 

• Neutron Reflectometry 

• Inelastic neutron scattering 

• neutron triple-axis spectrometry 

• neutron time-of-flight scattering 

• neutron backscattering 

• neutron spin echo 

• neutron resonance spin echo 



Neutron scattering 91 

• Neutron scattering facilities 

Applications 

Neutron scattering has been used to study various vibration modes, including low-frequency collective motion in 
proteins and DNA, [2] [3] [4] [5] [6] as reviewed by Dr. P. Martel in 1992. [1] 

References 

[1] Martel, P. (1992) Biophysical aspects of neutron scattering from vibrational modes of proteins. Prog Biophys Mol Biol, 57, 129-179. 

[2] Chou, K.C. (1983) Low-frequency vibrations of helical structures in protein molecules. Biochemical Journal, 209, 573-580. 

[3] Chou, K.C. (1985) Low-frequency motions in protein molecules: beta-sheet and beta-barrel. Biophysical Journal, 48, 289-297. 

[4] Chou, K.C, Maggiora, G.M. and Mao, B. (1989) Quasi-continuum models of twist-like and accordion-like low-frequency motions in DNA. 

Biophysical Journal, 56, 295-305. 
[5] Kuo-Chen Chou (1988) Review: Low-frequency collective motion in biomacromolecules and its biological functions. Biophysical Chemistry, 

30, 3-48. 
[6] Chou, K.C. (1989) Low-frequency resonance and cooperativity of hemoglobin. Trends in Biochemical Sciences, 14, 212. 

External links 

• Neutron Scattering - A primer (http://knocknick.files.wordpress.com/2008/04/ 
neutrons-a-primer-by-rogen-pynn.pdf) ( LANL-hosted black and white version (http://library.lanl.gov/cgi-bin/ 
getfile700326651.pdf)) - An introductory article written by Roger Pynn (Los Alamos National Laboratory) 

• Podcast Interview with two ILL scientists about neutron science/scattering at the ILL (http://omegataupodcast. 
net/20 10/03/28-neutron-science-at-the-ill/) 



Inelastic neutron scattering 



Inelastic neutron scattering is an experimental technique commonly used in condensed matter research to study 
atomic and molecular motion as well as magnetic and crystal field excitations. It distinguishes itself from other 
neutron scattering techniques by resolving the change in kinetic energy that occurs when the collision between 
neutrons and the sample is an inelastic one. Results are generally communicated as the dynamic structure factor (also 
called inelastic scattering law) S(q,co), sometimes also as the dynamic susceptibility x(l< m ) where the scattering 
vector q is the difference between incoming and outgoing wave vector, and fuj is the energy change experienced by 
the sample (negative that of the scattered neutron). When results are plotted as function of w, they can often be 
interpreted in the same way as spectra obtained by conventional spectroscopic techniques; insofar as inelastic 
neutron scattering can be seen as a special spectroscopy. 



Inelastic neutron scattering 



92 




Inelastic scattering experiments 
normally require a 

monochromatization of the incident or 
outgoing beam and an energy analysis 
of the scattered neutrons. This can be 
done either through time-of-flight 
techniques (neutron time-of-flight 
scattering) or through Bragg reflection 
from single crystals (neutron 
triple-axis spectroscopy, neutron 
backscattering). Monochromatization 
is not needed in echo techniques 
(neutron spin echo, neutron resonance 
spin echo), which use the quantum 
mechanical phase of the neutrons in addition to their amplitudes 



monitor 



D 



sample 

o 



V 



2" 



analyzer 







El 



detector 
Generic layout of an inelastic neutron scattering experiment 



beam 
stop 



o 



Types of Inelastic Neutron Scattering 

• neutron triple-axis scattering 

• neutron time-of-flight scattering 

• neutron backscattering 

• neutron spin echo 

Further Information 

Literature: 

• G L Squires Introduction to the Theory of Thermal Neutron Scattering Dover 1997 (reprint?) 



External links 

• Joachim Wuttke: Introduction to Inelastic Crystal Spectrometers 



[l] 



References 



[1] http://iffwww.iff.kfa-juelich.de/~wuttke/doku/lib/exe/fetch.php?id=spheres%3Aprinciple&cache=cache&media=spheres:np9v05.pdf 



Ionization cooling 93 



Ionization cooling 



Physical Principle 



Ionization cooling is a process by which the beam emittance of a beam of particles may be reduced .In ionization 
cooling, particles are passed through some material. The momentum of the particles is reduced as they ionize atomic 
electrons in the material. Thus the normalised beam emittance is reduced. By re-accelerating the beam, for example 
in an RF cavity, the longitudinal momentum may be restored without replacing transverse momentum. Thus overall 
the angular spread and hence the geometric emittance in the beam will be reduced. 

Ionization cooling can be spoiled by stochastic physical processes. Multiple Coulomb scattering in muons as well as 
nuclear scattering in protons and ions can reduce the cooling or even lead to net heating transverse to the direction of 
beam motion. In addition, energy straggling can cause heating parallel to the direction of beam motion. 

Muon Cooling 

The primary use of ionization cooling is envisaged to be for cooling of muon beams. This is because ionization 
cooling is the only technique that works on the timescale of the muon lifetime. Ionization cooling channels have 
been designed for use in a Neutrino Factory and a Muon Collider. Muon ionization cooling is expected to be 
demonstrated for the first time by the Proof of Principle International Muon Ionization Cooling Experiment. Other 
PoP muon ionization cooling experiments have been devised. 

Other Particles 

Ionization cooling has also been proposed for use in low energy ion beams and proton beams. 

References 

[1] G.I. Budker, in: Proceedings of 15th International Conference on High Energy Physics, Kiev, 1970 A.N. Skrinsky, Intersecting storage rings 
at Novosibirsk, in: Proceedings of Morges Seminar, 1971 Report CERN/D.PH II/YGC/mng 



Deep inelastic scattering 



94 



Deep inelastic scattering 



Deep inelastic scattering is the name given to a 
process used to probe the insides of hadrons 
(particularly the baryons, such as protons and 
neutrons), using electrons, muons and neutrinos. It 
provided the first convincing evidence of the reality of 
quarks, which up until that point had been considered 
by many to be a purely mathematical phenomenon. It is 
a relatively new process, first attempted in the 1960s 
and 1970s. It is conceptually similar to Rutherford 
Scattering, but with important differences. The reason 
why this type of scattering is described as "deep" and 

"inelastic" is discussed at the Oxford University 

[l] 
page. 



x/ 






Is 

Y* 


q^r 






\Q 


h g \ 












-► 


X 


Deep inelastic scattering of a lepton on a 


hadron, 


at leading order in 


perturbative expansion 







Quarks 

The Standard Model of physics, particularly given the 

work of Murray Gell-Mann in the 1960s, had been 

successful in uniting much of the previously disparate concepts in particle physics into one, relatively 

straightforward, scheme. In essence, there were three types of particles. 

• The leptons, which were light (as in not particularly massive) particles such as electrons, neutrinos and their 
antiparticles. They have integer charge 

• The bosons, which were particles that exchange forces. These ranged from the massless, easy-to-detect photon 
(the carrier of the electro-magnetic force) to the exotic (though still massless) gluons that carry the strong nuclear 
force 

• The quarks, which were massive particles that carried fractional charges. They are the "building blocks" of the 
hadrons. They are also the only particles to be affected by the strong interaction 

The leptons had been detected since 1897, when J. J. Thomson had shown that electric current is a flow of electrons. 
Some bosons were being routinely detected, although the W , W" and Z particles of the electroweak force were only 
categorically seen in the early 1980s, and gluons were only firmly pinned down at DESY in Hamburg at about the 
same time. Quarks, however, were still elusive. 



The experiments 

Drawing on Rutherford's groundbreaking experiments in the early years of the Twentieth century, ideas for detecting 
quarks were formulated. Rutherford had proven that atoms had a small, massive, charged nucleus at their centre by 
firing alpha particles at atoms in gold. Most had gone through with little or no deviation, but a few were deflected 
through large angles or came right back. This suggested that atoms had internal structure, and a lot of empty space. 

In order to enter baryons (where quarks were theoretically to be found), a small, penetrating (ie easily accelerated; in 
reality this meant charged) and easily produced particle needed to be found. Electrons were considered ideal for the 
role, and in a series of remarkable technological and engineering leaps, electrons were fired at protons and neutrons 
in atomic nuclei. As an added bonus, the electrostatic attraction of the positively charged nucleus and the negatively 
charged electron increased the speed. Later experiments were conducted with muons, but the same principles apply. 



Deep inelastic scattering 95 

The collision absorbs some kinetic energy, and as such it is inelastic (this compares to Rutherford scattering which is 
elastic, with no loss of kinetic energy, taking into account recoils of the nuclei). The electron emerges from the 
nucleus, and its trajectory and velocity can be detected. 

Analysis of the results led to the following conclusions: 

• The hadrons do have internal structure 

• In baryons, there are three points of deflection (i.e. baryons consist of three quarks) 

• In mesons, there are two points of deflection (i.e. mesons consist of a quark and an anti-quark. The reason they do 
not consist of two quarks is to do with their colour; see the quark article for more explanation) 

• Quarks appear to be point charges, as electrons appear to be, with the fractional charges suggested by the 
Standard Model 

The experiments were important because, not only did they confirm the physical reality of quarks but also proved 
again that the Standard Model was the correct avenue of research for particle physicists to pursue. 

References 

[1] Deep inelastic scattering (http://www.physics.ox.ac.uk/documents/PUS/dis/DIS.htm). Oxford University Physics Department, 2003. 



Timeline of microphysics 



Timeline of atomic and subatomic physics 

Early beginnings 

• 585 BC Buddha states that there were indivisible particles of mind and matter which vibrated 3 trillion times in 
the blink of an eye which he calls "kalapas" 

• 440 BC Democritus speculates about fundamental indivisible particles — calls them "atoms" 

The beginning of chemistry 

1766 Henry Cavendish discovers and studies hydrogen 

1778 Carl Scheele and Antoine Lavoisier discover that air is composed mostly of nitrogen and oxygen 

1781 Joseph Priestley creates water by igniting hydrogen and oxygen 

1800 William Nicholson and Anthony Carlisle use electrolysis to separate water into hydrogen and oxygen 

1803 John Dal ton introduces atomic ideas into chemistry and states that matter is composed of atoms of different 

weights 

1805 Thomas Young conducts Double-slit experiment (approximate time) 

1811 Amedeo Avogadro claims that equal volumes of gases should contain equal numbers of molecules 

1832 Michael Faraday states his laws of electrolysis 

1871 Dmitri Ivanovich Mendeleev systematically examines the periodic table and predicts the existence of 

gallium, scandium, and germanium 

1873 Johannes van der Waals introduces the idea of weak attractive forces between molecules 

1885 Johann B aimer finds a mathematical expression for observed hydrogen line wavelengths 

1887 Heinrich Hertz discovers the photoelectric effect 

1894 Lord Rayleigh and William Ramsay discover argon by spectroscopically analyzing the gas left over after 
nitrogen and oxygen are removed from air 

1895 William Ramsay discovers terrestrial helium by spectroscopically analyzing gas produced by decaying 
uranium 



Timeline of microphysics 

• 1896 Antoine Becquerel discovers the radioactivity of uranium 

• 1896 Pieter Zeeman studies the splitting of sodium D lines when sodium is held in a flame between strong 
magnetic poles 

• 1897 J.J. Thomson discovers the electron 

• 1898 William Ramsay and Morris Travers discover neon, and negatively charged beta particles 

The age of quantum mechanics 

1900 Paul Villard discovers gamma-rays while studying uranium decay 

1900 Johannes Rydberg refines the expression for observed hydrogen line wavelengths 

1900 Max Planck states his quantum hypothesis and blackbody radiation law 

1902 Philipp Lenard observes that maximum photoelectron energies are independent of illuminating intensity but 

depend on frequency 

1902 Theodor Svedberg suggests that fluctuations in molecular bombardment cause the Brownian motion 

1905 Albert Einstein explains the photoelectric effect 

1906 Charles Barkla discovers that each element has a characteristic X-ray and that the degree of penetration of 
these X-rays is related to the atomic weight of the element 

1909 Hans Geiger and Ernest Marsden discover large angle deflections of alpha particles by thin metal foils 
1909 Ernest Rutherford and Thomas Royds demonstrate that alpha particles are doubly ionized helium atoms 
1911 Ernest Rutherford explains the Geiger-Marsden experiment by invoking a nuclear atom model and derives 
the Rutherford cross section 

1911 Jean Perrin proves the existence of atoms and molecules 

1912 Max von Laue suggests using crystal lattices to diffract X-rays 

1912 Walter Friedrich and Paul Knipping diffract X-rays in zinc blende 

1913 William Henry Bragg and William Lawrence Bragg work out the Bragg condition for strong X-ray 
reflection 

1913 Henry Moseley shows that nuclear charge is the real basis for numbering the elements 
1913 Niels Bohr presents his quantum model of the atom 
1913 Robert Millikan measures the fundamental unit of electric charge 

1913 Johannes Stark demonstrates that strong electric fields will split the Balmer spectral line series of hydrogen 

1914 James Franck and Gustav Hertz observe atomic excitation 

1914 Ernest Rutherford suggests that the positively charged atomic nucleus contains protons 

1915 Arnold Sommerfeld develops a modified Bohr atomic model with elliptic orbits to explain relativistic fine 
structure 

1916 Gilbert N. Lewis and Irving Langmuir formulate an electron shell model of chemical bonding 

1917 Albert Einstein introduces the idea of stimulated radiation emission 

1921 Alfred Lande introduces the Lande g-factor 

1922 Arthur Compton studies X-ray photon scattering by electrons 

1922 Otto Stern and Walther Gerlach show "space quantization" 

1923 Louis de Broglie suggests that electrons may have wavelike properties 

1923 Lise Meitner discovers the Auger process 

1924 John Lennard-Jones proposes a semiempirical interatomic force law 

1924 Satyendra Bose and Albert Einstein introduce Bose-Einstein statistics 

1925 Wolfgang Pauli states the quantum exclusion principle 
1925 George Uhlenbeck and Samuel Goudsmit postulate electron spin 
1925 Pierre Auger discovers the Auger process (2 years after Lise Meitner) 
1925 Werner Heisenberg, Max Born, and Pascual Jordan formulate quantum matrix mechanics 



Timeline of microphysics 97 

1926 Erwin Schrodinger states his nonrelativistic quantum wave equation and formulates quantum wave 

mechanics 

1926 Erwin Schrodinger proves that the wave and matrix formulations of quantum theory are mathematically 

equivalent 

1926 Oskar Klein and Walter Gordon state their relativistic quantum wave equation, now the Klein-Gordon 

equation 

1926 Enrico Fermi discovers the spin-statistics connection 

1926 Paul Dirac introduces Fermi-Dirac statistics 

1927 Clinton Davisson, Lester Germer, and George Paget Thomson confirm the wavelike nature of electrons 
1927 Werner Heisenberg states the quantum uncertainty principle 
1927 Max Born interprets the probabilistic nature of wavefunctions 

1927 Walter Heitler and Fritz London introduce the concepts of valence bond theory and apply it to the hydrogen 
molecule. 
1927 Thomas and Fermi develop the Thomas -Fermi model 

1927 Max Born and Robert Oppenheimer introduce the Born-Oppenheimer approximation 

1928 Chandrasekhara Raman studies optical photon scattering by electrons 
1928 Paul Dirac states his relativistic electron quantum wave equation 
1928 Charles G Darwin and Walter Gordon solve the Dirac equation for a Coulomb potential 

1928 Friedrich Hund and Robert S. Mulliken introduce the concept of molecular orbital 

1929 Oskar Klein discovers the Klein paradox 

1929 Oskar Klein and Yoshio Nishina derive the Klein-Nishina cross section for high energy photon scattering by 
electrons 

1929 Nevill Mott derives the Mott cross section for the Coulomb scattering of relativistic electrons 

1930 Paul Dirac introduces electron hole theory 
1930 Erwin Schrodinger predicts the zitterbewegung motion 

1930 Fritz London explains van der Waals forces as due to the interacting fluctuating dipole moments between 
molecules 

1931 John Lennard-Jones proposes the Lennard-Jones interatomic potential 
1931 Irene Joliot-Curie and Frederic Joliot observe but misinterpret neutron scattering in paraffin 
1931 Wolfgang Pauli puts forth the neutrino hypothesis to explain the apparent violation of energy conservation 
in beta decay 

1931 Linus Pauling discovers resonance bonding and uses it to explain the high stability of symmetric planar 
molecules 
1931 Paul Dirac shows that charge quantization can be explained if magnetic monopoles exist 

1931 Harold Urey discovers deuterium using evaporation concentration techniques and spectroscopy 

1932 John Cockcroft and Ernest Walton split lithium and boron nuclei using proton bombardment 
1932 James Chadwick discovers the neutron 
1932 Werner Heisenberg presents the proton-neutron model of the nucleus and uses it to explain isotopes 

1932 Carl D. Anderson discovers the positron 

1933 Ernst Stueckelberg (1932), Lev Davidovich Landau (1932), and Clarence Zener discover the Landau-Zener 
transition 

1933 Max Delbruck suggests that quantum effects will cause photons to be scattered by an external electric field 

1934 Irene Joliot-Curie and Frederic Joliot bombard aluminum atoms with alpha particles to create artificially 
radioactive phosphorus-30 

1934 Leo Szilard realizes that nuclear chain reactions may be possible 
1934 Enrico Fermi formulates his theory of beta decay 



Timeline of microphysics 

• 1934 Lev Davidovich Landau tells Edward Teller that nonlinear molecules may have vibrational modes which 
remove the degeneracy of an orbitally degenerate state (Jahn-Teller effect) 
1934 Enrico Fermi suggests bombarding uranium atoms with neutrons to make a 93 proton element 

1934 Pavel Alekseyevich Cherenkov reports that light is emitted by relativistic particles traveling in a 
nonscintillating liquid 

1935 Hideki Yukawa presents a theory of strong interactions and predicts mesons 
1935 Albert Einstein, Boris Podolsky, and Nathan Rosen put forth the EPR paradox 
1935 Henry Eyring develop the transition state theory 

1935 Niels Bohr presents his analysis of the EPR paradox 

1936 Eugene Wigner develops the theory of neutron absorption by atomic nuclei 

1936 Hermann Arthur Jahn and Edward Teller present their systematic study of the symmetry types for which the 
Jahn-Teller effect is expected 

1937 Hans Hellmann finds the Hellmann-Feynman theorem 

1937 Seth Neddermeyer, Carl Anderson, J.C. Street, and E.C. Stevenson discover muons using cloud chamber 
measurements of cosmic rays 

1939 Richard Feynman finds the Hellmann-Feynman theorem 

1939 Otto Hahn and Fritz Strassmann bombard uranium salts with thermal neutrons and discover barium among 
the reaction products 

1939 Lise Meitner and Otto Robert Frisch determine that nuclear fission is taking place in the Hahn-Strassmann 
experiments 
1942 Enrico Fermi makes the first controlled nuclear chain reaction 

1942 Ernst Stueckelberg introduces the propagator to positron theory and interprets positrons as negative energy 
electrons moving backwards through spacetime 

1943 Sin-Itiro Tomonaga publishes his paper on the basic physical principles of quantum electrodynamics 
1947 Willis Lamb and Robert Retheford measure the Lamb-Retheford shift 
1947 Cecil Powell, Cesar Lattes, and Giuseppe Occhialini discover the pi-meson by studying cosmic ray tracks 

1947 Richard Feynman presents his propagator approach to quantum electrodynamics 

1948 Hendrik Casimir predicts a rudimentary attractive Casimir force on a parallel plate capacitor 

1951 Martin Deutsch discovers positronium 

1952 David Bohm propose his interpretation of quantum mechanics 

1953 Robert Wilson observes Delbruck scattering of 1.33 MeV gamma-rays by the electric fields of lead nuclei 

1954 Chen Ning Yang and Robert Mills investigate a theory of hadronic isospin by demanding local gauge 
invariance under isotopic spin space rotations— first non-Abelian gauge theory 

1955 Owen Chamberlain, Emilio Segre, Clyde Wiegand, and Thomas Ypsilantis discover the antiproton 

1956 Frederick Reines and Clyde Cowan detect antineutrino 
1956 Chen Ning Yang and Tsung Lee propose parity violation by the weak nuclear force 

1956 Chien Shiung Wu discovers parity violation by the weak force in decaying cobalt 

1957 Gerhart Luders proves the CPT theorem 

1957 Richard Feynman, Murray Gell-Mann, Robert Marshak, and E.C.G Sudarshan propose a vector/axial vector 
(VA) Lagrangian for weak interactions 

1958 Marcus Sparnaay experimentally confirms the Casimir effect 

1959 Yakir Aharonov and David Bohm predict the Aharonov-Bohm effect 

1960 R.G Chambers experimentally confirms the Aharonov-Bohm effect 

1961 Murray Gell-Mann and Yuval Ne'eman discover the Eightfold Way patterns— SU(3) group 

1961 Jeffrey Goldstone considers the breaking of global phase symmetry 

1962 Leon Lederman shows that the electron neutrino is distinct from the muon neutrino 



Timeline of microphysics 99 

The formation and successes of the Standard Model 

1963 Murray Gell-Mann and George Zweig propose the quark/aces model 

1964 Peter Higgs considers the breaking of local phase symmetry 

1964 John Stewart Bell shows that all local hidden variable theories must satisfy Bell's inequality 
1964 Val Fitch and James Cronin observe CP violation by the weak force in the decay of K mesons 
1967 Steven Weinberg puts forth his electroweak model of leptons 

1969 John Clauser, Michael Home, Abner Shimony and Richard Holt propose a polarization correlation test of 
Bell's inequality 

1970 Sheldon Glashow, John Iliopoulos, and Luciano Maiani propose the charm quark 

1971 Gerard 't Hooft shows that the Glashow-Salam-Weinberg electroweak model can be renormalized 

1972 Stuart Freedman and John Clauser perform the first polarization correlation test of Bell's inequality 

1973 David Politzer proposes the asymptotic freedom of quarks 

1974 Burton Richter and Samuel Ting discover the psi meson implying the existence of the charm quark 

1974 Robert J. Buenker and Sigrid D. Peyerimhoff introduce the multireference configuration interaction method. 

1975 Martin Perl discovers the tau lepton 
1977 Steve Herb finds the upsilon resonance implying the existence of the beauty/bottom quark 

1982 Alain Aspect, J. Dalibard, and G Roger perform a polarization correlation test of Bell's inequality that rules 
out conspiratorial polarizer communication 

1983 Carlo Rubbia, Simon van der Meer, and the CERN UA-1 collaboration find the W and Z intermediate vector 
bosons 
1989 The Z intermediate vector boson resonance width indicates three quark-lepton generations 

1994 The CERN LEAR Crystal Barrel Experiment justifies the existence of glueballs (exotic meson). 

1995 after 18 years searching at Fermilab was discovered the top quark, it had very big mass 
1998 Super-Kamiokande (Japan) observes evidence for neutrino oscillations, implying that at least one neutrino 
has mass. 

2001 The Sudbury Neutrino Observatory (Canada) confirms the existence of neutrino oscillations. 
2005 At the RHIC accelerator of Brookhaven National Laboratory they have created a quark-gluon liquid of very 
low viscosity, perhaps the quark-gluon plasma 

2008 The Large Hadron Collider at CERN is scheduled to begin operation in this year. Its primary goal is to 
search for the Higgs boson, which has not yet been found. 



Automatic calculation of particle interaction or decay 100 

Automatic calculation of particle interaction or 
decay 

The automatic calculation of particle interaction or decay is part of the computational particle physics branch. It 
refers to computing tools that help calculating the complex particle interactions as studied in high-energy physics, 
astroparticle physics and cosmology. The goal of the automation is to handle the full sequence of calculations in an 
automatic (programmed) way: from the Lagrangian expression describing the physics model up to the cross-sections 
values and to the event generator software. 

Particle accelerator or colliders produce collisions (interactions) of particle (like the electron or the proton). The 
colliding particles form the Initial State. In the collision, particles can be annihilated or/and exchanged producing 
possibly different sets of particles, the Final States. The Initial and Final States of the interaction relate through the 
so-called scattering matrix (S-matrix). 

For example at LEP, e + e~ — > e + e~, or e + e~ — > u, + \i~ are processes where the initial state is an electron and 
a positron colliding to produce an electron and a positron or two muons of opposite charge: the final states. In these 
simple cases, no automatic packages are needed and cross-section analytical expression can be easily derived at least 
for the lowest approximation: the Born approximation also called the leading order or the tree level (as Feynman 
diagrams have only trunk and branches, no loops). 

But particle physics is now requiring much more complex calculations like at LHC Pp — ► ^jets where pare 
protons and ^jetsis the number of jets of particles initiated by proton constituents (quarks and gluons). The number 
of subprocesses describing a given process is so large that automatic tools have been developed to mitigate the 
burden of hand calculations. 

Interactions at higher energies open a large spectrum of possible final states and consequently increase the number of 
processes to compute. 

High precision experiments impose the calculation of higher order calculation, namely the inclusion of subprocesses 
where more than one virtual particle can be created and annihilated during the interaction lapse creating so-called 
loops which induce much more involved calculations. 

Finally new theoretical models like the supersymmetry model (MSSM in its minimal version) predict a flurry of new 
processes. 

The automatic packages, once seen as mere teaching support, have become, this last 10 years an essential component 
of the data simulation and analysis suite for all experiments. They help constructing event generators and are 
sometime viewed as generators of event generators or Meta- generators. 

A particle physics model is essentially described by its Lagrangian. To simulate the production of events through 
event generators, 3 steps have to be taken. The Automatic Calculation project is to create the tools to make those 
steps as automatic (or programmed) as possible: 

I Feynman rules, coupling and mass generation 

LanHEP is an example of Feynman rules generation. Some model needs an additional step to compute, based on 
some parameters, the mass and coupling of new predicted particles. 

II Matrix element code generation: Various methods are used to automatically produce the Matrix element 
expression in a computer language (Fortran, C/C++). They use values (i.e. for the masses) or expressions (i.e. for the 
couplings) produced by step I or model specific libraries constructed by hands (usually heavily relying on Computer 
algebra languages). When this expression is integrated (usually numerically) over the internal degrees of freedom it 
will provide the total and differential cross-sections for a given set of initial parameters like the initial state particle 
energies and polarization. 



Automatic calculation of particle interaction or decay 101 

III Event generator code generation: This code must them be interfaced to other packages to fully provide the actual 
final state. The various effects or phenomenon that need to be implemeted are: 

Initial state radiation and beamstrahlung for e+e- initial states. Parton distribution functions describing the actual 
content in terms of gluons and quarks of the p or p-bar initial state particles Parton showering describing the way 
final state quarks or gluons due to the QCD confinement generate additional quark/gluon pairs generating a so-called 
shower of partons before transforming into hadrons. Hadronization describing how the final quark pairs/triplets form 
the visible and detectable hadrons. Underlying event takes care of the way the rest, in term of constituent, of the 
initial protons also contribute to any given event. 

The interplay or matching of the precise matrix element calculation and the approximations resulting from the 
simulation of the parton shower gives rise to further complications, either within a given level of precision like at 
leading order (LO) for the production of n jets or between two levels of precision when tempting to connect matrix 
element computed at next-to-leading (NLO) (1-loop) or next-to-next-leading order (NNLO) (2-loops) with LO 
partons shower package. 

Several methods have been developed for this matching: 

• Subtraction methods 

• 

But the only correct way is to match packages at the same level theoretical accuracy like the NLO matrix element 
calculation with NLO parton shower packages. This is currently in development. 

History 

The idea of automation of the calculations in high-energy physics is not new. It dates back to the 1960s when 
packages such as SCHOONSCHIP and then REDUCE had been developed. 

These are symbolic manipulation codes that automatize the algebraic parts of a matrix element evaluation, like traces 
on Dirac matrices and contraction of Lorentz indices. Such codes have evolved quite a lot with applications not only 
optimized for high-energy physics like FORM but also more general purpose programs like Mathematica and Maple. 

Generation of QED Feynman graphs at any order in the coupling constant was automatized in the late 70's[15]. One 
of the first major application of these early developments in this field was the calculation of the anomalous magnetic 
moments of the electron and the muon[16]. The first automatic system incorporating all the steps for the calculation 
of a cross section, from Feynman graph generation, amplitude generation through a REDUCE source code that 
produces a FORTRAN code, phase space integration and event generation with BASES/SPRING[17] is 
GRAND[18]. It was limited to tree-level processes in QED. In the early nineties, a few groups started to develop 
packages aiming at the automation in the SM[19]. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] 

Matrix element calculation methods 
Helicity amplitude 

Feynman amplitudes are written in terms of spinor products of wave functions for massless fermions, and then 
evaluated numerically before the amplitudes are squared. Taking into account fermion masses implies that Feynman 
amplitudes are decomposed into vertex amplitudes by splitting the internal lines into wave function of fermions and 
polarization vectors of gauge bosons. 

All helicity configuration can be computed independently. 



Automatic calculation of particle interaction or decay 102 

Helicity amplitude squared 

The method is similar to the previous one, but the numerical calculation is performed after squaring the Feynman 
Amplitude. The final expression is shorter and therefore faster to compute, but independent helicity information are 
not anymore available. 

Dyson- Sch winger recursive equations 

The scattering amplitude is evaluated recursively through a set of Dyson-Schwinger equations. The computational 
cost of this algorithm grows asymptotically as 3 , where n is the number of particles involved in the process, 
compared to n! in the traditional Feynman graphs approach. Unitary gauge is used and mass effects are available as 
well. Additionally, the color and helicity structures are appropriately transformed so the usual summation is replaced 
by the Monte Carlo techniques. 

Higher order calculations 

[12] 

Additional package for Event generation 

The integration of the "matrix element" over the multidimensional internal parameters phase space provides the total 
and differential cross-sections. Each point of this phase space is associated to an event probability. This is used to 
randomly generate events closely mimicking experimental data. This is called event generation, the first step in the 
complete chain of event simulation. The initial and final state particles can be elementary particles like electrons, 
muons, or photons but also partons (protons and neutrons). 

More effects must then be implemented to reproduce real life events as those detected at the colliders. 

The initial electron or positron may undergo radiation before they actually interact: initial state radiation and 
beamstrahlung. 

The bare partons that do not exist in nature (they are confined inside the hadrons) must be so to say dressed so that 
they form the known hadrons or mesons. They are made in two steps: parton shower and hadronization. 

When the initial state particles are protons at high energy, it is only their constituents which interact. Therefore the 
specific parton that will experience the "hard interaction" has to be selected. Structure functions must therefore be 
implemented. The other parton may interact "softly" must be also be simulated as they contribute to the complexity 
of the event: Underlying event 



Automatic calculation of particle interaction or decay 103 

Initial state radiation and beamstrahlung 

(to be written) 

Parton shower and Hadronization 

(to be written) 

At leading Order (LO) 

(to be written) 

At Next-to-Leading order (NLO) 

(to be written) 

Structure and Fragmentation Functions 

(to be written) 

Underlying event 

(to be written) 

Model specific packages 

(to be written) 

Related computational issues 

(to be written) 

Multi-dimensional integrators 

(to be written) 

Ultra-High Precision Numerical computation 

(to be written) 

Existing Packages 
Feynman rules generators 

• FeynRules 

• LanHEP 



Tree Level Packages 



Automatic calculation of particle interaction or decay 



104 



Name 


Model 


Max 
FS 


Tested 

FS 


Short description 


Publication 


Method 


Output 


Status 


MadGraph5 
[14] ' 


Any Model 


l/2->n 


2->8 


complete, massive, helicity, color, 
decay chain 


what is MG5 

[15] 


HA (automatic 
generation) 


Output 


PD 

[14] 


Grace 


SM/MSSM 


2->n 


2->6 


complete.massive, helicity, color 


Manual v2.0 
[16] 


HA 


Output 


PD 

[17] 


CompHEP 


Model 


MaxFS 


Tested 
FS 


Short description 


Publication 


method 


Output 


Status 


CalcHEP 


Model 


MaxFS 


Tested 
FS 


Short description 


Publication 


Method 


Output 


Status 


Sherpa 


SM/MSSM 


2->n 


2->8 


massive 


publication 
[18] 


HA/DS 


Output 


PD 

[19] 


GenEva 


Model 


MaxFS 


Tested 
FS 


Short description 


Publication 


Method 


Output 


Status 


HELAC 


Model 


MaxFS 


Tested 
FS 


Short description 


Publication 


Method 


Output 


Status 


Name 


Model 


MaxFS 


Tested 
FS 


Short description 


Publication 


Method 


Output 


Status 



Status: PD: Public Domain, 

Model: SM: Standard Model, MSSM: Minimal Supersymmetric Standard Model 

Method: HA: Helicity Amplitude, DS: Dyson Schwinger 

Output: ME: Matrix Element, CS: Cross-Sections, PEG: Parton level Event Generation, FEG: Full particle level 

Event Generation 

Higher-order Packages 



Name 



Model 



Order tested 



MaxFS 



Tested FS 



Short description 



Publication 



Method Status 



Grace L-l SM/MSSM 
Name Order 



1-loop 
Model 



2->n 
MaxFS 



2->4 complete, massive, helicity, color NA 

Tested FS Short description Publication 



Method NA 
Method Status 



References 

[1] Kaneko, T. (1990). "Automatic calculation of Feynman amplitudes" (http://www.slac.stanford.edu/spires/find/hep?key=5471150). . 

pp. 555. . 
[2] Boos, E.E; et al. (1994). "Automatic calculation in high-energy physics by Grace/Chanel and CompHEP.". International Journal of Modern 

Physics C 5 (4): 615. doi:10.1142/S0129183194000787. 
[3] Wang, J.-X. (1993). "Automatic calculation of Feynman loop-diagrams I. Generation of a simplified form of the amplitude". Computer 

Physics Communications 11 (2): 263. doi: 10.1016/0010-4655(93)90010- A. 
[4] Kaneko, T.; Nakazawa, N. (1995). "Automatic calculation of two loop weak corrections to muon anomalous magnetic moment" (http:// 

www. slac. Stanford. edu/spires/find/hep?key=3147525). . pp. 173. arXiv:hep-ph/9505278. . 
[5] Jimbo, M.; et al. (Minami-Tateya Collaboration) (1995). "Automatic calculation of SUSY particle production" (http://www.slac. Stanford. 

edu/spires/find/hep?key=3360695). . p. 155. arXiv:hep-ph/9605414. . 
[6] Franzkowski, J. (1997). "Automatic calculation of massive two-loop self-energies with XLOOPS". Nuclear Instruments and Methods in 

Physics Research A 389 (1-2): 333. doi:10.1016/S0168-9002(97)00121-6. arXiv:hep-ph/9611378. 
[7] Brucher, L. (2000). "Automatic Feynman diagram calculation with xloops: A Short overview" (http://www.slac.stanford.edu/spires/find/ 

hep?key=4306120). arXiv:hep-ph/0002028 [hep-ph]. . 
[8] Perret-Gallix, D. (1999). "Automatic amplitude calculation and event generation for collider physics: GRACE and CompHEP" (http://www. 

slac. Stanford. edu/spires/find/hep?key=4515617). . pp. 270. . 



Automatic calculation of particle interaction or decay 105 

[9] Belanger, G.; et al. (2006). "Automatic calculations in high energy physics and GRACE at one-loop". Physics Reports 430 (3): 117. 

doi: 10.1016/j.physrep.2006.02.001. arXiv:hep-ph/0308080. 
[10] Fujimoto, J.; et al. (2004). "Automatic one-loop calculation of MSSM processes with GRACE". Nuclear Instruments and Methods in 

Physics Research A 534 (1-2): 246. doi:10.1016/j.nima.2004.07.095. arXiv:hep-ph/0402145. 
[11] Kanaki, A.; Papadopoulos, C.G. (2000). "HELAC: A Package to compute electroweak helicity amplitudes". Computer Physics 

Communications 132: 306. doi:10.1016/S0010-4655(00)00151-X. arXiv:hep-ph/0002082. 
[12] Belanger, G.; et al. (2006). "Automatic calculations in high energy physics and Grace at one-loop". Physics Reports 430: 1 17. 

doi: 10.1016/j.physrep.2006.02.001. arXiv:hep-ph/0308080. 
[13] https://server06.fynu.ucl.ac.be/projects/feynrules 
[14] https://launchpad.net/madgraph5 
[15] https://server06.fynu.ucl.ac.be/projects/madgraph 
[16] http://minami-home.kek.jp/grace/gracedoc.ps 
[17] http://minami-home.kek.jp 

[18] http://www.slac.stanford.edu/spires/find/hep/www ?rawcmd=FIND+EPRINT+HEP-PH%2F03 11263 
[19] http://www.sherpa-mc.de 



S-matrix 



Scattering matrix redirects here. For the meaning in linear electrical networks, see scattering parameters. 

For the 1960's approach to particle physics, see S-matrix theory. 

In physics, the scattering matrix (or S-matrix) relates the initial state and the final state of a physical system 
undergoing a scattering process. It is used in quantum mechanics, scattering theory and quantum field theory. 

More formally, the S-matrix is defined as the unitary matrix connecting asymptotic particle states in the Hilbert 
space of physical states (scattering channels). While the S-matrix may be defined for any background (spacetime) 
that is asymptotically solvable and has no horizons, it has a simple form in the case of the Minkowski space. In this 
special case, the Hilbert space is a space of irreducible unitary representations of the inhomogeneous Lorentz group; 
the S-matrix is the evolution operator between time equal to minus infinity, and time equal to plus infinity. It is 
defined only in the limit of zero energy density (or infinite particle separation distance). It can be shown that if a 
quantum field theory in Minkowski space has a mass gap, the state in the asymptotic past and in the asymptotic 
future are both described by Fock spaces. 

History 

The S-matrix was first introduced by John Archibald Wheeler in the 1937 paper '"On the Mathematical Description 
of Light Nuclei by the Method of Resonating Group Structure'". In this paper Wheeler introduced a scattering 
matrix - a unitary matrix of coefficients connecting "the asymptotic behaviour of an arbitrary particular solution [of 

[21 

the integral equations] with that of solutions of a standard form". 

In the 1940s Werner Heisenberg developed, independently, the idea of the S-matrix. Due to the problematic 
divergences present in quantum field theory at that time Heisenberg was motivated to isolate the essential features of 
the theory that would not be affected by future changes as the theory developed. In doing so he was led to introduce 

[2] 

a unitary "characteristic" S-matrix. 



S -matrix 106 

Motivation 

In high-energy particle physics we are interested in computing the probability for different outcomes in scattering 
experiments. These experiments can be broken down into three stages: 

1. Collide together a collection of incoming particles (usually two particles with high energies). 

2. Allowing the incoming particles to interact. These interactions may change the types of particles present (e.g. if an 
electron and a positron annihilate they may produce two photons). 

3. Measuring the resulting outgoing particles. 

The process by which the incoming particles are transformed (through their interaction) into the outgoing particles is 
called scattering. For particle physics a physical theory of these processes must be able to compute the probability 
for different outgoing particles when we collide different incoming particles with different energies. The S-matrix in 
quantum field theory is used to do exactly this. It is assumed that the small-energy-density approximation is valid in 
these cases. 

Use of S-matrices 

The S-matrix is closely related to the transition probability amplitude in quantum mechanics and to cross sections of 
various interactions; the elements (individual numerical entries) in the S-matrix are known as scattering 
amplitudes. Poles of the S-matrix in the complex-energy plane are identified with bound states, virtual states or 
resonances. Branch cuts of the S-matrix in the complex-energy plane are associated to the opening of a scattering 
channel. 

In the Hamiltonian approach to quantum field theory, the S-matrix may be calculated as a time-ordered exponential 
of the integrated Hamiltonian in the interaction picture; it may be also expressed using Feynman's path integrals. In 
both cases, the perturbative calculation of the S-matrix leads to Feynman diagrams. 

In scattering theory, the S-matrix is an operator mapping free particle in-states to free particle out-states (scattering 
channels) in the Heisenberg picture. This is very useful because we cannot describe exactly the interaction (at least, 
the most interesting ones). 

Mathematical definition 

In Dirac notation, we define IfJ) as the vacuum quantum state. If a) (k)is a creation operator, its hermitian 
conjugate (destruction or annihilation operator) acts on the vacuum as follows: 

a(k) |0) = 0. 
Now, we define two kinds of creation/destruction operators, acting on different Hilbert spaces (IN space i, OUT 
space/), a t(/ c )and a |(fc). 

So now 

Tim = span{|7, k± . . . k n ) = a\(ki) ■ ■ ■ a\(k n ) \I, 0)}, 

Hout = span{|F,pi . ..p n ) = aj-(pi) ■ ■■a\(p n ) \F,0)}. 

It is possible to play the trick assuming that I J 0} and \F 0) are both invariant under translation and that the 

states \I,ki . . . k n ) and \F,pi . . .p n ) are eigenstates of the momentum operator / pi i , by adiabatically turning 

on and off the interaction. 

In the Heisenberg picture the states are time-independent, so we can expand initial states on a basis of final states (or 

vice versa) as follows: 

Where \(J I is the probability that the interaction transforms 1/ k\ . . . k n ) into \F,pi . . .p m ) 

According to Wigner's theorem, gtmist be a unitary operator such that (J, /3\ S \I , a) = S a R = (F, j3\I, a) ■ 

Moreover, g leaves the vacuum state invariant and transforms IN-space fields in OUT-space fields: 



S -matrix 107 

S\0) = \0) 

4>f = s^fas 

If S describes an interaction correctly, these properties must be also true: 

If the system is made up with a single particle in momentum eigenstate \k) , then S \k) = lit) 
The S-matrix element must be nonzero if and only if momentum is conserved. 

S-matrix and evolution operator U 

a(k,t) =U- 1 (t)a i (k)U(t) 
fa = f/- 1 (oo)^C/(oo) = S^fcS. 
Therefore S = e la U(oo) where 
(0|f/(oo)|0)- 



e ia '-i^'-->i"\- J 



because 

510) = |0). 

Substituting the explicit expression for U we obtain: 

Q = H-ifdrViir) 

(0|t/(oo)|0) 
By inspection it can be seen that this formula is not explicitly covariant. 

Dyson series 

The most widely used expression for the S-matrix is the Dyson series. This expresses the S-matrix operator as the 
series: 

where: 

• TV ■ ■] denotes time-ordering, 

• Hi n Ax) denotes the interaction Hamiltonian which describes the interactions in the theory. 

See also 

• Feynman diagram 

• LSZ reduction formula 

• Wick's theorem 

Notes 

[1] John A. Wheeler, ' On the Mathematical Description of Light Nuclei by the Method, of Resonating Group Structure (http://link.aps.org/ 

abstract/PR/v52/pll07)' Phys. Rev. 52, 1107 - 1122 (1937) 
[2] Jagdish Mehra, Helmut Rechenberg, The Historical Development of Quantum Theory (Pages 990 and 1031) Springer, 2001 ISBN 

0387950869, 9780387950860 

References 

• Barut, A.O. (1967). The Theory of the Scattering Matrix. 

• Tony Philips (11 2001). "Finite-dimensional Feynman Diagrams" (http://www.math.sunysb.edu/~tony/ 
whatsnew/column/feynman-1101/feynmanl.html). What's New In Math. American Mathematical Society. 
Retrieved 2007-10-23. 



List of materials analysis methods 108 

List of materials analysis methods 

List of materials analysis methods: 

Contents: Top-0-9-ABCDEFGHIJKLMNOPQRSTUVWXYZ 

• uSR - see Muon spin spectroscopy 

• / - see Magnetic susceptibility 



Analytical ultracentrifugation - Analytical ultracentrifugation 

AAS - Atomic absorption spectroscopy 

AED - Auger electron diffraction 

AES - Auger electron spectroscopy 

AFM - Atomic force microscopy 

AFS - Atomic fluorescence spectroscopy 

APFIM - Atom probe field ion microscopy 

APS - Appearance potential spectroscopy 

ARPES - Angle resolved photoemission spectroscopy 

ARUPS - Angle resolved ultraviolet photoemission spectroscopy 

ATR - Attenuated total reflectance 



B 



BET - BET surface area measurement (BET from Brunauer, Emmett, Teller) 
BiFC - Bimolecular fluorescence complementation 
BKD - Backscatter Kikuchi diffraction, see EBSD 
BRET - Bioluminescence resonance energy transfer 
BSED - Back scattered electron diffraction, see EBSD 



CAICISS - Coaxial impact collision ion scattering spectroscopy 

CARS - Coherent anti-Stokes Raman spectroscopy 

CBED - Convergent beam electron diffraction 

CCM - Charge collection microscopy 

CDI - Coherent diffraction imaging 

CE - Capillary electrophoresis 

CET - Cryo-electron tomography 

CL - Cathodoluminescence 

CLSM - Confocal laser scanning microscopy 

COSY - Correlation spectroscopy 

Cryo-EM - Cryo-electron microscopy 

CV - Cyclic voltammetry 



List of materials analysis methods 109 

D 

DE(T)A - Dielectric thermal analysis 

dHvA - De Haas-van Alphen effect 

DIC - Differential interference contrast microscopy 

Dielectric spectroscopy - Dielectric spectroscopy 

DLS - Dynamic light scattering 

DLTS - Deep-level transient spectroscopy 

DMA - Dynamic mechanical analysis 

DPI - Dual polarisation interferometry 

DRS - Differential reflectance spectroscopy 

DSC - Differential scanning calorimetry 

DTA - Differential thermal analysis 

DVS - Dynamic vapour sorption 



E 



EBIC - Electron beam induced current (and see IBIC: ion beam induced charge) 

EBS - Elastic (non-Rutherford) backscattering spectrometry (see RBS) 

EBSD - Electron backscatter diffraction 

ECOSY - Exclusive correlation spectroscopy 

ECT - Electrical capacitance tomography 

EDAX - Energy-dispersive analysis of x-rays 

EDMR - Electrically Detected Magnetic Resonance, see ESR or EPR 

EDS - Energy Dispersive Spectroscopy 

EDX - Energy dispersive X-ray spectroscopy 

EELS - Electron energy loss spectroscopy 

EFTEM - Energy filtered transmission electron microscopy 

EID - Electron induced desorption 

EIT and ERT - Electrical impedance tomography and Electrical resistivity tomography 

EL - Electroluminescence 

Electron crystallography - Electron crystallography 

ELS - Electrophoretic light scattering 

ENDOR - Electron nuclear double resonance, see ESR or EPR 

EPMA - Electron probe microanalysis 

EPR - Electron paramagnetic resonance spectroscopy 

ERD or ERDA - Elastic recoil detection or Elastic recoil detection analysis 

ESCA - Electron spectroscopy for chemical analysis* see XPS 

ESD - Electron stimulated desorption 

ESEM - Environmental scanning electron microscopy 

ESI-MS or ES-MS - Electrospray ionization mass spectrometry or Electrospray mass spectrometry 

ESR - Electron spin resonance spectroscopy 

ESTM - Electrochemical scanning tunneling microscopy 

EXAFS - Extended X-ray absorption fine structure 

EXSY - Exchange spectroscopy 



List of materials analysis methods 110 



FCS - Fluorescence correlation spectroscopy 

FCCS - Fluorescence cross-correlation spectroscopy 

FEM - Field emission microscopy 

FIB - Focused ion beam microscopy 

FIM-AP - Field ion microscopy— atom probe 

Flow birefringence - Flow birefringence 

Fluorescence anisotropy - Fluorescence anisotropy 

FLIM - Fluorescence lifetime imaging 

Fluorescence microscopy - Fluorescence microscopy 

FRET - Fluorescence resonance energy transfer 

FRS - Forward Recoil Spectrometry, a synonym of ERD 

FTICR or FT-MS - Fourier transform ion cyclotron resonance or Fourier transform mass spectrometry 

FTIR - Fourier transform infrared spectroscopy 



GC-MS - Gas chromatography-mass spectrometry 

GDMS - Glow discharge mass spectrometry 

GDOS - Glow discharge optical spectroscopy 

GISAXS - Grazing incidence small angle X-ray scattering 

GIXD - Grazing incidence X-ray diffraction 

GIXR - Grazing incidence X-ray reflectivity 

GLC - Gas-liquid chromatography 



H 



HAADF - high angle annular dark-field imaging 

HAS - Helium atom scattering 

HPLC - High performance liquid chromatography 

HREELS - High resolution electron energy loss spectroscopy 

HREM - High-resolution electron microscopy 

HRTEM - High-resolution transmission electron microscopy 



IAES - Ion induced Auger electron spectroscopy 

IBA - Ion beam analysis 

IBIC - Ion beam induced charge microscopy 

ICP-AES - Inductively_coupled_plasma_atomic_emission_spectroscopy 

ICP-MS - Inductively coupled plasma mass spectrometry 

Immunofluorescence - Immunofluorescence 

ICR - Ion cyclotron resonance 

IETS - Inelastic electron tunneling spectroscopy 

IGA - Intelligent gravimetric analysis 

IIX - Ion induced X-ray analysis: See Particle induced X-ray emission 

INS - Ion neutralization spectroscopy 

Inelastic neutron scattering 



List of materials analysis methods 111 

• IRS - Infrared spectroscopy 

• ISS - Ion scattering spectroscopy 

• ITC - Isothermal titration calorimetry 

• IVEM - Intermediate voltage electron microscopy 



List of materials analysis methods (deliberate self-link) 

LALLS - Low-angle laser light scattering 

LC-MS - Liquid chromatography-mass spectrometry 

LEED - Low-energy electron diffraction 

LEEM - Low-energy electron microscopy 

LEIS - Low -energy ion scattering 

LIBS - Laser induced breakdown spectroscopy 

LOES - Laser optical emission spectroscopy 

LS - Light (Raman) scattering 



M 



MALDI - Matrix-assisted laser desorption/ionization 

MBE - Molecular beam epitaxy 

MEIS - Medium energy ion scattering 

MFM - Magnetic force microscopy 

MIT - Magnetic induction tomography 

MPM - Multiphoton fluorescence microscopy 

MRFM - Magnetic resonance force microscopy 

MRI - Magnetic resonance imaging 

MS - Mass spectrometry 

MS/MS - Tandem mass spectrometry 

Mossbauer spectroscopy - Mossbauer spectroscopy 

MTA - Microthermal analysis 



N 



NAA - Neutron activation analysis 

Nanovid microscopy - Nanovid microscopy 

ND - Neutron diffraction 

NDP - Neutron depth profiling 

NEXAFS - Near edge X-ray absorption fine structure 

NIS - Nuclear inelastic scattering/absorption 

NMR - Nuclear magnetic resonance spectroscopy 

NOESY - Nuclear Overhauser effect spectroscopy 

NRA - Nuclear reaction analysis 

NSOM - Near-field optical microscopy 



List of materials analysis methods 112 

o 

• OBIC - Optical beam induced current 

• ODNMR - Optically detected magnetic resonance, see ESR or EPR 

• OES - Optical emission spectroscopy 

• Osmometry - Osmometry 



PAS - Positron annihilation spectroscopy 

Photoacoustic spectroscopy - Photoacoustic spectroscopy 

PAT or PACT - Photoacoustic tomography or photoacoustic computed tomography 

PAX - Photoemission of adsorbed xenon 

PC or PCS - Photocurrent spectroscopy 

Phase contrast microscopy - Phase contrast microscopy 

PhD - Photoelectron diffraction 

PD - Photodesorption 

PDEIS - Potentiodynamic electrochemical impedance spectroscopy 

PDS - Photothermal deflection spectroscopy 

PED - Photoelectron diffraction 

PEELS - parallel electron energy loss spectroscopy 

PES - Photoelectron spectroscopy 

PINEM - photon-induced near-field electron microscopy 

PIGE - Particle (or proton) induced gamma-ray spectroscopy, see Nuclear reaction analysis 

PIXE - Particle (or proton) induced X-ray spectroscopy 

PL - Photoluminescence 

Porosimetry - Porosimetry 

Powder diffraction - Powder diffraction 

PTMS - Photothermal microspectroscopy 

PTS - Photothermal spectroscopy 



Q 

• QENS - Quasi-elastic neutron scattering 



R 



Raman - Raman spectroscopy 

RAXRS - Resonant anomalous X-ray scattering 

RBS - Rutherford backscattering spectrometry 

REM - Reflection electron microscopy 

RDS - Reflectance Difference Spectroscopy 

RHEED - Reflection high energy electron diffraction 

RIXS - Resonant inelastic X-ray scattering 

RR spectroscopy - Resonance Raman spectroscopy 



List of materials analysis methods 113 



SAD - Selected area diffraction 

SAED - Selected area electron diffraction 

SAM - Scanning Auger microscopy 

SANS - Small angle neutron scattering 

SAXS - Small angle X-ray scattering 

SCANIIR - Surface composition by analysis of neutral species and ion-impact radiation 

SCEM - Scanning confocal electron microscopy 

SE - Spectroscopic ellipsometry 

SEC - Size exclusion chromatography 

SEIRA - Surface enhanced infrared absorption spectroscopy 

SEM - Scanning electron microscopy 

SERS - Surface enhanced Raman spectroscopy 

SERRS - Surface enhanced resonance Raman spectroscopy 

SEXAFS - Surface extended X-ray absorption fine structure 

SICM - Scanning ion-conductance microscopy 

SIL - Solid immersion lens 

SIM - Solid immersion mirror 

SIMS - Secondary ion mass spectrometry 

SNMS - Sputtered neutral species mass spectroscopy 

SNOM - Scanning near-field optical microscopy 

SPECT - Single photon emission computed tomography 

SPM - Scanning probe microscopy 

SRM-CE/MS - Selected-reaction-monitoring capillary-electrophoresis mass-spectrometry 

SSNMR - Solid-state nuclear magnetic resonance 

Stark spectroscopy - Stark spectroscopy 

STED - Stimulated Emission Depletion microscopy 

STEM - Scanning transmission electron microscopy 

STM - Scanning tunneling microscopy 

STS - Scanning tunneling spectroscopy 

SXRD - Surface X-ray Diffraction (SXRD) 



TAT or TACT - Thermoacoustic tomography or thermoacoustic computed tomography (see also photoacoustic 

tomography - PAT) 

TEM - transmission electron microscope/microscopy 

TGA - Thermogravimetric analysis 

TIKA - Transmitting ion kinetic analysis 

TIRFM - Total internal reflection fluorescence microscopy 

TLS - Photothermal lens spectroscopy, a type of Photothermal spectroscopy 

TMA - Thermomechanical analysis 

TOF-MS - Time-of- flight mass spectrometry 

Two-photon excitation microscopy - Two-photon excitation microscopy 

TXRF - Total reflection X-ray fluorescence analysis 



List of materials analysis methods 1 14 

u 

• Ultrasound attenuation spectroscopy - Ultrasound attenuation spectroscopy 

• Ultrasonic testing - Ultrasonic testing 

• UPS - UV-photoelectron spectroscopy 



VEDIC - Video-enhanced differential interference contrast microscopy 
Voltammetry - Voltammetry 



w 



WAXS - Wide angle X-ray scattering 

WDX or WDS - Wavelength dispersive X-ray spectroscopy 



X 

XAES - X-ray induced Auger electron spectroscopy 

XANES - XANES, synonymous with NEXAFS (Near edge X-ray absorption fine structure) 

XAS - X-ray absorption spectroscopy 

X-CTR - X-ray crystal truncation rod scattering 

X-ray crystallography - X-ray crystallography 

XDS - X-ray diffuse scattering 

XPEEM - X-ray photoelectron emission microscopy 

XPS - X-ray photoelectron spectroscopy 

XRD - X-ray diffraction 

XRES - X-ray resonant exchange scattering 

XRF - X-ray fluorescence analysis 

XRR - X-ray reflectivity 

XRS - X-ray Raman scattering 

XSW - X-ray standing wave technique 

References 

• Callister, WD (2000). Materials Science and Engineering - An Introduction. John Wiley and Sons : London. 
ISBN 0-471-32013-7. 

• Yao, N, ed (2007). Focused Ion Beam Systems: Basics and Applications. Cambridge University Press : 
Cambridge, UK. ISBN 978-052183-1994. 



List of neutrino experiments 



115 



List of neutrino experiments 



This is a list of neutrino experiments, neutrino detectors, and neutrino telescopes. 



Abbreviation 


Full name 


Sensitivity 


Type 


Induced 
reaction 


Type of 
reaction 


Detector 


Type of 
detector 


Threshold 
energy 


Location 


Operation 


Home 
page 


ANTARES 


Astronomy with 
a Neutrino 
Telescope and 
Abyss 

Environmental 
RESearch 


ATM, CR, 
AGN, PUL 


v , v , v 

e [i t 












Mediterranean 
Sea, France 


2006- 


[1] 


BOREXINO 


BORon 
Experiment 


LS 


V 


v + e~ 
— > v + 
e~ 


ES 


LOS 

shielded by 
water 


Scintillation 


250-665 
keV 


Gran Sasso, 
Italy 


May 2007- 


[2] 
[3] 


CLEAN 


Cryogenic 
Low-Energy 
Astrophysics with 
Neon 


LS, SN, 
WIMP 


V 


v + e~ 
— > v + 
e~ 
v + 

V + 

2 Ve 


ES 
ES 


Liquid Ne 
(lOt) 


Scintillation 






f untie 


[4] 


Day a Bay 


Day a Bay 
Reactor Neutrino 
Experiment 


R 


V 


v + p 
-^ e + + 
n 


CC 


Gd-doped 
LOS 


Scintillation 


1.8 MeV 


Daya Bay, 
China 


2009- 


[5] 


Double CHOOZ 


Double Chooz 
Reactor Neutrino 
Experiment 


R 


V 


v + p 
-^ e + + 
n 


CC 


Gd-doped 
LOS 


Scintillation 


1.8 MeV 


Chooz, 
France 


2008- 


[6] 


EXO-200 


Enriched Xenon 








BB 


LXe 






WIPP, New 


2009- 


[7] 




Observatory 
















Mexico 




GALLEX 


GALLium 
Experiment 


LS 


V 


v + 

7l Ga^ 
7l Ge + 
e~ 


CC 


GaCl (30 t) 


Radiochemical 


233.2 
keV 


Gran Sasso, 
Italy 


1991-1997 


[8] 


GNO 


Gallium Neutrino 
Observatory 


LS 


V 


v + 
Ga-> 
Ge + 

e 


CC 


GaCl (30 t) 


Radiochemical 


233.2 
keV 


Gran Sasso, 
Italy 


May 1998-Ian 2002 


[9] 


HERON 


Helium Roton 
Observation of 

Neutrinos 


LS 


V 

(mainly) 


v + e~ 

-^ v + 
e~ 


NC 


Superfluid 
He 


Rotational 
excitation 


1 MeV 




future 


[10] 


HOMESTAKE-CHLORINE 


Homestake 
chlorine 
experiment 


S 


V 


37 C1 + 
V -^ 
37 Ar* + 
e~ 

37 Ar* 
^ 37 C1 
+ e + + v 


CC 


C CI (615 t) 

2 4 V ' 


Radiochemical 


814 keV 


Homestake 
Mine, South 
Dakota 


1967-1998 


[11] 



List of neutrino experiments 



116 



HOMESTAKE-IODINE 


Homestake 
iodine experiment 


S 


V 


v + e 

— > V + 

e 

127 T 
V + I 

— > 
127,, 

Xe + 
e 


ES 

cc 


Nal in water 


Radiochemical 


789 keV 


Homestake 
Mine, South 
Dakota 


future 


[11] 


ICARUS 


Imaging Cosmic 
And Rare 
Underground 
Signal 


S, ATM, 
GSN 


V , V , V 
e [i t 


v + e~ 
— > v + 
e~ 


ES 


Liquid Ar 


Cherenkov 


5.9 MeV 


Gran Sasso, 
Italy 




[12] 


IceCube 


IceCube Neutrino 
Detector 


S, ATM, 
CR, ? 


V , V , V 
e [i t 


v + e~ 
-^ v + 
e~ 


ES 


Water ice 
(1 km 3 ) 


Cherenkov 


-10 MeV 


South Pole, 
Antarctica 


2006- 


[13] 


Kamiokande 


Kamioka 
Nucleon Decay 
Experiment 


S,ATM 


V 


v + e~ 
-^ v + 
e~ 


ES 


Water (H^O) 


Cherenkov 


7.5 MeV 


Kamioka, 
Japan 


1986-1995 


[14] 


KamLAND 


Kamioka Liquid 
Scintillator 
Antineutrino 
Detector 
















Kamioka, 
Japan 


2002- 


[15] 


KM3NeT 


KM3 Neutrino 
Telescope 
















Mediterranean 
Sea,? 


2009- 


[16] 


LENS 


Low Energy 
Neutrino 
Spectroscopy 


LS 


V 


v + 

115 T 

In -^ 

115 

Sn + 

v +2y 


CC 


In-doped 
LOS 


Scintillation 


120 keV 






[17] 
[18] 


Majorana 


Neutrinoless 
Double Beta 
Decay in Ge to 
measure lepton 
number violation 
and neutrino mass 
scale 


BB 


V 


76 Ga^ 
76 As + 
e~+ e~ 


BB 


HPGe 


Semiconductor 


2039 keV 


Homestake 
Mine, South 
Dakota 


construction start 
2010 


[19] 


MOON 


Molybdenum 
Observatory Of 

Neutrinos 


LS, LSN 


V 


v + 

-> 100 Tc 
+ e~ 


CC 


UK).. ,, , .. 

Mo (1 kt) 
+ MoF6 (gas) 


Scintillation 


168 keV 


Washington, 
USA 




[20] 


MiniBooNE 


Mini Booster 
Neutrino 
Experiment 


AC 


V , V 
e (i 


12~ 

v + C 
— > e~ + 
X 


CC 


Mineral oil 
(1 kton) 


Cherenkov 


-100 keV 


Illinois, USA 


2002- 


[21] 


NEMO Experiment 


Neutrino Ettore 
Majorana 
Observatory 
















Frejus Road 

Tunnel, 

Italy/France 


2003- 


[22] 


NEMO Telescope 


NEutrino 
Mediterranean 
Observatory 
















Mediterranean 
Sea, Italy 


2007- 


[23] 



List of neutrino experiments 



117 



NEVOD 


Cherenkov water 
detector NEVOD 


ATM, CR 


V 


v + n 

n 

— > [i~ + 

P 

v + p 
n 

— > \i~ + 

n 


CC 


Water (H.,0) 


Cherenkov 


~2GeV 


Moscow, 

Russia 


1993- 


[24] 


NOvA 


NuMI Off-Axis v 
Appearance 
















Illinois and 
Minnesota, 
United States 


2011- 


[25] 


OPERA 


Oscillation 
Project with 
Emulsion-tRacking 
Apparatus 




V 












LNGS (Italy) 
and CERN 


2008- 


[26] 


SAGE 


Soviet— American 

Gallium 

Experiment 


LS 


V 


v + 

71< Ga^ 
71 Ge + 
e~ 


CC 


GaCl 

3 


Radiochemical 


233.2 
keV 


Baksan 
Valley, 

Russia 


1990-2006 


[27] 


SciBooNE 


SciBar 

(Scintillator Bar) 
Booster Neutrino 
Experiment 


AC 


V 


v + I2 C 
n 

->u~ + 

X 


CC,NC 


Plastic 
(CH,10 ton) 


Scintillation 


-100 keV 


Illinois, USA 


2007-2008 


[28] 


SNO 


Sudbury Neutrino 


S, ATM, 


V , V , V 
e [i t 


v + 2 D 


CC 


Heavy water 


Cherenkov 


3.5 MeV 


Creighton 


1999-2006 


T29] 




Observatory 


GSN 




-> 2p + 

e~ 

v + 2 D 

-^ v + 
n + p 
v + e~ 
-^ v + 
e~ 


NC 
ES 


(lktD 2 0) 






Mine, Ontario 






SNO+ 


SNO with liquid 
















Creighton 


2009- 


[29] 




scintillator 
















Mine, Ontario 




SK 


Super- Kamiokande 


S, ATM, 
GSN 


V , V , V 
e [i t 


v + e 
-^ v + 
e 
v + n 

-^ e~ + 

P 

v + p 
^ e + + 
n 


ES 
CC 
CC 


Water (H.O) 


Cherenkov 




Kamioka, 
Japan 


1996- 


[30] 
[31] 


UNO 


Underground 

Nucleon decay 
and neutrino 
Observatory 


S, ATM, 
GSN, RSN 


V , V , V 
e [i t 


v + e~ 
^ v + 
e~ 


ES 


Water 

(440 kt H 2 0) 


Cherenkov 




Henderson 
Mine, 

Colorado 


f ut ure 


[32] 



[a] Solar neutrino (S), Low-energy solar neutrino (LS), Reactor neutrino (R), Terrestrial neutrino (T), Atmospheric 
neutrino (ATM), Accelerator neutrino (AC), Cosmic ray neutrino (CR), Supernova neutrino (SN), Low-energy 
supernova neutrino (LSN), Active galactic nuclei neutrino (AGN), Pulsar neutrino (PUL) 

[b] Elastic scattering (ES), Neutral current (NC), Charged current (CC), Double beta decay (BB) 



List of neutrino experiments 



118 



References 

[1] http://antares.in2p3.fr/index.html 

[2] http://www.ge.infn.it/borexino/ 

[3] http://borex.lngs.infn.it/ 

[4] http://mckinseygroup.physics.yale.edu/publications/CLEAN.pdf 

[5] http://dayawane.ihep.ac.cn/ 

[6] http://doublechooz.in2p3.fr/ 

[7] http://www-project. slac. stanford.edu/exo/ 

[8] http://www.mpi-hd.mpg.de/nuastro/gallex.html 

[9] http://www.lngs.infn.it/lngs_infn/contents/lngs_en/research/experiments_scientific_info/experiments/past/gno/ 

//www. physics. brown.edu/physics/researchpages/cme/heron/LTD_home. html 

//www-spires, dur.ac.uk/cgi-bin/spiface/find/experiments/ www2?rawcmd=fin+expt+homestake 

//www. aquila.infn.it/icarus/ 

//icecube, wisc.edu/ 

//www-sk.icrr.u-tokyo.ac.jp/doc/kam/index.html 

//www. awa. tohoku. ac.jp/KamLAND/ 

//www. km3net.org/ 

//www. phys.vt.edu/~lens/ 

//lens. in2p3.fr/ 

//maj orana. npl. Washington, edu/ 

//ewi. npl. Washington, edu/moon/ 

//www-boone. fnal.gov 

//nemo. in2p3.fr/nemow3/ 

//nemoweb. Ins. infn.it 

//www. nevod.mephi.ru/English/index.htm 

//www-nova.fnal.gov 

//operaweb. lngs.infn.it/ 

//ewi. npl. washington.edu/SAGE/sage. html 

//www-sciboone. fnal.gov 

//www.sno.phy.queensu.ca/ 

//neutrino. phys.washington.edu/~superk/ 

//www-sk. icrr.u-tokyo.ac.jp/sk/index_e. html 

//ale. physics, sunysb.edu/uno/ 



[10] 


http 


[II] 


http 


[12] 


http 


[13] 


http 


[14] 


http 


[15] 


http 


[16] 


http 


[17] 


http 


[18] 


http 


[19] 


http 


[20] 


http 


[21] 


http 


[22] 


http 


[23] 


http 


[24] 


http 


[25] 


http 


[26] 


http 


[27] 


http 


[28] 


http 


[29] 


http 


[30] 


http 


[31] 


http 


[32] 


http 



119 



Instruments 



Optical microscope 




Notable experiments Discovery of cells 



Inventor 



Related items 



Hans Lippershey 
Zacharias Janssen 

Microscope 
Electron microscope 



The optical microscope, often referred to as 
the "light microscope", is a type of 
microscope which uses visible light and a 
system of lenses to magnify images of small 
samples. Optical microscopes are the oldest 
design of microscope and were designed 
around 1600. Basic optical microscopes can 
be very simple, although there are many 
complex designs which aim to improve 
resolution and sample contrast. Historically 
optical microscopes were easy to develop 
and are popular because they use visible 
light so the sample can be directly observed 
by eye. 




A modem microscope with a mercury bulb for fluorescence microscopy. The 
microscope has a digital camera, and is attached to a computer. 



Optical microscope 120 

The image from an optical microscope can be captured by normal light-sensitive cameras to generate a micrograph. 
Originally images were captured by photographic film but modern developments in CMOS and later charge-coupled 
device (CCD) cameras allow the capture of digital images. Purely Digital microscopes are now available which just 
use a CCD camera to examine a sample, and the image is shown directly on a computer screen without the need for 
eye-pieces. 

Alternatives to optical microscopy which do not use visible light include scanning electron microscopy and 
transmission electron microscopy. 

Optical configurations 

There are two basic configurations of the conventional optical microscope, the simple (one lens) and compound 
(many lenses). The vast majority of modern research microscopes are compound microscopes while some cheaper 
commercial digital microscopes are simple single lens microscopes. A magnifying glass is, in essence, a basic single 
lens microscope. In general microscope optics are static; to focus at different focal depths the lens to sample distance 
is adjusted and to get a wider or narrower field of view a different magnification objective lens must be used. Most 
modern research microscopes also have a separate set of optics for illuminating the sample. 

Single lens (simple) microscope 

A simple microscope is a microscope that uses only one lens for magnification, and is the original design of light 
microscope. Van Leeuwenhoek's microscopes consisted of a small, single converging lens mounted on a brass plate, 
with a screw mechanism to hold the sample or specimen to be examined. Demonstrations by British microscopist 
have images from such basic instruments. Though now considered primitive, the use of a single, convex lens for 
viewing is still found in simple magnification devices, such as the magnifying glass, and the loupe. 

Compound microscope 

A compound microscope is a microscope which uses multiple lenses to collect light from the sample and then a 
separate set of lenses to focus the light into the eye or camera. Compound microscopes are heavier, larger and more 
expensive than simple microscopes due to the increased number of lenses used in construction. The main advantages 
of multiple lenses are improved numerical aperture (see resolution limit below), reduced chromatic aberration and 
exchangeable objective lenses to adjust the magnification. A compound microscope also makes more advanced 
illumination setups, such as phase contrast. 



Optical microscope 

History 



121 



Deftrivjtitt 




i i r „„,„./, 

i *-Jf<eehemoJ<'t*ilfiUi 
U On. ' 
■ I'.r-.irU.lf, 



7 liyiZSrunrltstirpvTn' to tculto.o 
S Tf/u fan la Imyua ryiif . j i Gamku d, 
0/4L partr ' 



The oldest published image known to have been 

made with a microscope: bees by Francesco 

Stelluti, 1630 [2] 



Invention 

It is difficult to say who invented the compound microscope. Dutch 
spectacle-makers Hans Janssen and his son Zacharias Janssen are often 
said to have invented the first compound microscope in 1590, but this 
was a declaration made by Zacharias Janssen himself during the mid 
17th century. The date is unlikely, as it has been shown that Zacharias 
Janssen actually was born around 1590. Another favorite for the title of 
'inventor of the microscope' was Galileo Galilei. He developed an 
occhiolino or compound microscope with a convex and a concave lens 
in 1609. Galileo's microscope was celebrated in the Accademia dei 
Lincei in 1624 and was the first such device to be given the name 
"microscope" a year later by fellow Lincean Giovanni Faber. Faber 
coined the name from the Greek words /.ukqov (micron) meaning 
"small", and okokelv (skopein) meaning "to look at", a name meant to 
be analogous with "telescope", another word coined by the Linceans. 

Christiaan Huygens, another Dutchman, developed a simple 2-lens 
ocular system in the late 17th century that was achromatically 
corrected, and therefore a huge step forward in microscope 
development. The Huygens ocular is still being produced to this day, 
but suffers from a small field size, and other minor problems. 



Popularisation 

Anton van Leeuwenhoek (1632—1723) is credited with bringing the microscope to the attention of biologists, even 
though simple magnifying lenses were already being produced in the 16th century. Van Leeuwenhoek's home-made 
microscopes were very small simple instruments, with a single, yet strong lens. They were awkward in use, but 
enabled van Leeuwenhoek to see detailed images. It took about 150 years of optical development before the 
compound microscope was able to provide the same quality image as van Leeuwenhoek's simple microscopes, due to 
difficulties in configuring multiple lenses. Still, despite widespread claims, van Leeuwenhoek is not the inventor of 
the microscope. 

Lighting techniques 

While basic microscope technology and optics have been available for over 400 years it is much more recently that 
techniques in sample illumination were developed to generate the high quality images seen today. 

In August 1893 August Kohler developed Kohler illumination. This method of sample illumination gives rise to 
extremely even lighting and overcomes many limitations of older techniques of sample illumination. Before 
development of Kohler illumination the image of the light source, for example a lightbulb filament, was always 
visible in the image of the sample. 

The Nobel Prize in physics was awarded to Fritz Zernike in 1953 for his development of phase contrast illumination 
which allows imaging of transparent samples. By using interference rather than absorption of light, extremely 
transparent samples, such as live mammalian cells, can be imaged without having to use staining techniques. Just 
two years later, in 1955, George Nomarski published the theory for differential interference contrast microscopy, 
another interference-based technique for imaging transparent samples. 



Optical microscope 



122 



Fluorescence microscopy 

Modern biological microscopy depends heavily on the development of fluorescent probes for specific structures 
within a cell. In contrast to normal transilluminated light microscopy in fluorescence microscopy the sample is 
illuminated through the objective lens with a narrow set of wavelengths of light. This light interacts with 
fluorophores in the sample which then emit light of a longer wavelength. It is this emitted light which makes up the 
image. 

Since the mid 20th century chemical fluorescent stains, such as DAPI which binds to DNA, have been used to label 
specific structures within the cell. More recent developments include immunofluorescence, which uses fluorescently 
labelled antibodies to recognise specific proteins within a sample, and fluorescent proteins like GFP which a live cell 
can express making it fluorescent. 



Components 

All modern optical microscopes designed for viewing 
samples by transmitted light share the same basic 
components of the light path, listed here in the order the 
light travels through them: 

• Light source, a light or a mirror (7) 

• Diaphragm and condenser lens (8) 

• Objective (3) 

• Ocular lens (eyepiece) (1) 

In addition the vast majority of microscopes have the 
same 'structural' components: 

• Objective turret (to hold multiple objective lenses) (2) 

• Stage (to hold the sample) (9) 

• Focus wheel to move the stage (4 - coarse adjustment, 
5 - fine adjustment) 

These entries are numbered according to the image on 
the right. 



Ocular (eyepiece) 

The ocular, or eyepiece, is a cylinder containing two or 

more lenses; its function is to bring the image into focus 

for the eye. The eyepiece is inserted into the top end of 

the body tube. Eyepieces are interchangeable and many 

different eyepieces can be inserted with different degrees 

of magnification. Typical magnification values for eyepieces include 2x, 5x and lOx. In some high performance 

microscopes, the optical configuration of the 




Basic optical transmission microscope elements(1990's) 



Optical microscope 



123 




Two Leica oil immersion microscope objective lenses; left lOOx, right 40x. 



objective lens and eyepiece are matched to 
give the best possible optical performance. 
This occurs most commonly with 
apochromatic objectives. 

Objective 

The objective is a cylinder containing one or 

more lenses that are typically made of glass; 

its function is to collect light from the 

sample. At the lower end of the microscope 

tube one or more objective lenses are 

screwed into a circular nose piece which 

may be rotated to select the required 

objective lens. Typical magnification values 

of objective lenses are 4x, 5x, lOx, 20x, 40x, 50x, 60x and lOOx. Some high performance objective lenses may 

require matched eyepieces to deliver the best optical performance. 

Stage 

The stage is a platform below the objective which supports the specimen being viewed. In the center of the stage is a 
hole through which light passes to illuminate the specimen. The stage usually has arms to hold slides (rectangular 
glass plates with typical dimensions of 25 mm by 75 mm, on which the specimen is mounted). 

Light source 

Many sources of light can be used. At its simplest, daylight is directed via a mirror. Most microscopes, however, 
have their own controllable light source - normally a halogen lamp. 

Condenser 

The condenser is a lens designed to focus light from the illumination source onto the sample. The condenser may 
also include other features, such as a diaphragm and/or filters, to manage the quality and intensity of the 
illumination. For illumination techniques like dark field, phase contrast and differential interference contrast 
microscopy additional optical components must be precisely aligned in the light path. 



Frame 

The whole of the optical assembly is traditionally attached to a rigid arm which in turn is attached to a robust U 
shaped foot to provide the necessary rigidity. The arm angle may be adjustable to allow the viewing angle to be 
adjusted. 

The frame provides a mounting point for various microscope controls. Normally this will include controls for 
focusing, typically a large knurled wheel to adjust coarse focus, together with a smaller knurled wheel to control fine 
focus. Other features may be lamp controls and/or controls for adjusting the condenser. 



Optical microscope 124 

Objective lenses 

On a typical compound optical microscope there are a selection of lenses available for different applications. Many 
different objective lenses with different properties and magnification are available. 

Typically there will be around three objective lenses: a low power lens for scanning the sample, a medium power 
lens for normal observation and a high power lens for detailed observation. The typical magnification of objective 
lenses depends on the intended application, normal groups of lens magnificaitons may be [4x, lOx, 20x] for low 
magnification work and [lOx, 40x, lOOx] for high magnification work. 

Objective lenses with higher magnifications normally have a higher numerical aperture and a shorter depth of field in 
the resulting image. 

Oil immersion objective 

Some microscopes make use of oil immersion lens. These objectives must be used with oil (immersion oil) between 
the objective lens and the sample. The refractive index of the immersion oil is higher than air and this allows the 
objective lens to have a larger numerical aperture. The larger numerical aperture allows collection of more light 
making detailed observation of faint details possible. 

Immersion lenses are designed so that the refractive index of the oil and of the cover slip are closely matched so that 
the light is transmitted from the specimen to the outer face of the objective lens with minimal refraction. An oil 
immersion lens usually has a magnification of 40 to lOOx. 

Magnification 

The actual power or magnification of a compound optical microscope is the product of the powers of the ocular 
(eyepiece) and the objective lens. The maximum normal magnifications of the occular and objective are lOx and 
lOOx respectively giving a final magnification of lOOOx. 

Magnification and micrographs 

When using a camera to capture a micrograph the effective magnification of the image must take into account the 
size of the image. This is independent of whether it is on a print from a film negative or displayed digitally on a 
computer screen. 

In the case of photographic film cameras the calculation is simple; the final magnification is the product of: the 
objective lens magnification, the camera optics magnification and the enlargement factor of the film print relative to 
the negative. A typical value of the enlargement factor is around 5x (for the case of 35mm film and a 6x4 inch print). 

In the case of digital cameras the size of the pixels in the CMOS or CCD detector and the size of the pixels on the 
screen have to be known. The enlargement factor from the detector to the pixels on screen can then be calculated. As 
with a film camera the final magnification is the product of: the objective lens magnification, the camera optics 
magnification and the enlargement factor. 



Optical microscope 



125 



Operation 



tubus 
length 



tubus intermediate [ 
_ length image 
1 1 plane 



loupe (eye lens) 
marginal 

/ ray 




Optical path in a typical microscope 



The optical components of a modern 
microscope are very complex and for a 
microscope to work well, the whole 
optical path has to be very accurately 
set up and controlled. Despite this, the 
basic operating principles of a 
microscope are quite simple. 

The objective lens is, at its simplest, a 

very high powered magnifying glass 

i.e. a lens with a very short focal 

length. This is brought very close to 

the specimen being examined so that the light from the specimen comes to a focus about 160 mm inside the 

microscope tube. This creates an enlarged image of the subject. This image is inverted and can be seen by removing 

the eyepiece and placing a piece of tracing paper over the end of the tube. By carefully focusing a brightly lit 

specimen, a highly enlarged image can be seen. It is this real image that is viewed by the eyepiece lens that provides 

further enlargement. 

In most microscopes, the eyepiece is a compound lens, with one component lens near the front and one near the back 
of the eyepiece tube. This forms an air-separated couplet. In many designs, the virtual image comes to a focus 
between the two lenses of the eyepiece, the first lens bringing the real image to a focus and the second lens enabling 
the eye to focus on the virtual image. 

In all microscopes the image is intended to be viewed with the eyes focused at infinity (mind that the position of the 
eye in the above figure is determined by the eye's focus). Headaches and tired eyes after using a microscope are 
usually signs that the eye is being forced to focus at a close distance rather than at infinity. 

The essential principle of the microscope is that an objective lens with very short focal length (often a few mm) is 
used to form a highly magnified real image of the object. Here, the quantity of interest is linear magnification, and 
this number is generally inscribed on the objective lens casing. In practice, today, this magnification is carried out by 
means of two lenses: the objective lens which creates an image at infinity, and a second weak tube lens which then 



forms a real image in its focal plane 



[4] 



Optical microscope 



126 



Illumination techniques 

Many techniques are available which modify the light path to generate an improved contrast image from a sample. 
Major techniques for generating increased contrast from the sample include cross-polarized light, dark field, phase 
contrast and differential interference contrast illumination. A recent technique (Sarfus) combines cross-polarized 
light and specific contrast-enhanced slides for the visualization of nanometric samples. 

Four examples of transilumination techniques used to generate contrast in a sample of [[tissue 

paper]]. 1.559 ^im/pixel. 



V /'\ / 







-- ' 



Bright field illumination, sample 

contrast comes from absorbance 

of light in the sample. 






Cross-polarized light 

illumination, sample contrast 

comes from rotation of polarized 

light through the sample. 




Dark field illumination, sample 
contrast comes from light 
scattered by the sample. 



Phase contrast illumination, 
sample contrast comes from 
interference of different path 
lengths of light through the 
sample. 



Other techniques 

Modern microscopes allow more than just observation of transmitted light image of a sample; there are many 
techniques which can be used to extract other kinds of data. Most of these require additional equipment in addition to 
a basic compound microscope. 

• Reflected light, or incident, illumnation (for analysis of surface structures) 

• Fluorescence microscopy, both: 

• Epifluorescence microscopy 

• Confocal microscopy 

• Sample spectroscopy 

• Automation (for automatic scanning of a large sample or image capture) 



Optical microscope 



127 



Applications 



Optical microscopy is used extensively in microelectronics, nanophysics, biotechnology, pharmaceutic research and 
microbiology. 

Optical microscopy is used for medical diagnosis, the field being termed histopathology when dealing with tissues, 
or in smear tests on free cells or tissue fragments. 



Optical microscope variants 



There are many variants of the basic 
compound optical microscope design for 
specialized purposes. Some of these are 
physical design differences allowing 
specialization for certain purposes: 

• Stereo microscope, a low powered 
microscope which provides a 
stereoscopic view of the sample, 
commonly used for dissection. 

• Comparison microscope, which has two 
separate light paths allowing direct 
comparison of two samples via one 
image in each eye. 

• Inverted microscope, for studying 
samples from below; useful for cell 
cultures in liquid. 

• Student microscope, designed for low cost, durability, and ease of use. 
Other microscope variants are designed for different illumination techniques: 

• Petrographic microscope, whose design usually includes a polarizing filter, rotating stage and gypsum plate to 
facilitate the study of minerals or other crystalline materials whose optical properties can vary with orientation. 

• Polarizing microscope, similar to the petrographic microscope. 

• Phase contrast microscope, which applies the phase contrast illumination method. 

• Epifluorescence microscope, designed for analysis of samples which include fluorophores. 

• Confocal microscope, a widely used variant of epifluorescent illumination which uses a scanning laser to 
illuminate a sample for fluorescence. 




Digital microscope 

A digital microscope is a microscope equipped with a digital camera allowing observation of a sample via a 
computer. Microscopes can also be partly or wholly computer-controlled with various levels of automation. Digital 
microscopy allows greater analysis of a microscope image, for example measurements of distances and areas and 
quantitaton of a fluorescent or histological stain. Low-powered digital microscopes, USB microscopes, are also 
commercially available. These are essentially webcams with a high-powered macro lens and generally do not use 
transillumination. The camera attached directly to the USB port of a computer, so that the images are shown directly 
on the monitor. They offer modest magnifications (up to about 200x) without the need to use eyepieces, and at very 
low cost. The lack of illumination optics limits their use in a similar manner to stereo microscopes. 

) 



Optical microscope 128 

Limitations 

At very high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by diffraction 
rings. These are called Airy disks. The resolving power of a microscope is taken as the ability to distinguish between 
two closely spaced Airy disks (or, in other words the ability of the microscope to reveal adjacent structural detail as 
distinct and separate). It is these impacts of diffraction that limit the ability to resolve fine details. The extent and 
magnitude of the diffraction patterns are affected by both the wavelength of light ( \ ), the refractive materials used 
to manufacture the objective lens and the numerical aperture (NA) of the objective lens. There is therefore a finite 
limit beyond which it is impossible to resolve separate points in the objective field, known as the diffraction limit. 
Assuming that optical aberrations in the whole optical set-up are negligible, the resolution d, can be stated as: 

a x 



2NA 

Usually a wavelength of 550 nm is assumed, which corresponds to green light. With air as the external medium, the 
highest practical NA is 0.95, and with oil, up to 1.5. In practice the lowest value of d obtainable is about 200 nm. 

Surpassing the resolution limit 

Multiple techniques are available for reaching resolutions higher than the transmitted light limit described above. 
Techniques for surpassing the resolution limit for bright field microscopy include ultraviolet microscopes, which use 
shorter wavelengths of light so the diffraction limit is lower. Holographic techniques, as described by Courjon and 
Bulabois in 1979, are also capable of breaking this resolution limit, although resolution was restricted in their 
experimental analysis. 

Using fluorescent samples more techniques are available. Examples include Vertico SMI, near field scanning optical 
microscopy which uses evanescent waves, and stimulated emission depletion. In 2005, a microscope capable of 
detecting a single molecule was described as a teaching tool. 

While most techniques focus on increases in lateral resolution there are also some techniques which aim to allow 
analysis of extremely thin samples. For example sarfus methods place the thin sample on a contrast-enhancing 
surface and thereby allows to directly visualize films as thin as 0.3 nanometers. 

STED 

Stimulated emission depletion is a simple example of how higher resolution surpassing the diffraction limit is 
possible, but it has major limitations. STED is a fluorescence microscopy technique which uses a combination of 
light pulses to induce fluorescence in a small sub-population of fluorescent molecules in a sample. Each molecule 
produces a diffraction-limited spot of light in the image, and the centre of each of these spots corresponds to the 
location of the molecule. As the number of fluorescing molecules is low the spots of light are unlikely to overlap and 
therefore can be placed accurately. This process is then repeated many times to generate the image. Stefan Hell of 
the Max Planck Institute for Biophysical Chemistry was awarded the 10th German Future Prize in 2006 for his 

ro] 

development of the STED microscope. 



Optical microscope 129 

Alternatives 

In order to overcome the limitations set by the diffraction limit of visible light other microscopes have been designed 
which use other waves. 

• Atomic Force Microscope (AFM) 

• Scanning Electron Microscope (SEM) 

• Scanning Ion-Conductance Microscope (SICM) 

• Scanning Tunneling Microscope (STM) 

• Transmission Electron Microscope (TEM) 

• X-ray microscope 

The use of electrons and x-rays in place of light allows much higher resolution - the wavelength of the radiation is 
shorter so the diffraction limit is lower. To make the short-wavelength probe non-destructive, the atomic beam 
imaging system (atomic nanoscope) has been proposed and widely discussed in the literature, but it is not yet 
competitive with conventional imaging systems. 

STM and AFM are scanning probe techniques using a small probe which is scanned over the sample surface. 
Resolution in these cases is limited by the size of the probe; micromachining techniques can produce probes with tip 
radii of 5-10 nm. 

Additionally, methods such as electron or X-ray microscopy use a vacuum or partial vacuum, which limits their use 
for live and biological samples (with the exception of ESEM). The specimen chambers needed for all such 
instruments also limits sample size, and sample manipulation is more difficult. Color cannot be seen in images made 
by these methods, so some information is lost. They are however, essential when investigating molecular or atomic 
effects, such as age hardening in aluminium alloys, or the microstructure of polymers. 

See also 

• Digital microscope 

• Kohler illumination 

• Microscope slide 

• Objective 

References 

[1] http://www.brianjford.com/wavrbcs.htm 

[2] "The Lying stones of Marrakech" , by Stephen Jay Gould, 2000 

[3] Fi.it (http://brunelleschi.imss.fi.it/esplora/microscopio/dswmedia/risorse/testi_completi.pdf), "II microscopio di Galileo" 

[4] Stephen G. Lipson, Ariel Lipson, Henry Lipson, Optical Physics 4th Edition, Cambridge University Press, ISBN 978052 149345 1 

[5] Ol Optical Microscopy (http://www.fy.chalmers.se/microscopy/students/imagecourse/01.pdf) By Katarina Logg. Chalmers Dept. 

Applied Physics. 2006-01-20 
[6] D. Courjon and J. Bulabois (1979). "Real Time Holographic Microscopy Using a Peculiar Holographic Illuminating System and a Rotary 

Shearing Interferometer". +Journal of Optic 10 (3). 
[7] "Demonstration of a Low-Cost, Single-Molecule Capable, Multimode Optical Microscope" (http://chemeducator.org/bibs/0010004/ 

1040269mk.htm). . Retrieved February 25, 2009. 
[8] "German Future Prize for crossing Abbe's Limit" (http://www.heise.de/english/newsticker/news/81528). . Retrieved Feb 24, 2009. 



Optical microscope 



130 



Further reading 

• "Metallographic and Materialographic Specimen Preparation, Light Microscopy, Image Analysis and Hardness 
Testing", Kay Geels in collaboration with Struers A/S, ASTM International 2006. 

External links 

• Antique Microscopes.com (http://www.antique-microscopes.com) A collection of early microscopes 

• Historical microscopes (http://www.musoptin.com/mikrol.html), an illustrated collection with more than 
3000 photos of scientific microscopes by European makers (German) 

• The Golub Collection (http://golubcollection.berkeley.edu), A collection of 17th through 19th Century 
microscopes, including extensive descriptions 

• Molecular Expressions (http://micro.magnet.fsu.edu/primer/anatomy/anatomy.html), concepts in optical 
microscopy 

• Online tutorial of practical optical microscopy (http://www.doitpoms.ac.uk/tlplib/optical-microscopy/index. 
php) 

• Open WetW are (http://openwetware.org/wiki/Microscopy) 

• Cell Centered Database (http://ccdb.ucsd. edu/sand/main?stype=lite&keyword=light microscopy& 
Submit=Go&event=display&start=l) 

• Antonie van Leeuwenhoek: Father of Microscopy and Microbiology (http://www.juliantrubin.com/bigten/ 
leeuwenhoek_microscope. html) 



Confocal microscope 



Confocal microscopy is an optical imaging 
technique used to increase optical resolution 
and contrast of a micrograph by using point 
illumination and a spatial pinhole to 
eliminate out-of-focus light in specimens 



[l] 



It 



that are thicker than the focal plane 
enables the reconstruction of 

three-dimensional structures from the 
obtained images. This technique has gained 
popularity in the scientific and industrial 
communities and typical applications are in 
life sciences, semiconductor inspection and 
material science. 



Light source 



Beam splitter , 




Principle of confocal microscopy 



Confocal microscope 



131 



Basic concept 



,P] 




FIG. 3. 



INVENTOR. 



Confocal point sensor principle from Minsky's 
patent 



The principle of confocal imaging was patented by Marvin Minsky 

and aims to overcome some limitations of traditional wide-field 

fluorescence microscopes. In a conventional (i.e., wide-field) 

fluorescence microscope, the entire specimen is flooded evenly in light 

from a light source. All parts of the specimen in the optical path are 

excited at the same time and the resulting fluorescence is detected by 

the microscope's photodetector or camera including a large unfocused 

background part. In contrast, a confocal microscope uses point 

illumination (see Point Spread Function) and a pinhole in an optically 

conjugate plane in front of the detector to eliminate out-of-focus signal - the name "confocal" stems from this 

configuration. As only light produced by fluorescence very close to the focal plane can be detected the image optical 

resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes. However, 

as much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of 

decreased signal intensity so long exposures are often required. 

As only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster 
(i.e. a rectangular pattern of parallel scanning lines) in the specimen. The achievable thickness of the focal plane is 
defined mostly by the wavelength of the used light divided by the numerical aperture of the objective lens, but also 
by the optical properties of the specimen. The thin optical sectioning possible make these types of microscopes 
particularly good at 3D imaging and surface profiling of samples. 



Types 

Three types of confocal microscopes are commercially available: 

• Confocal laser scanning microscopes 

• Spinning-disk (Nipkow disk) confocal microscopes 

• Programmable Array Microscopes (PAM) 

Each of these classes of confocal microscope have particular advantages and disadvantages, most systems are either 
optimised for resolution or high recording speed (i.e. video capture). Confocal laser scanning microscopes can have a 
programmable sampling density while Nipkow and PAM use a fixed sampling density defined by the camera 
resolution. Imaging frame rates are typically very slow for laser scanning systems (e.g. less than 3 frames/second). 
Commercial spinning-disk confocal microscopes achieve frame rates of over 50 per second - a desirable feature for 
dynamic observations such as live cell imaging. So the spinning-disk as well as the programmble array microscopes 
- can be seen as parallel versions of the confocal scanning principle. Cutting edge development of confocal laser 
scanning microscopy now allows better than video rate (60 frames/second) imaging by using multiple 
microelectromechanical systems based scanning mirrors. 

Confocal x-ray fluorescence imaging is a newer technique that allows control over depth, in addition to horizontal 
and vertical aiming, for example, when analyzing buried layers in a painting. 



Confocal microscope 



132 



Images 




|3-tubulin in 

Tetrahymena (a ciliated 

protozoan). 




Partial surface profile of a 

1-Euro coin, measured 

with a Nipkow Disk 

Confocal Microscope. 




Reflection data for 
1-Euro coin. 





D 


/~~\_^-^ 




-10 


/ ~^^\ 




S-20 
N" 30 


/ \ 




-40 


\ 




-50 


^-___ 




60 






C 


100 200 300 400 500 600 700 BOO 

x/fim 


Cross section at y=200 through 


profile of 1-Euro coin. 



References 

[1] Pawley JB (editor) (2006). Handbook of Biological Confocal Microscopy (3rd ed.). Berlin: Springer. ISBN 038725921X. 

[2] Filed in 1957 and granted 1961. US 3013467 (http://v3.espacenet.com/textdoc?DB=EPODOC&IDX=US3013467) 

[3] "Data Sheet of NanoFocus psutf spinning disk confocal white light microscope" (http://www.nanofocus-us.com/fileadmin/user_upload/ 

download/Produkte/NanoFocus-usurf_explore_.pdf) (pdf). . 
[4] "Data Sheet of Sensofar 'PLu neox Dual Technology sensor head combining Confocal and Interferometry techniques, as well as 

Spectroscopic Reflectometry" (http://www.sensofar.com/products/products_neox.html). . 
[5] Vincze L (2005). "Confocal X-ray Fluorescence Imaging and XRF Tomography for Three Dimensional Trace Element Microanalysis" (http:/ 

/journals. Cambridge. org/ action/displayFulltext?type=l&fid=326128&jid=MAM&volumeId=ll&issueId=S02&aid=326127). 

Microscopy and Microanalysis 11 (Supplement 2). doi:10.1017/S1431927605503167. . 



External links 

• Molecular Expressions: (http://micro.magnet.fsu.edu) Laser Scanning Confocal Microscopy (http://micro. 
magnet.fsu.edu/primer/techniques/confocal/index.html) 

• Nikon's MicroscopyU (http://www.microscopyu.com/articles/confocal/confocalintrobasics.html). 
Comprehensive introduction to confocal microscopy. 

• Emory's Physics Department (http://www.physics.emory.edu/~weeks/confocal/). Introduction to confocal 
microscopy and fluorescence. 

• The Science Creative Quarterly's overview of confocal microscopy (http://www.scq.ubc. ca/?p=278) - high res 
images also available. 

• Programmable Array Microscope (http://spiedl.aip.org/getabs/servlet/GetabsServlet?prog=normal& 
id=PSISDG00644100000164410S000001&idtype=cvips&gifs=yes) - Confocal Microscope Capabilities. 



Atomic force microscope 



133 



Atomic force microscope 



Atomic force microscopy (AFM) or scanning force 
microscopy (SFM) is a very high-resolution type of 
scanning probe microscopy, with demonstrated resolution 
on the order of fractions of a nanometer, more than 1000 
times better than the optical diffraction limit. The 
precursor to the AFM, the scanning tunneling 
microscope, was developed by Gerd Binnig and Heinrich 
Rohrer in the early 1980s at IBM Research - Zurich, a 
development that earned them the Nobel Prize for Physics 
in 1986. Binnig, Quate and Gerber invented the first 
atomic force microscope (also abbreviated as AFM) in 
1986. The first commercially available atomic force 
microscope was introduced in 1989. The AFM is one of 
the foremost tools for imaging, measuring, and 
manipulating matter at the nanoscale. The information is 
gathered by "feeling" the surface with a mechanical 
probe. Piezoelectric elements that facilitate tiny but 
accurate and precise movements on (electronic) command 
enable the very precise scanning. In some variations, 
electric potentials can also be scanned using conducting 
cantilevers. In newer more advanced versions, currents 
can even be passed through the tip to probe the electrical 
conductivity or transport of the underlying surface, but 
this is much more challenging with very few groups 
reporting reliable data. 



Basic principles 





A commercial AFM setup 



Detector and 

Feedback 

Electronics 




Sample Surface 



Cantilever & Tip 



PZT Scanner 



Block diagram of atomic force microscope 



Electron micrograph of a used AFM cantilever image width -100 micrometers. 




and -30 micrometers 



Atomic force microscope 134 

The AFM consists of a cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface. The 
cantilever is typically silicon or silicon nitride with a tip radius of curvature on the order of nanometers. When the tip 
is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the 
cantilever according to Hooke's law. Depending on the situation, forces that are measured in AFM include 
mechanical contact force, van der Waals forces, capillary forces, chemical bonding, electrostatic forces, magnetic 
forces (see magnetic force microscope, MFM), Casimir forces, solvation forces, etc. Along with force, additional 
quantities may simultaneously be measured through the use of specialized types of probe (see scanning thermal 
microscopy, scanning joule expansion microscopy, photothermal microspectroscopy, etc.). Typically, the deflection 
is measured using a laser spot reflected from the top surface of the cantilever into an array of photodiodes. Other 
methods that are used include optical interferometry, capacitive sensing or piezoresistive AFM cantilevers. These 
cantilevers are fabricated with piezoresistive elements that act as a strain gauge. Using a Wheatstone bridge, strain in 
the AFM cantilever due to deflection can be measured, but this method is not as sensitive as laser deflection or 
interferometry. 

If the tip was scanned at a constant height, a risk would exist that the tip collides with the surface, causing damage. 
Hence, in most cases a feedback mechanism is employed to adjust the tip-to-sample distance to maintain a constant 
force between the tip and the sample. Traditionally, the sample is mounted on a piezoelectric tube, that can move the 
sample in the z direction for maintaining a constant force, and the x and y directions for scanning the sample. 
Alternatively a 'tripod' configuration of three piezo crystals may be employed, with each responsible for scanning in 
the x,y and z directions. This eliminates some of the distortion effects seen with a tube scanner. In newer designs, the 
tip is mounted on a vertical piezo scanner while the sample is being scanned in X and Y using another piezo block. 
The resulting map of the area s = f(x,y) represents the topography of the sample. 

The AFM can be operated in a number of modes, depending on the application. In general, possible imaging modes 
are divided into static (also called contact) modes and a variety of dynamic (or non-contact) modes where the 
cantilever is vibrated. 

Imaging modes 

The primary modes of operation for an AFM are static mode and dynamic mode. In static mode, the cantilever is 
"dragged" across the surface of the sample and the contours of the surface are measured directly using the deflection 
of the cantilever. In the dynamic mode, the cantilever is externally oscillated at or close to its fundamental resonance 
frequency or a harmonic. The oscillation amplitude, phase and resonance frequency are modified by tip-sample 
interaction forces. These changes in oscillation with respect to the external reference oscillation provide information 
about the sample's characteristics. 

Contact mode 

In the static mode operation, the static tip deflection is used as a feedback signal. Because the measurement of a 
static signal is prone to noise and drift, low stiffness cantilevers are used to boost the deflection signal. However, 
close to the surface of the sample, attractive forces can be quite strong, causing the tip to "snap-in" to the surface. 
Thus static mode AFM is almost always done in contact where the overall force is repulsive. Consequently, this 
technique is typically called "contact mode". In contact mode, the force between the tip and the surface is kept 
constant during scanning by maintaining a constant deflection. 



Atomic force microscope 



135 



Fiitititiack Loop Maintains Const;in1 
Osnillminn Ampliturjp nr Frpqnpnr.y 



Non-contact mode 

In this mode, the tip of the cantilever 
does not contact the sample surface. 
The cantilever is instead oscillated at a 
frequency slightly above its resonance 
frequency where the amplitude of 
oscillation is typically a few 
nanometers (<10nm). The van der 
Waals forces, which are strongest from 
1 nm to 10 nm above the surface, or 
any other long range force which 
extends above the surface acts to 
decrease the resonance frequency of 
the cantilever. This decrease in 
resonance frequency combined with 
the feedback loop system maintains a 
constant oscillation amplitude or 
frequency by adjusting the average 
tip-to-sample distance. Measuring the 
tip-to-sample distance at each (x,y) 
data point allows the scanning software to construct a topographic image of the sample surface. 



Controller 

Eiactmmcc 




MuiHhirnu 
One Hla:lG r 
■\rrslitudc or 



Saiviple 



AFM - non-contact mode 



Non-contact mode AFM does not suffer from tip or sample degradation effects that are sometimes observed after 
taking numerous scans with contact AFM. This makes non-contact AFM preferable to contact AFM for measuring 
soft samples. In the case of rigid samples, contact and non-contact images may look the same. However, if a few 
monolayers of adsorbed fluid are lying on the surface of a rigid sample, the images may look quite different. An 
AFM operating in contact mode will penetrate the liquid layer to image the underlying surface, whereas in 
non-contact mode an AFM will oscillate above the adsorbed fluid layer to image both the liquid and surface. 

Schemes for dynamic mode operation include frequency modulation and the more common amplitude modulation. 
In frequency modulation, changes in the oscillation frequency provide information about tip-sample interactions. 
Frequency can be measured with very high sensitivity and thus the frequency modulation mode allows for the use of 
very stiff cantilevers. Stiff cantilevers provide stability very close to the surface and, as a result, this technique was 
the first AFM technique to provide true atomic resolution in ultra-high vacuum conditions. 

In amplitude modulation, changes in the oscillation amplitude or phase provide the feedback signal for imaging. In 
amplitude modulation, changes in the phase of oscillation can be used to discriminate between different types of 
materials on the surface. Amplitude modulation can be operated either in the non-contact or in the intermittent 
contact regime. In dynamic contact mode, the cantilever is oscillated such that the separation distance between the 
cantilever tip and the sample surface is modulated. 

Amplitude modulation has also been used in the non-contact regime to image with atomic resolution by using very 
stiff cantilevers and small amplitudes in an ultra-high vacuum environment. 



Atomic force microscope 



136 



Tapping mode 

In ambient conditions, most samples develop a liquid meniscus 
layer. Because of this, keeping the probe tip close enough to the 
sample for short-range forces to become detectable while preventing 
the tip from sticking to the surface presents a major problem for 
non-contact dynamic mode in ambient conditions. Dynamic contact 
mode (also called intermittent contact or tapping mode) was 
developed to bypass this problem 



[3] 




Single polymer chains (0.4 nm thick) recorded in a 

tapping mode under aqueous media with different 

pH. [2] 



In tapping mode, the cantilever is driven to oscillate up and down at 

near its resonance frequency by a small piezoelectric element 

mounted in the AFM tip holder similar to non-contact mode. 

However, the amplitude of this oscillation is greater than 10 nm, 

typically 100 to 200 nm. Due to the interaction of forces acting on 

the cantilever when the tip comes close to the surface, Van der 

Waals force, dipole-dipole interaction, electrostatic forces, etc. cause 

the amplitude of this oscillation to decrease as the tip gets closer to 

the sample. An electronic servo uses the piezoelectric actuator to 

control the height of the cantilever above the sample. The servo 

adjusts the height to maintain a set cantilever oscillation amplitude as the cantilever is scanned over the sample. A 

tapping AFM image is therefore produced by imaging the force of the intermittent contacts of the tip with the sample 

surface. 

This method of "tapping" lessens the damage done to the surface and the tip compared to the amount done in contact 
mode. Tapping mode is gentle enough even for the visualization of supported lipid bilayers or adsorbed single 
polymer molecules (for instance, 0.4 nm thick chains of synthetic polyelectrolytes) under liquid medium. With 
proper scanning parameters, the conformation of single molecules can remain unchanged for hours. 



AFM cantilever deflection measurement 



Solid State LaserDiode 



Output 



m 



Laser light from a solid state diode is 

reflected off the back of the cantilever 

and collected by a position sensitive 

detector (PSD) consisting of two 

closely spaced photodiodes whose 

output signal is collected by a 

differential amplifier. Angular 

displacement of cantilever results in 

one photodiode collecting more light 

than the other photodiode, producing 

an output signal (the difference 

between the photodiode signals 

normalized by their sum) which is 

proportional to the deflection of the 

cantilever. It detects cantilever deflections <10 nm (thermal noise limited). A long beam path (several centimeters) 

amplifies changes in beam angle. 



Split Photodiode Detector 




CantileverardTip 



AFM beam deflection detection 



Atomic force microscope 



137 



Force spectroscopy 

Another major application of AFM (besides imaging) is force spectroscopy, the direct measurement of tip-sample 
interaction forces as a function of the gap between the tip and sample (the result of this measurement is called a 
force-distance curve). For this method, the AFM tip is extended towards and retracted from the surface as the 
deflection of the cantilever is monitored as a function of piezoelectric displacement. These measurements have been 
used to measure nanoscale contacts, atomic bonding, Van der Waals forces, and Casimir forces, dissolution forces in 
liquids and single molecule stretching and rupture forces. Furthermore, AFM was used to measure in aqueous 
environment dispersion force due to polymer adsorbed on the substrate. Forces of the order of a few piconewtons 
can now be routinely measured with a vertical distance resolution of better than 0.1 nanometers. Force spectroscopy 
can be performed with either static or dynamic modes. In dynamic modes, information about the cantilever vibration 
is monitored in addition to the static deflection. 

Problems with the technique include no direct measurement of the tip-sample separation and the common need for 
low stiffness cantilevers which tend to 'snap' to the surface. The snap-in can be reduced by measuring in liquids or by 
using stiffer cantilevers, but in the latter case a more sensitive deflection sensor is needed. By applying a small dither 
to the tip, the stiffness (force gradient) of the bond can be measured as well 



[7] 



Identification of individual surface atoms 

The AFM can be used to image and manipulate atoms and structures on a variety of surfaces. The atom at the apex 
of the tip "senses" individual atoms on the underlying surface when it forms incipient chemical bonds with each 
atom. Because these chemical interactions subtly alter the tip's vibration frequency, they can be detected and 
mapped. This principle was used to distinguish between atoms of silicon, tin and lead on an alloy surface, by 
comparing these 'atomic fingerprints' to values obtained from large-scale density functional theory (DFT) 
simulations. 

The trick is to first measure these forces precisely for each type of atom expected in the sample, and then to compare 
with forces given by DFT simulations. The team found that the tip interacted most strongly with silicon atoms, and 
interacted 23% and 41% less strongly with tin and lead atoms, respectively. Thus, each different type of atom can be 
identified in the matrix as the tip is moved across the surface. 

Such a technique has been used now in biology and extended recently to cell biology. Forces corresponding to (i) the 
unbinding of receptor ligand couples (ii) unfolding of proteins (iii) cell adhesion at single cell scale have been 
gathered. 



Advantages and disadvantages 

Just like any other tool, an AFM's usefulness has limitations. When 
determining whether or not analyzing a sample with an AFM is 
appropriate, there are various advantages and disadvantages that must 
be considered. 

Advantages 

AFM has several advantages over the scanning electron microscope 
(SEM). Unlike the electron microscope which provides a 
two-dimensional projection or a two-dimensional image of a sample, 
the AFM provides a three-dimensional surface profile. Additionally, 







The first atomic force microscope 



Atomic force microscope 138 

samples viewed by AFM do not require any special treatments (such as metal/carbon coatings) that would 
irreversibly change or damage the sample. While an electron microscope needs an expensive vacuum environment 
for proper operation, most AFM modes can work perfectly well in ambient air or even a liquid environment. This 
makes it possible to study biological macromolecules and even living organisms. In principle, AFM can provide 
higher resolution than SEM. It has been shown to give true atomic resolution in ultra-high vacuum (UHV) and, more 
recently, in liquid environments. High resolution AFM is comparable in resolution to scanning tunneling microscopy 
and transmission electron microscopy. 

Disadvantages 

A disadvantage of AFM compared with the scanning electron microscope (SEM) is the single scan image size. In 
one pass, the SEM can image an area on the order of square millimeters with a depth of field on the order of 
millimeters. Whereas the AFM can only image a maximum height on the order of 10-20 micrometers and a 
maximum scanning area of about 150x150 micrometers. One method of improving the scanned area size for AFM is 
by using parallel probes in a fashion similar to that of millipede data storage. 

The scanning speed of an AFM is also a limitation. Traditionally, an AFM cannot scan images as fast as a SEM, 
requiring several minutes for a typical scan, while a SEM is capable of scanning at near real-time, although at 
relatively low quality. The relatively slow rate of scanning during AFM imaging often leads to thermal drift in the 
image making the AFM microscope less suited for measuring accurate distances between topographical 

features on the image. However, several fast-acting designs were suggested to increase microscope scanning 

productivity including what is being termed videoAFM (reasonable quality images are being obtained with 
videoAFM at video rate: faster than the average SEM). To eliminate image distortions induced by thermal drift, 
several methods have been introduced. 

ri3i 

AFM images can also be affected by hysteresis of the piezoelectric material and cross-talk between the x, y, z 
axes that may require software enhancement and filtering. Such filtering could "flatten" out real topographical 
features. However, newer AFMs utilize closed-loop scanners which practically eliminate these problems. Some 
AFMs also use separated orthogonal scanners (as opposed to a single tube) which also serve to eliminate part of the 
cross-talk problems. 

As with any other imaging technique, there is the possibility of image artifacts, which could be induced by an 
unsuitable tip, a poor operating environment, or even by the sample itself. These image artifacts are unavoidable 
however, their occurrence and effect on results can be reduced through various methods. 

Due to the nature of AFM probes, they cannot normally measure steep walls or overhangs. Specially made 
cantilevers and AFMs can be used to modulate the probe sideways as well as up and down (as with dynamic contact 
and non-contact modes) to measure sidewalls, at the cost of more expensive cantilevers, lower lateral resolution and 
additional artifacts. 

Piezoelectric scanners 

AFM scanners are made from piezoelectric material, which expands and contracts proportionally to an applied 
voltage. Whether they elongate or contract depends upon the polarity of the voltage applied. The scanner is 
constructed by combining independently operated piezo electrodes for X, Y, and Z into a single tube, forming a 
scanner which can manipulate samples and probes with extreme precision in 3 dimensions. 

Scanners are characterized by their sensitivity which is the ratio of piezo movement to piezo voltage, i.e., by how 
much the piezo material extends or contracts per applied volt. Because of differences in material or size, the 
sensitivity varies from scanner to scanner. Sensitivity varies non-linearly with respect to scan size. Piezo scanners 
exhibit more sensitivity at the end than at the beginning of a scan. This causes the forward and reverse scans to 
behave differently and display hysteresis between the two scan directions. This can be corrected by applying a 



Atomic force microscope 139 

non-linear voltage to the piezo electrodes to cause linear scanner movement and calibrating the scanner 
accordingly. 

The sensitivity of piezoelectric materials decreases exponentially with time. This causes most of the change in 
sensitivity to occur in the initial stages of the scanner's life. Piezoelectric scanners are run for approximately 48 
hours before they are shipped from the factory so that they are past the point where they may have large changes in 
sensitivity. As the scanner ages, the sensitivity will change less with time and the scanner would seldom require 
recalibration. 

See also 

• Frictional force mapping 

• Scanning tunneling microscope 

• Scanning probe microscopy 

• Scanning voltage microscopy 

• Surface force apparatus 

References 

[I] Giessibl, Franz J. (2003). "Advances in atomic force microscopy". Reviews of Modern Physics 75: 949. doi:10.1103/RevModPhys.75.949. 
[2] Roiter, Y; Minko, S (Nov 2005). "AFM single molecule experiments at the solid-liquid interface: in situ conformation of adsorbed flexible 

polyelectrolyte chains". Journal of the American Chemical Society 127 (45): 15688-9. doi:10.1021/ja0558239. ISSN 0002-7863. 

PMID 16277495. 
[3] Zhong, Q; Inniss, D; Kjoller, K; Elings, V (1993). "Fractured polymer/silica fiber surface studied by tapping mode atomic force microscopy". 

Surface Science Letters 290: L688. doi:10.1016/0167-2584(93)90906-Y. 
[4] Hinterdorfer, P; Dufrene, Yf (May 2006). "Detection and localization of single molecular recognition events using atomic force microscopy". 

Nature methods 3 (5): 347-55. doi:10.1038/nmeth871. ISSN 1548-7091. PMID 16628204. 
[5] J Colloid Interface Sci. 2010 Jul 1;347(1): 15-24. Epub 2010 Mar 7. Interaction of cement model systems with superplasticizers investigated 

by atomic force microscopy, zeta potential, and adsorption measurements. Ferrari L., Kaufmann J., Winnefeld F., Plank J., 
[6] Butt, H; Cappella, B; Kappl, M (2005). "Force measurements with the atomic force microscope: Technique, interpretation and applications". 

Surface Science Reports 59: 1-152. doi:10.1016/j.surfrep.2005.08.003. 
[7] M. Hoffmann, Ahmet Oral, Ralph A. G, Peter (2001). "Direct measurement of interatomic force gradients using an ultra-low-amplitude 

atomic force microscope". Proceedings of the Royal Society a Mathematical Physical and Engineering Sciences 457: 1161. 

doi:10.1098/rspa.2000.0713. 
[8] Sugimoto, Y; Pou, P; Abe, M; Jelinek, P; Perez, R; Morita, S; Custance, O (Mar 2007). "Chemical identification of individual surface atoms 

by atomic force microscopy". Nature 446 (7131): 64-7. doi:10.1038/nature05530. ISSN 0028-0836. PMID 17330040. 
[9] R. V. Lapshin (2004). "Feature-oriented scanning methodology for probe microscopy and nanotechnology" (http://www.nanoworld.org/ 

homepages/lapshin/publications.htm#feature2004) (PDF). Nanotechnology (UK: IOP) 15 (9): 1135-1151. doi: 10. 1088/0957-4484/15/9/006. 

ISSN 0957-4484. . 
[10] R. V. Lapshin (2007). "Automatic drift elimination in probe microscope images based on techniques of counter-scanning and topography 

feature recognition" (http://www.nanoworld.Org/homepages/lapshin/publications.htm#automatic2007) (PDF). Measurement Science and 

Technology (UK: IOP) 18 (3): 907-927. doi: 10.1088/0957-0233/18/3/046. ISSN 0957-0233. . 

[II] G. Schitter, M. J. Rost (2008). "Scanning probe microscopy at video-rate" (http://www.materialstoday.com/view/2194/ 
scanning-probe-microscopy-at-videorate/) (PDF). Materials Today (UK: Elsevier) 11 (special issue): 40—48. 
doi:10.1016/S1369-7021(09)70006-9. ISSN 1369-7021. . 

[12] R. V. Lapshin, O. V. Obyedkov (1993). "Fast-acting piezoactuator and digital feedback loop for scanning tunneling microscopes" (http:// 

www.nanoworld.org/homepages/lapshin/publications.htm#fastl993) (PDF). Review of Scientific Instruments (USA: AIP) 64 (10): 

2883-2887. doi: 10. 1063/1. 1144377. ISSN 0034-6748. . 
[13] R. V. Lapshin (1995). "Analytical model for the approximation of hysteresis loop and its application to the scanning tunneling microscope" 

(http://www.nanoworld.Org/homepages/lapshin/publications.htm#analyticall995) (PDF). Review of Scientific Instruments (USA: AIP) 66 

(9): 4718-4730. doi: 10.1063/1. 1145314. ISSN 0034-6748. . ( is available). 
[14] R. V. Lapshin (1998). "Automatic lateral calibration of tunneling microscope scanners" (http://www.nanoworld.org/homepages/lapshin/ 

publications.htm#automaticl998) (PDF). Review of Scientific Instruments (USA: AIP) 69 (9): 3268-3276. doi: 10.1063/1.1 149091. 

ISSN 0034-6748. . 



Atomic force microscope 



140 



External links 

• ME 597/PHYS 570: Fundamentals of Atomic Force Microscopy (http://nanohub.org/resources/7320) 

• DoITPoMS Teaching and Learning Package - Atomic Force Microscopy (http://www.doitpoms.ac.uk/tlplib/ 
afm/index.php) 

• SPM gallery: surface scans, collages, artworks, desktop wallpapers (http://www.nanoworld.org/homepages/ 
lapshin/gallery.htm) 

Further reading 

• SPM - Scanning Probe Microscopy Website (http://www.mobot.org/jwcross/spm/) 

• Atomic Force Microscopy resource library (http://www.afmuniversity.org) 

• R. W. Carpick and M. Salmeron, Scratching the surface: Fundamental investigations of tribology with atomic 
force microscopy (http://dx.doi.org/10.1021/cr960068q), Chemical Reviews, vol. 97, iss. 4, pp. 1163—1194 
(2007). 



Electron microscope 



An electron microscope is a type of microscope that produces an 
electronically-magnified image of a specimen for detailed 
observation. The electron microscope (EM) uses a particle beam of 
electrons to illuminate the specimen and create a magnified image of 
it. The microscope has a greater resolving power than a 
light-powered optical microscope, because it uses electrons that have 
wavelengths about 100,000 times shorter than visible light (photons), 
and can achieve magnifications of up to 2,000,000x, whereas 
ordinary, non-confocal light microscopes are limited to 2000x 
magnification. 




High voltage 
Electrongun 

First condenser lens 

Condenser aperture 
Second condenser lens 

Condenser aperture 
Specimen holdcrand air-lock 
Objective lenses and aperture 

Electron beam 

Fluorescent screen andcamera 



Transmission Electron Microscope 

Diagram of a transmission electron microscope 



The electron microscope uses electrostatic and electromagnetic 
"lenses" to control the electron beam and focus it to form an image. 

These lenses are analogous to, but different from the glass lenses of an optical microscope that form a magnified 
image by focusing light on or through the specimen. In transmission, the electron beam is first diffracted by the 
specimen, and then, the electron microscope "lenses" re-focus the beam into a Fourier-transformed image of the 
diffraction pattern for the selected area of investigation. The real image thus formed is a highly "magnified' image by 
a factor of several million, and can be then recorded on a special photographic plate, or viewed on a detecting screen. 
Electron microscopes are used to observe a wide range of biological and inorganic specimens including 
microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, the electron microscope is 
primarily used for quality control and failure analysis in semiconductor device fabrication. 



Election microscope 



141 



An electron microscope's advantages over X-ray crystallography are 
that the specimen need not be a single crystal or even a polycrystalline 
powder, and also that the Fourier transform reconstruction of the 
object's magnified structure occurs physically and thus avoids the need 
for solving the phase problem faced by the X-ray crystalographers after 
obatining their X-ray diffraction patterns of a single crystal or 
polycrystalline powder. The transmission electron microscope's major 
"disadvantage' is the need for extremely thin sections of the specimens, 
typically less than 10 nanometers. For biological specimens it also 
requires biological sample special "staining' with heavy atom labels in 
order to achieve the required contrast, and then chemical fixation as 
well as encasing with a polymer resin to stabilize the biological 
specimen which is thin sectioned. 




A 1973 Siemens electron microscope, Musee des 
Arts et Metiers, Paris 



History 

In 1931, the German physicist Ernst Ruska and German electrical 
engineer Max Knoll constructed the prototype electron microscope, 
capable of four-hundred-power magnification; the apparatus was a 
practical application of the principles of electron microscopy. Two 
years later, in 1933, Ruska built an electron microscope that exceeded 
the resolution attainable with an optical (lens) microscope. 
Moreover, Reinhold Rudenberg, the scientific director of 
Siemens-Schuckertwerke, obtained the patent for the electron 
microscope in May 1931. Family illness compelled the electrical 
engineer to devise an electrostatic microscope, because he wanted to 
make visible the poliomyelitis virus. 

In 1937, the Siemens company financed the development work of 
Ernst Ruska and Bodo von Borries, and employed Helmut Ruska 
(Ernst's brother) to develop applications for the microscope, especially 

with biologic specimens. Also in 1937, Manfred von Ardenne 

T31 
pioneered the scanning electron microscope. The first practical 

electron microscope was constructed in 1938, at the University of 

Toronto, by Eli Franklin Burton and students Cecil Hall, James Hillier, 

and Albert Prebus; and Siemens produced the first commercial 

Transmission Electron Microscope (TEM) in 1939. Although 

contemporary electron microscopes are capable of two million-power 

magnification, as scientific instruments, they remain based upon 

Ruska's prototype. 




Electron microscope constructed by Ernst Ruska 
in 1933 



Electron microscope 



142 



Types 



Transmission electron microscope (TEM) 

The original form of electron microscope, the transmission electron microscope (TEM) uses a high voltage electron 
beam to create an image. The electrons are emitted by an electron gun, commonly fitted with a tungsten filament 
cathode as the electron source. The electron beam is accelerated by an anode typically at +100 keV (40 to 400 keV) 
with respect to the cathode, focused by electrostatic and electromagnetic lenses, and transmitted through the 
specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the 
specimen, the electron beam carries information about the structure of the specimen that is magnified by the 
objective lens system of the microscope. The spatial variation in this information (the "image") is viewed by 
projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator 
material such as zinc sulfide. The image can be photographically recorded by exposing a photographic film or plate 
directly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a 
fibre optic light-guide to the sensor of a CCD (charge-coupled device) camera. The image detected by the CCD may 
be displayed on a monitor or computer. 

Resolution of the TEM is limited primarily by spherical aberration, but a new generation of aberration correctors 
have been able to partially overcome spherical aberration to increase resolution. Hardware correction of spherical 
aberration for the High Resolution TEM (HRTEM) has allowed the production of images with resolution below 0.5 
Angstrom (50 picometres) at magnifications above 50 million times. The ability to determine the positions of 



atoms within materials has made the HRTEM an important tool for nano-technologies research and development 



[7] 



Scanning electron microscope (SEM) 



Unlike the TEM, where electrons of the high voltage beam carry the 
image of the specimen, the electron beam of the Scanning Electron 

ro] 

Microscope (SEM) does not at any time carry a complete image of 
the specimen. The SEM produces images by probing the specimen 
with a focused electron beam that is scanned across a rectangular area 
of the specimen (raster scanning). At each point on the specimen the 
incident electron beam loses some energy, and that lost energy is 
converted into other forms, such as heat, emission of low-energy 
secondary electrons, light emission (cathodoluminescence) or x-ray 
emission. The display of the SEM maps the varying intensity of any of 
these signals into the image in a position corresponding to the position 
of the beam on the specimen when the signal was generated. In the 
SEM image of an ant shown at right, the image was constructed from 
signals produced by a secondary electron detector, the normal or 
conventional imaging mode in most SEMs. 

Generally, the image resolution of an SEM is about an order of magnitude poorer than that of a TEM. However, 
because the SEM image relies on surface processes rather than transmission, it is able to image bulk samples up to 
many centimetres in size and (depending on instrument design and settings) has a great depth of field, and so can 
produce images that are good representations of the three-dimensional shape of the sample. 




An image of an ant in a scanning electron 
microscope 



Election microscope 



143 



Reflection electron microscope (REM) 

In the Reflection Electron Microscope (REM) as in the TEM, an electron beam is incident on a surface, but instead 
of using the transmission (TEM) or secondary electrons (SEM), the reflected beam of elastically scattered electrons 
is detected. This technique is typically coupled with Reflection High Energy Electron Diffraction (RHEED) and 
Reflection high-energy loss spectrum (RHELS). Another variation is Spin-Polarized Low-Energy Electron 



Microscopy (SPLEEM), which is used for looking at the microstructure of magnetic domains 



[9] 



Scanning transmission electron microscope (STEM) 

The STEM rasters a focused incident probe across a specimen that (as with the TEM) has been thinned to facilitate 
detection of electrons scattered through the specimen. The high resolution of the TEM is thus possible in STEM. The 
focusing action (and aberrations) occur before the electrons hit the specimen in the STEM, but afterward in the 
TEM. The STEMs use of SEM-like beam rastering simplifies annular dark-field imaging, and other analytical 
techniques, but also means that image data is acquired in serial rather than in parallel fashion. 

Low voltage electron microscope (LVEM) 

The low voltage electron microscope (LVEM) is a combination of SEM, TEM and STEM in one instrument, which 
operates at relatively low electron accelerating voltage of 5 kV. Low voltage increases image contrast which is 
especially important for biological specimens. This increase in contrast significantly reduces, or even eliminates the 
need to stain. Sectioned samples generally need to be thinner than they would be for conventional TEM (20-65 nm). 
Resolutions of a few nm are possible in TEM, SEM and STEM modes. 



Sample preparation 



Materials to be viewed under an electron microscope may require 
processing to produce a suitable sample. The technique required varies 
depending on the specimen and the analysis required: 

• Chemical fixation for biological specimens aims to stabilize the 
specimen's mobile macromolecular structure by chemical 
crosslinking of proteins with aldehydes such as formaldehyde and 
glutaraldehyde, and lipids with osmium tetroxide. 

• Cryofixation — freezing a specimen so rapidly, to liquid nitrogen or 
even liquid helium temperatures, that the water forms vitreous 
(non-crystalline) ice. This preserves the specimen in a snapshot of 
its solution state. An entire field called cryo-electron microscopy 
has branched from this technique. With the development of 
cryo-electron microscopy of vitreous sections (CEMOVIS), it is 
now possible to observe samples from virtually any biological 
specimen close to its native state. 

• Dehydration — freeze drying, or replacement of water with organic 

solvents such as ethanol or acetone, followed by critical point drying or infiltration with embedding resins. 

• Embedding, biological specimens — after dehydration, tissue for observation in the transmission electron 
microscope is embedded so it can be sectioned ready for viewing. To do this the tissue is passed through a 
'transition solvent' such as epoxy propane and then infiltrated with a resin such as Araldite epoxy resin; tissues 
may also be embedded directly in water-miscible acrylic resin. After the resin has been polymerised (hardened) 
the sample is thin sectioned (ultrathin sections) and stained - it is then ready for viewing. 

• Embedding, materials - after embedding in resin, the specimen is usually ground and polished to a mirror-like 
finish using ultra-fine abrasives. The polishing process must be performed carefully to minimize scratches and 




An insect coated in gold for viewing with a 
scanning electron microscope. 



Electron microscope 144 

other polishing artifacts that reduce image quality. 

• Sectioning — produces thin slices of specimen, semitransparent to electrons. These can be cut on an 
ultramicrotome with a diamond knife to produce ultrathin slices about 60-90 nm thick. Disposable glass knives 
are also used because they can be made in the lab and are much cheaper. 

• Staining — uses heavy metals such as lead, uranium or tungsten to scatter imaging electrons and thus give contrast 
between different structures, since many (especially biological) materials are nearly "transparent" to electrons 
(weak phase objects). In biology, specimens are can be stained "en bloc" before embedding and also later after 
sectioning. Typically thin sections are stained for several minutes with an aqueous or alcoholic solution of uranyl 
acetate followed by aqueous lead citrate. 

• Freeze-fracture or freeze-etch — a preparation method particularly useful for examining lipid membranes and 
their incorporated proteins in "face on" view. The fresh tissue or cell suspension is frozen rapidly (cryofixed), 
then fractured by simply breaking or by using a microtome while maintained at liquid nitrogen temperature. The 
cold fractured surface (sometimes "etched" by increasing the temperature to about —100 °C for several minutes to 
let some ice sublime) is then shadowed with evaporated platinum or gold at an average angle of 45° in a high 
vacuum evaporator. A second coat of carbon, evaporated perpendicular to the average surface plane is often 
performed to improve stability of the replica coating. The specimen is returned to room temperature and pressure, 
then the extremely fragile "pre-shadowed" metal replica of the fracture surface is released from the underlying 
biological material by careful chemical digestion with acids, hypochlorite solution or SDS detergent. The 
still-floating replica is thoroughly washed from residual chemicals, carefully fished up on fine grids, dried then 
viewed in the TEM. 

• Ion Beam Milling — thins samples until they are transparent to electrons by firing ions (typically argon) at the 
surface from an angle and sputtering material from the surface. A subclass of this is Focused ion beam milling, 
where gallium ions are used to produce an electron transparent membrane in a specific region of the sample, for 
example through a device within a microprocessor. Ion beam milling may also be used for cross-section polishing 
prior to SEM analysis of materials that are difficult to prepare using mechanical polishing. 

• Conductive Coating — an ultrathin coating of electrically-conducting material, deposited either by high vacuum 
evaporation or by low vacuum sputter coating of the sample. This is done to prevent the accumulation of static 
electric fields at the specimen due to the electron irradiation required during imaging. Such coatings include gold, 
gold/palladium, platinum, tungsten, graphite etc. and are especially important for the study of specimens with the 
scanning electron microscope. Another reason for coating, even when there is more than enough conductivity, is 
to improve contrast, a situation more common with the operation of a FESEM (field emission SEM). 



Election microscope 



145 



Disadvantages 

Electron microscopes are expensive to build and maintain, but the 
capital and running costs of confocal light microscope systems now 
overlaps with those of basic electron microscopes. They are dynamic 
rather than static in their operation, requiring extremely stable 
high-voltage supplies, extremely stable currents to each 
electromagnetic coil/lens, continuously-pumped high- or 
ultra-high-vacuum systems, and a cooling water supply circulation 
through the lenses and pumps. As they are very sensitive to vibration 
and external magnetic fields, microscopes designed to achieve high 
resolutions must be housed in stable buildings (sometimes 
underground) with special services such as magnetic field cancelling 
systems. Some desktop low voltage electron microscopes have TEM 
capabilities at very low voltages (around 5 kV) without stringent 
voltage supply, lens coil current, cooling water or vibration isolation 
requirements and as such are much less expensive to buy and far easier 
to install and maintain, but do not have the same ultra-high (atomic 
scale) resolution capabilities as the larger instruments. 




False-color SEM image of the filter setae of an 
Antarctic krill. (Raw electron microscope images 

carry no color information.) 

Pictured: First degree filter setae with V-shaped 

second degree setae pointing towards the inside 

of the feeding basket. The purple ball is 1 um in 

diameter. 



The samples largely have to be viewed in vacuum, as the molecules 

that make up air would scatter the electrons. One exception is the environmental scanning electron microscope, 

which allows hydrated samples to be viewed in a low-pressure (up to 20 Torr/2.7 kPa), wet environment. 

Scanning electron microscopes usually image conductive or semi-conductive materials best. Non-conductive 
materials can be imaged by an environmental scanning electron microscope. A common preparation technique is to 
coat the sample with a several-nanometer layer of conductive material, such as gold, from a sputtering machine; 
however, this process has the potential to disturb delicate samples. 

Small, stable specimens such as carbon nanotubes, diatom frustules and small mineral crystals (asbestos fibres, for 
example) require no special treatment before being examined in the electron microscope. Samples of hydrated 
materials, including almost all biological specimens have to be prepared in various ways to stabilize them, reduce 
their thickness (ultrathin sectioning) and increase their electron optical contrast (staining). These processes may 
result in artifacts, but these can usually be identified by comparing the results obtained by using radically different 
specimen preparation methods. It is generally believed by scientists working in the field that as results from various 
preparation techniques have been compared and that there is no reason that they should all produce similar artifacts, 
it is reasonable to believe that electron microscopy features correspond with those of living cells. In addition, 
higher-resolution work has been directly compared to results from X-ray crystallography, providing independent 
confirmation of the validity of this technique. Since the 1980s, analysis of cryofixed, vitrified specimens has also 
become increasingly used by scientists, further confirming the validity of this technique. 



Electron microscope 



146 



Applications 



Semiconductor and data storage 

• Circuit edit 

• Defect analysis 

• Failure analysis 

Biology and life sciences 

Diagnostic electron microscopy 

Cryobiology 

Protein localization 

Electron tomography 

Cellular tomography 

Cryo-electron microscopy 

Toxicology 

Biological production and viral load monitoring 

Particle analysis 

Pharmaceutical QC 

Structural biology 

3D tissue imaging 

Virology 

Vitrification 



Research 

• Electron beam-induced deposition 

• Materials qualification 

• Materials and sample preparation 

• Nanoprototyping 

• Nanometrology 

• Device testing and characterization 

Industry 

• High-resolution imaging 

• 2D & 3D micro-characterization 

• Macro sample to nanometer metrology 

• Particle detection and characterization 

• Direct beam-writing fabrication 

• Dynamic materials experiments 

• Sample preparation 

• Forensics 

• Mining (mineral liberation analysis) 

• Chemical/Petrochemical 



See also 

Category:Electron microscope images 

Electron energy loss spectroscopy (EELS) 

Energy filtered transmission electron microscopy (EFTEM) 

Field emission microscope 

HiRISE 

High-resolution transmission electron microscopy (HRTEM) 

Scanning tunneling microscope 

Scanning confocal electron microscopy 

Scanning electron microscope (SEM) 

Scanning transmission electron microscope (STEM) 

Transmission Electron Aberration-corrected Microscope 

Electron diffraction 

X-ray diffraction 

X-ray microscope 

X-ray crystallography 

X-ray photoelectron spectroscopy (XPS) 

Microscope image processing 

Microscopy 

Acronyms in microscopy 

Nanoscience 

Nanotechnology 

Surface science 

Ultramicroscopy (journal) 



Electron microscope 147 

References 

[I] Ernst Ruska (1986). "Ernst Ruska Autobiography" (http://nobelprize.org/nobel_prizes/physics/laureates/1986/ruska-autobio.html). 
Nobel Foundation. . Retrieved 2010-01-31. 

[2] Kruger DH, Schneck P, Gelderblom HR (May 2000). "Helmut Ruska and the visualisation of viruses" (http://linkinghub.elsevier.com/ 

retrieve/pii/S0140673600022509). Lancet 355 (9216): 1713-7. doi:10.1016/S0140-6736(00)02250-9. PMID 10905259. . 
[3] M von Ardenne and D Beischer (1940). "Untersuchung von metalloxyd-rauchen mit dem universal-elektronenmikroskop" (in German). 

Zeitschrift Electrochemie 46: 270—277. 
[4] "James Hillier" (http://web.mit.edu/Invent/iow/hillier.html). Inventor of the Week: Archive. 2003-05-01. . Retrieved 2010-01-31. 
[5] Erni, Rolf; Rossell, MD; Kisielowski, C; Dahmen, U (2009). "Atomic-Resolution Imaging with a Sub-50-pm Electron Probe". Physical 

Review Letters 102 (9): 096101. doi:10.1103/PhysRevLett.l02.096101. PMID 19392535. 
[6] "The Scale of Things" (http://www.sc.doe.gov/bes/scale_of_things.html). Office of Basic Energy Sciences, U.S. Department of Energy. 

2006-05-26. . Retrieved 2010-01-31. 
[7] O'Keefe MA, Allard LF (pdf). Sub-Angstrom Electron Microscopy for Sub-Angstrom Nano-Metrology (http://www.osti.gov/bridge/ 

servlets/purl/821768-E3YVgN/native/821768.pdf). Information Bridge: DOE Scientific and Technical Information - Sponsored by OSTI. . 

Retrieved 2010-01-31. 
[8] McMullan D (1993). "Scanning Electron Microscopy, 1928 - 1965" (http://www-g.eng.cam.ac.uk/125/achievements/mcmullan/mcm. 

htm). . Cincinnati, OH. . Retrieved 2010-01-31. 
[9] "SPLEEM" (http://ncem.lbl.gov/frames/spleem.html). National Center for Electron Microscopy (NCEM). . Retrieved 2010-01-31. 
[10] Nebesafoval, Jana; Vancova, Marie (2007). "How to Observe Small Biological Objects in Low Voltage Electron Microscope" (http:// 

journals.cambridge.org/abstract_S143192760708124X). Microscopy and Microanalysis 13 (3): 248-249. . 

[II] Drummy, Lawrence, F.; Yang, Junyan; Martin, David C. (2004). "Low-voltage electron microscopy of polymer and organic molecular thin 
films". Ultramicroscopy 99 (4): 247-256. doi:10.1016/j.ultramic.2004.01.011. PMID 15149719. 

[12] Adrian, Marc; Dubochet, Jacques; Lepault, Jean; McDowall, Alasdair W. (1984). "Cryo-electron microscopy of viruses". Nature 308 

(5954): 32-36. doi:10.1038/308032a0. PMID 6322001. 
[13] Sabanay, I.; Arad, T.; Weiner, S.; Geiger, B. (1991). "Study of vitrified, unstained frozen tissue sections by cryoimmunoelectron 

microscopy" (http://jcs.biologists.Org/cgi/content/abstract/100/l/227). Journal of Cell Science 100 (1): 227-236. PMID 1795028. . 
[14] Kasas, S.; Dumas, G.; Dietler, G.; Catsicas, S.; Adrian, M. (2003). "Vitrification of cryoelectron microscopy specimens revealed by 

high-speed photographic imaging". Journal of Microscopy 211 (1): 48-53. doi:10.1046/j. 1365-2818.2003.01 193.x. 

External links 

• Science Aid: Electron Microscopy (http://scienceaid.co.uk/biology/cell/analysingcells.html) High School 
(GCSE, A Level) resource 

• Cell Centered Database - Electron microscopy data (http://ccdb.ucsd. edu/sand/main?typeid=4& 
event=showMPByType&start=l) 

General 

• Nanohedron.comlNano image gallery (http://www.nanohedron.com/) beautiful images generated with electron 
microscopes. 

• electron microscopy (http://www.microscopy.ethz.ch) Website of the ETH Zurich: Very good graphics and 
images, which illustrate various procedures. 

• Environmental Scanning Electron Microscope (ESEM) (http://www.danilatos.com) 

• X-ray element analysis in electron microscope (http://www.microanalyst.net/index_e.phtml) — Information 
portal with X-ray microanalysis and EDX contents 



Election microscope 



148 



History 

• John H.L. Watson: Very early Electron Microscopy in the Department of Physics, the University of Toronto — A 
personal recollection (http://www.physics.utoronto.ca/overview/history/microsco) 

• Rubin Borasky Electron Microscopy Collection, 1930-1988 (http://americanhistory.si.edu/archives/d8452. 
htm) Archives Center, National Museum of American History, Smithsonian Institution. 

Other 

• The Royal Microscopical Society, Electron Microscopy Section (UK) (http://www.rms.org.uk/em.shtml) 

• Albert Lleal micrograph. Scanning Electron Micrograph Coloured SEM (http://www.albertlleal.com/ 
microphotography . html) 



Synchrotron 




A synchrotron is a particular type of cyclic 
particle accelerator in which the magnetic 
field (to turn the particles so they circulate) 
and the electric field (to accelerate the 
particles) are carefully synchronised with 
the travelling particle beam. The proton 
synchrotron was originally conceived by Sir 
Marcus Oliphant . The honour of being 
the first to publish the idea went to Vladimir 
Veksler, and the first electron synchrotron 
was constructed by Edwin McMillan. 

Characteristics 

While a cyclotron uses a constant magnetic field and a constant-frequency applied electric field (one of these is 
varied in the synchrocyclotron), both of these fields are varied in the synchrotron. By increasing these parameters 
appropriately as the particles gain energy, their path can be held constant as they are accelerated. This allows the 
vacuum chamber for the particles to be a large thin torus. In reality it is easier to use some straight sections between 
the bending magnets and some bent sections within the magnets giving the torus the shape of a round-cornered 
polygon. A path of large effective radius may thus be constructed using simple straight and curved pipe segments, 
unlike the disc-shaped chamber of the cyclotron type devices. The shape also allows and requires the use of multiple 
magnets to bend the particle beams. Straight sections are required at spacings around a ring for both radiofrequency 
cavities, and in third generation setups space is allowed for insertion of energy extraction devices such as wigglers 
and undulators. 

The maximum energy that a cyclic accelerator can impart is typically limited by the strength of the magnetic field(s) 
and the minimum radius (maximum curvature) of the particle path. 



Synchrotron 



149 




The interior of the Australian Synchrotron facility. Dominating the image is the storage 

ring, showing the optical diagnostic beamline at front right. In the middle of the storage 

ring is the booster synchrotron and linac 



In a cyclotron the maximum radius is 

quite limited as the particles start at the 

center and spiral outward, thus the 

entire path must be a self-supporting 

disc-shaped evacuated chamber. Since 

the radius is limited, the power of the 

machine becomes limited by the 

strength of the magnetic field. In the 

case of an ordinary electromagnet the 

field strength is limited by the 

saturation of the core (when all 

magnetic domains are aligned the field may not be further increased to any practical extent). The arrangement of the 

single pair of magnets the full width of the device also limits the economic size of the device. 

Synchrotrons overcome these limitations, using a narrow beam pipe which can be surrounded by much smaller and 
more tightly focusing magnets. The ability of this device to accelerate particles is limited by the fact that the particles 
must be charged to be accelerated at all, but charged particles under acceleration emit photons (light), thereby losing 
energy. The limiting beam energy is reached when the energy lost to the lateral acceleration required to maintain the 
beam path in a circle equals the energy added each cycle. More powerful accelerators are built by using large radius 
paths and by using more numerous and more powerful microwave cavities to accelerate the particle beam between 
corners. Lighter particles (such as electrons) lose a larger fraction of their energy when turning. Practically speaking, 
the energy of electron/positron accelerators is limited by this radiation loss, while it does not play a significant role 
in the dynamics of proton or ion accelerators. The energy of those is limited strictly by the strength of magnets and 
by the cost. 



Design and operation 

Particles are injected into the main ring at substantial energies by either a linear accelerator or by an intermediate 
synchrotron which is in turn fed by a linear accelerator. The "linac" is in turn fed by particles accelerated to 
intermediate energy by a simple high voltage power supply, typically a Cockcroft-Walton generator. 

Starting from an appropriate initial value determined by the injection velocity the magnetic field is then increased. 
The particles pass through an electrostatic accelerator driven by a high alternating voltage. At particle speeds not 
close to the speed of light the frequency of the accelerating voltage can be made roughly proportional to the current 
in the bending magnets. A finer control of the frequency is performed by a servo loop which responds to the 
detection of the passing of the traveling group of particles. At particle speeds approaching light speed the frequency 
becomes more nearly constant, while the current in the bending magnets continues to increase. The maximum energy 
that can be applied to the particles (for a given ring size and magnet count) is determined by the saturation of the 
cores of the bending magnets (the point at which increasing current does not produce additional magnetic field). One 
way to obtain additional power is to make the torus larger and add additional bending magnets. This allows the 
amount of particle redirection at saturation to be less and so the particles can be more energetic. Another means of 
obtaining higher power is to use superconducting magnets, these not being limited by core saturation. 



Synchrotron 



150 



Large synchrotrons 

One of the early large synchrotrons, now 
retired, is the Bevatron, constructed in 1950 
at the Lawrence Berkeley Laboratory. The 
name of this proton accelerator comes from 
its power, in the range of 6.3 GeV (then 
called BeV for billion electron volts; the 
name predates the adoption of the SI prefix 
giga-). A number of heavy elements, unseen 
in the natural world, were first created with 
this machine. This site is also the location of 
one of the first large bubble chambers used 
to examine the results of the atomic 
collisions produced here. 

Another early large synchrotron is the Cosmotron built at Brookhaven National Laboratory which reached 3.3 GeV 
in 1953 




[2] 



Until August 2008, the highest energy synchrotron in the world was the Tevatron, at the Fermi National Accelerator 
Laboratory, in the United States. It accelerates protons and antiprotons to slightly less than 1 TeV of kinetic energy 
and collides them together. The Large Hadron Collider (LHC), which has been built at the European Laboratory for 
High Energy Physics (CERN), has roughly seven times this energy (so proton-proton collisions occur at roughly 14 
TeV). It is housed in the 27 km tunnel which formerly housed the Large Electron Positron (LEP) collider, so it will 
maintain the claim as the largest scientific device ever built. The LHC will also accelerate heavy ions (such as lead) 
up to an energy of 1.15 PeV. 

The largest device of this type seriously proposed was the Superconducting Super Collider (SSC), which was to be 
built in the United States. This design, like others, used superconducting magnets which allow more intense 
magnetic fields to be created without the limitations of core saturation. While construction was begun, the project 
was cancelled in 1994, citing excessive budget overruns — this was due to naive cost estimation and economic 
management issues rather than any basic engineering flaws. It can also be argued that the end of the Cold War 
resulted in a change of scientific funding priorities that contributed to its ultimate cancellation. While there is still 
potential for yet more powerful proton and heavy particle cyclic accelerators, it appears that the next step up in 
electron beam energy must avoid losses due to synchrotron radiation. This will require a return to the linear 
accelerator, but with devices significantly longer than those currently in use. There is at present a major effort to 
design and build the International Linear Collider (ILC), which will consist of two opposing linear accelerators, one 
for electrons and one for positrons. These will collide at a total center of mass energy of 0.5 TeV. 

However, synchrotron radiation also has a wide range of applications (see synchrotron light) and many 2nd and 3rd 
generation synchrotrons have been built especially to harness it. The largest of those 3rd generation synchrotron light 
sources are the European Synchrotron Radiation Facility (ESRF) in Grenoble, France, the Advanced Photon Source 
(APS) near Chicago, USA, and SPring-8 in Japan, accelerating electrons up to 6, 7 and 8 GeV, respectively. 

Synchrotrons which are useful for cutting edge research are large machines, costing tens or hundreds of millions of 
dollars to construct, and each beamline (there may be 20 to 50 at a large synchrotron) costs another two or three 
million dollars on average. These installations are mostly built by the science funding agencies of governments of 
developed countries, or by collaborations between several countries in a region, and operated as infrastructure 
facilities available to scientists from universities and research organisations throughout the country, region, or world. 
More compact models, however, have been developed, such as the Compact Light Source. 



Synchrotron 



151 



List of installations 



Synchrotron 


Location & Country 


Energy 

(GeV) 


Circumference 

(m) 


Commissioned 


Decommissioned 


Advanced Photon Source (APS) 


Argonne National Laboratory, USA 


7.0 


1104 


1995 




ALBA 


Cerdanyola del Valles near Barcelona, 
Spain 


3 


270 


2010 




ISIS 


Rutherford Appleton Laboratory, UK 


0.8 


163 


1985 




Australian Synchrotron 


Melbourne, Australia 


3 


216 


2006 




ANKA 


Karlsruhe Institute of Technology, 
Germany 


2.5 


110.4 


2000 




LNLS 


Campinas, Brazil 


1.37 


93.2 


1997 




SESAME 


Allaan, Jordan 


2.5 


125 


Under Design 




Bevatron 


Lawrence Berkeley Laboratory, USA 


6 


114 


1954 


1993 


Advanced Light Source 


Lawrence Berkeley Laboratory, USA 


1.9 


196.8 


1993 




Cosmotron 


Brookhaven National Laboratory, USA 


3 


72 


1953 


1968 


National Synchrotron Light 
Source 


Brookhaven National Laboratory, USA 


2.8 


170 


1982 




Nimrod 


Rutherford Appleton Laboratory, UK 


7 




1957 


1978 


Alternating Gradient Synchrotron 
(AGS) 


Brookhaven National Laboratory, USA 


33 


800 


1960 




Stanford Synchrotron Radiation 
Lightsource 


SLAC National Accelerator Laboratory, 
USA 


3 


234 


1973 




Synchrotron Radiation Center 
(SRC) 


Madison, USA 


1 


121 


1968 




Cornell High Energy Synchrotron 
Source (CHESS) 


Cornell University, USA 


5.5 


768 


1979 




Soleil 


Paris, France 


3 


354 


2006 




Shanghai Synchrotron Radiation 
Facility (SSRF) 


Shanghai, China 


3.5 


432 


2007 




Proton Synchrotron 


CERN, Switzerland 


28 


628.3 


1959 




Tevatron 


Fermi National Accelerator Laboratory, 
USA 


1000 


6300 


1983 




Swiss Light Source 


Paul Scherrer Institute, Switzerland 


2.8 


288 


2001 




Large Hadron Collider (LHC) 


CERN, Switzerland 


7000 


26659 


2008 




BESSY II 


Helmholtz-Zentrum Berlin in Berlin, 
Germany 


1.7 


240 


1998 




European Synchrotron Radiation 
Facility (ESRF) 


Grenoble, France 


6 


844 


1992 




MAX-I 


MAX-lab, Sweden 


0.55 


30 


1986 




MAX-II 


MAX-lab, Sweden 


1.5 


90 


1997 




MAX-III 


MAX-lab, Sweden 


0.7 


36 


2008 




ELETTRA 


Trieste, Italy 


2-2.4 


260 


1993 




Synchrotron Radiation Source 


Daresbury Laboratory, UK 


2 


96 


1980 


2008 



Synchrotron 



152 



ASTRID 


Aarhus University, Denmark 


0.58 


40 


1991 




Diamond Light Source 


Oxfordshire, UK 


3 


561.6 


2006 




DORIS III 


DESY, Germany 


4.5 


289 


1980 




PETRA II 


DESY, Germany 


12 


2304 


1995 


2007 


PETRA III 


DESY, Germany 


6.5 


2304 


2009 




Canadian Light Source 


University of Saskatchewan, Canada 


2.9 


171 


2002 




SPring-8 


RIKEN, Japan 


8 


1436 


1997 




KEK 


Tsukuba, Japan 


12 








National Synchrotron Radiation 
Research Center 


Hsinchu Science Park, Taiwan 


3.3 


518.4 


2008 




Synchrotron Light Research 
Institute (SLRI) 


Nakhon Ratchasima, Thailand 


1.2 


81.4 


2004 




Indus 1 


Raja Ramanna Centre for Advanced 
Technology, Indore, India 


0.45 


18.96 


1999 




Indus 2 


Raja Ramanna Centre for Advanced 
Technology, Indore, India 


2.5 


36 


2005 




Synchrophasotron 


JINR, Dubna, Russia 


10 


180 


1957 


2005 


U-70 synchrotron 


IHEP, Protvino, Russia 


70 




1967 




CAMD 


LSU, Louisiana, US 


1.5 


- 


- 




PLS 


PAL, Pohang, Korea 


2.5 


280.56 


1994 





Note: in the case of colliders, the quoted energy is often double what is shown here. The above table shows the 
energy of one beam but if two opposing beams collide head on, the centre of mass energy is double the beam 
energy shown. 



Applications 



Life sciences: protein and large molecule crystallography 

LIGA based microfabrication 

Drug discovery and research 

"Burning" computer chip designs into metal wafers 

Studying molecule shapes and protein crystals 

Analysing chemicals to determine their composition 

Observing the reaction of living cells to drugs 

Inorganic material crystallography and microanalysis 

Fluorescence studies 

Semiconductor material analysis and structural studies 

Geological material analysis 

Medical imaging 

Proton therapy to treat some forms of cancer 



Synchrotron 153 

See also 

• List of synchrotron radiation facilities 

• Synchrotron X-ray tomographic microscopy 

• Energy amplifier 

• Superconducting Radio Frequency 

References 

[1] Nature 407, 468 (28 September 2000) (http://www.nature.com/nature/journal/v407/n6803/full/407468a0.html). 
[2] The Cosmotron (http://www.bnl.gov/bnlweb/history/cosmotron.asp) 

External links 

Canadian Light Source (http://www.lightsource.ca) 

Australian Synchrotron (http://www.synchrotron.org.au) 

Diamond UK Synchrotron (http://www.diamond.ac.uk) 

Lightsources.org (http://www.lightsources.org/cms/) 

CERN Large Hadron Collider (http://lhc-new-homepage.web.cern.ch/lhc-new-homepage) 

Synchrotron Light Sources of the World (http://www-als.lbl.gov/als/synchrotron_sources.html) 

A Miniature Synchrotron: (http://www.technologyreview.com/Biotech/20149/) room-size synchrotron offers 

scientists a new way to perform high-quality x-ray experiments in their own labs, Technology Review, February 

04, 2008 

Brazilian Synchrotron Light Laboratory (http://www.lnls.br/lnls/cgi/cgilua.exe/sys/start. 

htm?UserActiveTemplate=lnls_2007_english&tpl=home) 

Podcast interview (http://omegataupodcast.net/2009/03/28/ll-synchrotron-radiation-science-at-esrf/) with a 

scientist at the European Synchrotron Radiation Facility 

Indian SRS (http://www.cat.gov.in/index.html) 



X-ray microscope 



154 



X-ray microscope 



An X-ray microscope uses electromagnetic radiation in the soft X-ray band to produce images of very small objects. 

Unlike visible light, X-rays do not reflect or refract easily, and they are invisible to the human eye. Therefore the 
basic process of an X-ray microscope is to expose film or use a charge-coupled device (CCD) detector to detect 
X-rays that pass through the specimen. It is a contrast imaging technology using the difference in absorption of soft 
x-ray in the water window region (wavelength region: 2.3 - 4.4 nm, photon energy region: 0.28 - 0.53 keV) by the 
carbon atom (main element composing the living cell) and the oxygen atom (main element for water). 

Early X-ray microscopes by Paul Kirkpatrick and Albert Baez used grazing-incidence reflective optics to focus the 
X-rays, which grazed X-rays off parabolic curved mirrors at a very high angle of incidence. An alternative method of 
focusing X-rays is to use a tiny fresnel zone plate of concentric gold or nickel rings on a silicon dioxide substrate. Sir 
Lawrence Bragg produced some of the first usable X-ray images with his apparatus in the late 1940's. 



In the 1950's Newberry produced a shadow X-ray microscope 
which placed the specimen between the source and a target plate, 
this became the basis for the first commercial X-ray microscopes 
from the General Electric Company. 

The Advanced Light Source (ALS)[1] in Berkeley CA is home to 
XM-1 (http://www.cxro.lbl.gov/BL612/), a full field soft 
X-ray microscope operated by the Center for X-ray Optics [2] and 
dedicated to various applications in modern nanoscience, such as 
nanomagnetic materials, environmental and materials sciences and 
biology. XM-1 uses an X-ray lens to focus X-rays on a CCD, in a 
manner similar to an optical microscope. XM-1 still holds the 
world record in spatial resolution with Fresnel zone plates down to 
15nm and is able to combine high spatial resolution with a 
sub-lOOps time resolution to study e.g. ultrafast spin dynamics. 




Indirect drive laser inertial confinement fusion uses a 

"hohlraum" which is irradiated with laser beam cones 

from either side on it its inner surface to bathe a fusion 

microcapsule inside with smooth high intensity X-rays. 

The highest energy X-rays which penetrate the 

hohlraum can be visualized using an X-ray microscope 

such as here, where X-radiation is represented in 

orange/red. 



The ALS is also home to the world's first soft x-ray microscope 

designed for biological and biomedical research. This new 

instrument, XM-2 was designed and built by scientists from the National Center for X-ray Tomography (http://ncxt. 

lbl.gov).XM-2 is capable of producing 3-Dimensional tomograms of cells. 

Sources of soft X-rays suitable for microscopy, such as synchrotron radiation sources, have fairly low brightness of 
the required wavelengths, so an alternative method of image formation is scanning transmission soft X-ray 
microscopy. Here the X-rays are focused to a point and the sample is mechanically scanned through the produced 
focal spot. At each point the transmitted X-rays are recorded with a detector such as a proportional counter or an 
avalanche photodiode. This type of Scanning Transmission X-ray Microscope (STXM) was first developed by 
researchers at Stony Brook University and was employed at the National Synchrotron Light Source at Brookhaven 
National Laboratory. 

The resolution of X-ray microscopy lies between that of the optical microscope and the electron microscope. It has 
an advantage over conventional electron microscopy in that it can view biological samples in their natural state. 
Electron microscopy is widely used to obtain images with nanometer level resolution but the relatively thick living 
cell cannot be observed as the sample has to be chemically fixed, dehydrated, embedded in resin, then sliced ultra 
thin. However, it should be mentioned that cryo-electron microscopy allows the observation of biological specimens 
in their hydrated natural state, albeit embedded in water ice. Until now, resolutions of 30 nanometer are possible 
using the Fresnel zone plate lens which forms the image using the soft x-rays emitted from a synchrotron. Recently, 
the use of soft x-rays emitted from laser-produced plasmas rather than synchrotron radiation is becoming more 



X-ray microscope 



155 



popular. 

Additionally, X-rays cause fluorescence in most materials, and these emissions can be analyzed to determine the 
chemical elements of an imaged object. Another use is to generate diffraction patterns, a process used in X-ray 
crystallography. By analyzing the internal reflections of a diffraction pattern (usually with a computer program), the 
three-dimensional structure of a crystal can be determined down to the placement of individual atoms within its 
molecules. X-ray microscopes are sometimes used for these analyses because the samples are too small to be 
analyzed in any other way. 



See also 

• Synchrotron X-ray tomographic microscopy 

• Electron microscope 

External links 

• Application of X-ray microscopy in analysis of 
living hydrated cells 

• Hard X-ray microbeam experiments with a 
sputtered-sliced Fresnel zone plate and its 



applications 



[4] 



Scientific applications of soft x-ray microscopy 



[5] 




A square beryllium foil mounted in a steel case to be used as a 

window between a vacuum chamber and an X-ray microscope. 

Beryllium, due to its low Z number is highly transparent to X-rays. 



References 

[1] http://www-als.lbl.gov 

[2] http://www.cxro.lbl.gov 

[3] http://www.ncbi. nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12379938 

[4] http://www.ncbi. nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=l 1972376 

[5] http://www.cxro.lbl.gov/BL612/index.php?content=research.html 



Field emission microscope 



156 



Field emission microscope 



Field emission microscopy (FEM) is an analytical technique used in materials science to investigate molecular 
surface structures and their electronic properties. Invented by Erwin Wilhelm Miiller in 1936, the FEM was one of 
the first surface analysis instruments that approached near-atomic resolution. 

Introduction 

Microscopy techniques are used to produce real space magnified images of a surface showing what it looks like. In 
general microscopy information concerns surface crystallography (i.e. how the atoms are arranged at the surface, 
surface morphology (i.e. the shape and size of topographic features making the surface), and surface composition 
(the elements and compounds the surface is composed of). 

Field emission microscopy (FEM) was invented by Erwin Miiller in 1936. In FEM, the phenomenon of field electron 
emission was used to obtain an image on the detector on the basis of the difference in work function of the various 
crystallographic planes on the surface. 



Design 

A Field Emission Microscope consists of a metallic sample in the form of a sharp tip and a conducting fluorescent 
screen enclosed in ultrahigh vacuum. The tip radius used is typically of the order of 100 nm. The sample is held at a 
large negative potential (1-10 kV) relative to the fluorescent screen. This gives the electric field near the tip apex to 
be the order of 10 V/m which is high enough for field emission of electrons to take place. Fig.l shows the 
experimental set up for FEM. 

The field emitted electrons travel along the field lines and produce 
bright and dark patches on the fluorescent screen giving a one-to-one 
correspondence with the crystal planes of the hemispherical emitter. 
The emission current varies strongly with the local work function in 
accordance with the Fowler-Nordheim equation; hence, the FEM 
image displays the projected work function map of the emitter surface. 
The closely packed faces have higher work functions than atomically 
rough regions and thus they show up in the image as dark spots on the 
brighter background. In short, the work function anisotropy of the 
crystal planes is mapped onto the screen as intensity variations. 

The magnification is given by the ratio M = LI R , where J{ is the tip apex radius and £ is the tip-screen 
distance. Linear magnifications of about 10 to 10 are attained. The spatial resolution of this technique is of the 
order of 2 nm and is limited by the momentum of the emitted electrons parallel to the tip surface, which is of the 
order of the Fermi velocity of the electron in metal. 

It is possible to set up an FEM with a probe hole in the phosphor screen and a Faraday cup collector behind it to 
collect the current emitted from a single plane. This technique allows the measurement of the variation of work 
function with orientation for a wide variety of orientations on a single sample. The FEM has also been used to study 
adsorption and surface diffusion processes, making use of the work function change associated with the adsorption 
process. 

Field emission requires a very good vacuum, and often, even in ultra high vacuum (UHV), emission is not due to the 
clean surface. A typical field emitter needs to be 'flashed' to clean it, usually by passing a current through a loop on 
which it is mounted. After flashing the emission current is high but unstable. The current decays with time and in the 
process becomes more stable due to the contamination of the tip, either from the vacuum, or more often from 




Field emission microscope 



157 



diffusion of adsorbed surface species to the tip. Thus the real nature of the FEM tips during use is somewhat 
unknown. 

Application of FEM is limited by the materials which can be fabricated in the shape of a sharp tip, can be used in a 
UHV environment, and can tolerate the high electrostatic fields. For these reasons, refractory metals with high 
melting temperature (for e.g. W, Mo, Pt, Ir) are conventional objects for FEM experiments. 

See also 

• Atom Probe 

• Electron microscope 

• Field ion microscope 

• List of surface analysis methods 

References 

[1] "Intro to Field Emission" (http://physics.unipune.ernet.in/~fem/intro-fem.htm). Field Emission / Ion Microscopy Laboratory, Purdue 
University, Dept. of Physics. . Retrieved 2007-05-10. 

• 2. K.Oura, V.G.Lifshits, A.ASaranin, A.V.Zotov and M.Katayama, Surface Science — An Introduction, 
(Springer- Verlag Berlin Heidelberg 2003). 

• 3. John B. Hudson, Surface Science - An Introduction, (BUTTERWORTH-Heinemann 1992). 



Scanning tunneling microscope 



A scanning tunneling microscope (STM) is an instrument for 
imaging surfaces at the atomic level. Its development in 1981 earned 
its inventors, Gerd Binnig and Heinrich Rohrer (at IBM Zurich), the 
Nobel Prize in Physics in 1986. For an STM, good resolution is 

considered to be 0.1 nm lateral resolution and 0.01 nm depth 
resolution. With this resolution, individual atoms within materials 
are routinely imaged and manipulated. The STM can be used not only 
in ultra high vacuum but also in air, water, and various other liquid or 
gas ambients, and at temperatures ranging from near zero kelvin to a 



few hundred degrees Celsius 



[4] 



The STM is based on the concept of quantum tunneling. When a 
conducting tip is brought very near to the surface to be examined, a 
bias (voltage difference) applied between the two can allow electrons 
to tunnel through the vacuum between them. The resulting tunneling 
current is a function of tip position, applied voltage, and the local 
density of states (LDOS) of the sample. Information is acquired by 
monitoring the current as the tip's position scans across the surface, and 
is usually displayed in image form. STM can be a challenging 
technique, as it requires extremely clean and stable surfaces, sharp tips, 
excellent vibration control, and sophisticated electronics. 




Image of reconstruction on a clean Gold(100) 
surface 






An STM image of single-walled carbon nanotube 



Scanning tunneling microscope 



158 




A close-up of a simple scanning tunneling microscope head using a 
platinum-iridium stylus. 



Procedure 

First, a voltage bias is applied and the tip is brought 
close to the sample by some coarse sample-to-tip 
control, which is turned off when the tip and sample are 
sufficiently close. At close range, fine control of the tip 
in all three dimensions when near the sample is 
typically piezoelectric, maintaining tip-sample 
separation W typically in the 4-7 A range, which is the 

equilibrium position between attractive (3<W<10A) 

° 141 

and repulsive (W<3A) interactions . In this situation, 

the voltage bias will cause electrons to tunnel between 

the tip and sample, creating a current that can be 

measured. Once tunneling is established, the tip's bias 

and position with respect to the sample can be varied 

(with the details of this variation depending on the 

experiment) and data is obtained from the resulting 

changes in current. 

If the tip is moved across the sample in the x-y plane, the changes in surface height and density of states cause 

changes in current. These changes are mapped in images. This change in current with respect to position can be 

141 
measured itself, or the height, z, of the tip corresponding to a constant current can be measured . These two modes 

are called constant height mode and constant current mode, respectively. In constant current mode, feedback 

electronics adjust the height by a voltage to the piezoelectric height control mechanism . This leads to a height 

variation and thus the image comes from the tip topography across the sample and gives a constant charge density 

surface; this means contrast on the image is due to variations in charge density . In constant height mode, the 

voltage and height are both held constant while the current changes to keep the voltage from changing; this leads to 

an image made of current changes over the surface, which can be related to charge density . The benefit to using a 

constant height mode is that it is faster, as the piezoelectric movements require more time to register the change in 

constant current mode than the voltage response in constant height mode . All images produced by STM are 

grayscale, with color optionally added in post-processing in order to visually emphasize important features. 

In addition to scanning across the sample, information on the electronic structure at a given location in the sample 
can be obtained by sweeping voltage and measuring current at a specific location . This type of measurement is 
called scanning tunneling spectroscopy (STS) and typically results in a plot of the local density of states as a function 
of energy within the sample. The advantage of STM over other measurements of the density of states lies in its 
ability to make extremely local measurements: for example, the density of states at an impurity site can be compared 



[7] 

Framerates of at least 1 Hz enable so called Video-STM (up to 50 Hz is possible). This can be used to scan 



to the density of states far from impurities 

Framerates of at lea; 
surface diffusion. 



Scanning tunneling microscope 



159 



Instrumentation 

The components of an STM include 
scanning tip, piezoelectric controlled 
height and x,y scanner, coarse 
sample-to-tip control, vibration 
isolation system, and computer . 

The resolution of an image is limited 
by the radius of curvature of the 
scanning tip of the STM. Additionally, 
image artifacts can occur if the tip has 
two tips at the end rather than a single 
atom; this leads to "double-tip 
imaging," a situation in which both tips 
contribute to the tunneling 
Therefore it has been essential to 
develop processes for consistently 
obtaining sharp, usable tips. Recently, 
carbon nanotubes have been used in 



this instance 



[11] 



:u:p| Control voltages for piezotube 



Distance control 
and scanning unit 




J— Tunneling s 

I voltage 



Data processing 
and display 



Schematic view of an STM 



[3] 



The tip is often made of tungsten or platinum-iridium, though gold is also used . Tungsten tips are usually made by 

T31 
electrochemical etching, and platinum-iridium tips by mechanical shearing . 

Due to the extreme sensitivity of tunnel current to height, proper vibration isolation or an extremely rigid STM body 
is imperative for obtaining usable results. In the first STM by Binnig and Rohrer, magnetic levitation was used to 
keep the STM free from vibrations; now mechanical spring or gas spring systems are often used . Additionally, 
mechanisms for reducing eddy currents are sometimes implemented. 

Maintaining the tip position with respect to the sample, scanning the sample and acquiring the data is computer 
controlled . The computer may also be used for enhancing the image with the help of image processing as 

well as performing quantitative measurements 



[14] 



Other STM related studies 



Many other microscopy techniques have been developed based upon 
STM. These include photon scanning microscopy (PSTM), which uses 

[3] 

an optical tip to tunnel photons ; scanning tunneling potentiometry 

[3] 

(STP), which measures electric potential across a surface ; spin 
polarized scanning tunneling microscopy (SPSTM), which uses a 
ferromagnetic tip to tunnel spin-polarized electrons into a magnetic 
sample, and atomic force microscopy (AFM), in which the force 
caused by interaction between the tip and sample is measured. 

Other STM methods involve manipulating the tip in order to change 
the topography of the sample. This is attractive for several reasons. 
Firstly the STM has an atomically precise positioning system which 
allows very accurate atomic scale manipulation. Furthermore, after the 




Nanomanipulation via STM of a self-assembled 
organic semiconductor monolayer (here: PTCDA 
molecules) on graphite, in which the logo of the 
Center for NanoScience (CeNS), LMU has been 
written. 



Scanning tunneling microscope 160 

surface is modified by the tip, it is a simple matter to then image with the same tip, without changing the instrument. 
IBM researchers developed a way to manipulate Xenon atoms absorbed on a nickel surface This technique has 
been used to create electron "corrals" with a small number of adsorbed atoms, which allows the STM to be used to 
observe electron Friedel oscillations on the surface of the material. Aside from modifying the actual sample surface, 
one can also use the STM to tunnel electrons into a layer of E-Beam photoresist on a sample, in order to do 
lithography. This has the advantage of offering more control of the exposure than traditional Electron beam 
lithography. Another practical application of STM is atomic deposition of metals (Au, Ag, W, etc.) with any desired 
(pre-programmed) pattern, which can be used as contacts to nanodevices or as nanodevices themselves. 

Recently groups have found they can use the STM tip to rotate individual bonds within single molecules. The 
electrical resistance of the molecule depends on the orientation of the bond, so the molecule effectively becomes a 
molecular switch. 

Principle of operation 

Tunneling is a functioning concept that arises from quantum mechanics. Classically, an object hitting an 
impenetrable barrier will not pass through. In contrast, objects with a very small mass, such as the electron, have 
wavelike characteristics which permit such an event, referred to as tunneling. 

Electrons behave as beams of energy, and in the presence of a potential U(z), assuming 1 -dimensional case, the 
energy levels ip (z) of the electrons are given by solutions to Schrodinger's equation, 

h 2 d^ n {z) 



2m dz 2 



+ U(z)4> n {z) = E$ n (z) 



where h is the reduced Planck s constant, z is the position, and m is the mass of an electron . If an electron of 
energy E is incident upon an energy barrier of height U(z), the electron wave function is a traveling wave solution, 

where 

y/2m(E - U(z)) 
k = 

h 

mi 
if E > U(z), which is true for a wave function inside the tip or inside the sample . Inside a barrier, E < U(z) so the 

wave functions which satisfy this are decaying waves, 

iP n {z) = ^n(0)e ±KZ , 
where 



k = 



2m(U - E) 



h 

quantifies the decay of the wave inside the barrier, with the barrier in the +z direction for — k . 

Knowing the wave function allows one to calculate the probability density for that electron to be found at some 
location. In the case of tunneling, the tip and sample wave functions overlap such that when under a bias, there is 
some finite probability to find the electron in the barrier region and even on the other side of the barrier . Let us 
assume the bias is V and the barrier width is W. This probability, P, that an electron at z=0 (left edge of barrier) can 
be found at z=W (right edge of barrier) is proportional to the wave function squared, 

Poc|^(0)| 2 e- 2 ^ [4] . 
If the bias is small, we can let U - E ~ q>M in the expression for k, where q>M, the work function, gives the minimum 
energy needed to bring an electron from an occupied level, the highest of which is at the Fermi level (for metals at 
T=0 kelvins), to vacuum level. When a small bias V is applied to the system, only electronic states very near the 
Fermi level, within eV (a product of electron charge and voltage, not to be confused here with electronvolt unit), are 



Scanning tunneling microscope 161 

excited . These excited electrons can tunnel across the barrier. In other words, tunneling occurs mainly with 
electrons of energies near the Fermi level. 

However, tunneling does require that there is an empty level of the same energy as the electron for the electron to 

tunnel into on the other side of the barrier. It is because of this restriction that the tunneling current can be related to 

the density of available or filled states in the sample. The current due to an applied voltage V (assume tunneling 

occurs sample to tip) depends on two factors: 1) the number of electrons between E and eV in the sample, and 2) the 

mi 
number among them which have corresponding free states to tunnel into on the other side of the barrier at the tip . 

The higher density of available states the greater the tunneling current. When V is positive, electrons in the tip tunnel 

into empty states in the sample; for a negative bias, electrons tunnel out of occupied states in the sample into the 

tip [4] . 

Mathematically, this tunneling current is given by 

iot £ ivao)i 2 e- 2 ^. 

E f -eV 

One can sum the probability over energies between E - eV and eV to get the number of states available in this 
energy range per unit volume, thereby finding the local density of states (LDOS) near the Fermi level . The LDOS 
near some energy E in an interval e is given by 

6 E-e 

and the tunnel current at a small bias V is proportional to the LDOS near the Fermi level, which gives important 

[41 
information about the sample . It is desirable to use LDOS to express the current because this value does not 

[41 
change as the volume changes, while probability density does . Thus the tunneling current is given by 



I^Vp s (0,E f )e 



-2kW 



[41 

where p (0,E ) is the LDOS near the Fermi level of the sample at the sample surface . This current can also be 
expressed in terms of the LDOS near the Fermi level of the sample at the tip surface, 

IcxV Ps (W,E f )V 

The exponential term in the above equations means that small variations in W greatly influence the tunnel current. If 
the separation is decreased by 1 A, the current increases by an order of magnitude, and vice versa. 

This approach fails to account for the rate at which electrons can pass the barrier. This rate should affect the tunnel 

current, so it can be treated using the Fermi's golden rule with the appropriate tunneling matrix element. John 

Bardeen solved this problem in his study of the metal-insulator-metal junction. He found that if he solved 

Schrodinger's equation for each side of the junction separately to obtain the wave functions ajj and x for each 

[41 
electrode, he could obtain the tunnel matrix, M, from the overlap of these two wave functions . This can be applied 

to STM by making the electrodes the tip and sample, assigning \p and x as sample and tip wave functions, 

respectively, and evaluating M at some surface S between the metal electrodes, where z=0 at the sample surface and 

[41 
z=W at the tip surface . 

Now, Fermi's Golden Rule gives the rate for electron transfer across the barrier, and is written 

2tt, 

T 

[41 
where 5(E — E ) restricts tunneling to occur only between electron levels with the same energy . The tunnel matrix 

element, given by 

M = — / (x * — - ^—-)dS , 
2vr Jz=zo oz az 

is a description of the lower energy associated with the interaction of wave functions at the overlap, also called the 

[4] 

resonance energy . 



w = Z_\ M \H( ElP - E x ), 



Scanning tunneling microscope 162 

Summing over all the states gives the tunneling current as 

[41 
where / is the Fermi function, p and p are the density of states in the sample and tip, respectively . The Fermi 

distribution function describes the filling of electron levels at a given temperature T. 

Early invention 

ri7i 
An earlier, similar invention, the Topografiner of R. Young, J. Ward, and F. Scire from the NIST , relied on field 

emission. However, Young is credited by the Nobel Committee as the person who realized that it should be possible 

MO] 

to achieve better resolution by using the tunnel effect. 

See also 

• Microscopy 

• Scanning probe microscopy 

• Scanning tunneling spectroscopy 

• Electrochemical scanning tunneling microscope 

• Atomic force microscope 

• Electron microscope 

• Spin polarized scanning tunneling microscopy 

References 

[I] G. Binnig, H. Rohrer (1986). "Scanning tunneling microscopy". IBM Journal of Research and Development 30: 4. 

[2] Press release for the 1986 Nobel Prize in physics (http://nobelprize.org/nobel_prizes/physics/laureates/1986/press.html) 

[3] C. Bai (2000). Scanning tunneling microscopy and its applications (http://books. google. com/?id=3Q08jRmmtrkC&pg=PA345). New 

York: Springer Verlag. ISBN 3540657150. . 
[4] C. Julian Chen (1993). Introduction to Scanning Tunneling Microscopy (http://www.columbia.edu/~jcc2161/documents/stm_R.pdf). 

Oxford University Press. ISBN 0195071506. . 
[5] K. Oura, V. G. Lifshits, A. A. Saranin, A. V. Zotov, and M. Katayama (2003). Surface science: an introduction (http://books.google.com/ 

?id=TTPMbOGqF-YC&pg=PPl). Berlin: Springer- Verlag. ISBN 3540005455. . 
[6] D. A. Bonnell and B. D. Huey (2001). "Basic principles of scanning probe microscopy". In D. A. Bonnell. Scanning probe microscopy and 

spectroscopy: Theory, techniques, and applications (2 ed.). New York: Wiley-VCH. ISBN 047124824X. 
[7] Pan, S. H.; Hudson, EW; Lang, KM; Eisaki, H; Uchida, S; Davis, JC (2000). "Imaging the effects of individual zinc impurity atoms on 

superconductivity in Bi2Sr2CaCu208+delta". Nature 403 (6771): 746-750. doi:10.1038/35001534. PMID 10693798. 
[8] G. Schitter, M. J. Rost (2008). "Scanning probe microscopy at video-rate" (http://www.materialstoday.com/view/2194/ 

scanning-probe-microscopy-at-videorate/) (PDF). Materials Today (UK: Elsevier) 11 (special issue): 40—48. 

doi:10.1016/S1369-7021(09)70006-9. ISSN 1369-7021. . 
[9] R. V. Lapshin, O. V. Obyedkov (1993). "Fast-acting piezoactuator and digital feedback loop for scanning tunneling microscopes" (http:// 

www.nanoworld.org/homepages/lapshin/publications.htm#fastl993) (PDF). Review of Scientific Instruments 64 (10): 2883—2887. 

doi: 10.1063/1.1144377.. 
[10] B. S. Swartzentruber (1996). "Direct measurement of surface diffusion using atom-tracking scanning tunneling microscopy". Physical 

Review Letters 76 (3): 459-462. doi: 10.1 103/PhysRevLett. 76.459. PMID 10061462. 

[II] "STM carbon nanotube tips fabrication for critical dimension measurements". Sensors and Actuators A 123-124: 655. 2005. 
doi:10.1016/j.sna.2005.02.036. 

[12] R. V. Lapshin (1995). "Analytical model for the approximation of hysteresis loop and its application to the scanning tunneling microscope" 

(http://www.nanoworld.Org/homepages/lapshin/publications.htm#analyticall995) (PDF). Review of Scientific Instruments 66 (9): 

4718-4730. doi: 10. 1063/1. 11453 14. . ( is available). 
[13] R. V. Lapshin (2007). "Automatic drift elimination in probe microscope images based on techniques of counter-scanning and topography 

feature recognition" (http://www.nanoworld.Org/homepages/lapshin/publications.htm#automatic2007) (PDF). Measurement Science and 

Technology 18 (3): 907-927. doi:10.1088/0957-0233/18/3/046. . 
[14] R. V. Lapshin (2004). "Feature-oriented scanning methodology for probe microscopy and nanotechnology" (http://www.nanoworld.org/ 

homepages/lapshin/publications.htm#feature2004) (PDF). Nanotechnology 15 (9): 1135-1151. doi: 10. 1088/0957-4484/15/9/006. . 
[15] R. Wiesendanger, I. V. Shvets, D. Burgler, G. Tarrach, H.-J. Guntherodt, and J.M.D. Coey (1992). "Recent advances in spin-polarized 

scanning tunneling microscopy". Ultramicroscopy 42-44: 338. doi:10.1016/0304-3991(92)90289-V. 



Scanning tunneling microscope 163 

[16] J. Bardeen (1961). "Tunneling from a many particle point of view". Phys. Rev. Lett. 6 (2): 57—59. doi: 10.1 103/PhysRevLett.6.57. 

[17] R. Young, J. Ward, F. Scire (1972). "The Topografiner: An Instrument for Measuring Surface Topography" (http://www.nanoworld.org/ 

museum/young2.pdf). Rev. Sci. lustrum. 43: 999. doi:10.1063/l. 1685846. . 
[18] "The Topografiner: An Instrument for Measuring Surface Microtopography" (http://nvl.nist.gov/pub/nistpubs/sp958-lide/214-218.pdf). 

NIST. . 

Further reading 

• Tersoff, J.: Hamann, D. R.: Theory of the scanning tunneling microscope, Physical Review B 31, 1985, p. 805 - 
813 (http://dx.doi.org/10.1103/PhysRevB.31.805). 

• Bardeen, J.: Tunnelling from a many-particle point of view, Physical Review Letters 6 (2), 1961, p. 57-59 (http:// 
dx.doi.org/ 10. 1 103/PhysRevLett.6.57). 

• Chen, C. J.: Origin of Atomic Resolution on Metal Surfaces in Scanning Tunneling Microscopy, Physical Review 
Letters 65 (4), 1990, p. 448-451 (http://dx.doi.org/10.1103/PhysRevLett.65.448) 

• G. Binnig, H. Rohrer, Ch. Gerber, and E. Weibel, Phys. Rev. Lett. 50, 120 - 123 (1983) (http://dx.doi.org/10. 
1 103/PhysRevLett.50. 120) 

• G Binnig, H. Rohrer, Ch. Gerber, and E. Weibel, Phys. Rev. Lett. 49, 57 - 61 (1982) (http://dx.doi.org/10. 
1103/PhysRevLett.49.57) 

• G Binnig, H. Rohrer, Ch. Gerber, and E. Weibel, Appl. Phys. Lett., Vol. 40, Issue 2, pp. 178-180 (1982) (http:// 
dx. doi. org/ 10.1 063/ 1 .92999) 

• R. V. Lapshin, Feature-oriented scanning methodology for probe microscopy and nanotechnology, 
Nanotechnology, volume 15, issue 9, pages 1135-1151, 2004 (http://stacks.iop.org/Nano/15/1135) 

• D. Fujita and K. Sagisaka, Topical review: Active nanocharacterization of nanofunctional materials by scanning 
tunneling microscopy Sci. Technol. Adv. Mater. 9, 013003(9pp) (2008) (http://dx.doi.org/10.1088/ 
1468-6996/9/1/013003) (free download). 

• Roland Wiesendanger (1994). Scanning probe microscopy and spectroscopy: methods and applications (http:// 
books. google. com/?id=EXae0pjS2vwC&printsec=frontcover). Cambridge University Press. 

ISBN 0521428475. 

• Theory ofSTM and Related Scanning Probe Methods. Springer Series in Surface Sciences, Band 3. Springer, 
Berlin 1998 

External links 

• A microscope is filming a microscope (http://www.fz-juelich.de/ibn/microscope_e) (Mpeg, AVI movies) 

• Zooming into the NanoWorld (http://www.nano.geo.uni-muenchen.de/SW/images/zoom.html) (Animation 
with measured STM images) 

• NobelPrize.org website about STM (http://nobelprize.org/educational_games/physics/microscopes/scanning/ 
index.html), including an interactive STM simulator. 

• SPM - Scanning Probe Microscopy Website (http://www.mobot.org/jwcross/spm/) 

• STM Image Gallery at IBM Almaden Research Center (http://www.almaden.ibm.com/vis/stm/gallery.html) 

• STM Gallery at Vienna University of technology (http://www.iap.tuwien.ac.at/www/surface/STM_Gallery/ 

) 

• Build a simple STM with a cost of materials less than $100.00 excluding oscilloscope (http://www.geocities. 
com/spm_stm/Project.html) 

• Nanotimes Simulation engine download page (http://www.nanotimes-corp.com/content/view/22/38/) 

• Structure and Dynamics of Organic Nanostructures discovered by STM (http://www.uni-ulm.de/~hhoster/ 
personal/self_assembly . htm) 

• Metal organic coordination networks of oligopyridines and Cu on graphite investigated by STM (http://www. 
uni-ulm.de/~hhoster/personal/metal_organic.htm) 



Scanning tunneling microscope 164 

• Surface Alloys discovered by STM (http://www.uni-ulm.de/~hhoster/personal/surface_alloys.html) 

• Animated illustration of tunneling and STM (http://molecularmodelingbasics.blogspot.com/2009/09/ 
tunneling-and-stm. html) 

• 60 second movie clip with an introduction to Scanning Tunneling Microscopy(STM) (http://nanohub.org/ 
resources/2620) 

Transmission Electron Aberration-corrected 
Microscope 

Transmission Electron Aberration-corrected Microscope or TEAM is a collaborative research project between 
four US laboratories and two companies. It is based at the Lawrence Berkeley National Laboratory in Berkeley, 
California and involves Argonne National Laboratory, Oak Ridge National Laboratory and Frederick Seitz Materials 
Research Laboratory at the University of Illinois at Urbana-Champaign, as well as FEI and CEOS companies, and is 
supported by the U.S. Department of Energy. The project's main activity is design and application of a transmission 
electron microscope (TEM) with a spatial resolution below 0.05 nanometers, which is roughly half the size of an 
atom of hydrogen. The project was started in 2004; the operational microscope was built in 2008 and achieved the 
0.05 nm resolution target in 2009. The microscope is a shared facility available to external users. 

Scientific background 

It has long been known that the best achievable spatial resolution of an optical microscope, that is the smallest 
feature it can observe, is of the order of the wavelength of the light X, which is about 550 nm for green light. One 
route to improve this resolution is to use particles with smaller X, such as high-energy electrons. Practical limitations 
set a convenient electron energy to 100-300 keV that corresponds to X = 3.7-2.0 pm. Unfortunately, the resolution of 
electron microscopes is limited not by the electron wavelength, but by intrinsic imperfections of electron lenses. 
These are referred to as spherical and chromatic aberrations because of their similarity to aberrations in optical 
lenses. Those aberrations are reduced by installing in a microscope a set of specially designed auxiliary "lenses" 
which are called aberration correctors. 

Hardware 

The TEAM is based on a commercial FEI Titan 80-300 electron microscope, which can be operated at voltages 
between 80 and 300 keV, both in TEM and STEM (that is scanning TEM) modes. To minimize the mechanical 
vibrations, the microscope is located in a separate room within a sound-proof enclosure and is operated remotely. 
The electron source is a Schottky type field emission gun with a relatively low energy spread of 0.8 eV at 300 keV. 
In order to reduce chromatic aberrations, this spread is further lowered to 0.13 eV at 300 keV and 0.08 eV at 80 kV 
using a Wien-filter type monochromator. Both the illumination lens, which is located above the sample and is 
conventionally called the condenser lens, and the collection lens (called the objective lens) are equipped with 
fifth-order spherical aberration correctors. The electrons are further energy filtered by a GIF filter and detected by a 
CCD camera. The filter makes it possible to select electrons scattered by specific chemical elements and so identify 
individual atoms in the sample being studied. 



Transmission Electron Aberration-corrected Microscope 165 

Applications 

The TEAM has been tested on various crystalline solids, resolving individual atoms in GaN ([211] orientation), 
germanium ([114]), gold ([111]) and others, and reaching the spatial resolution below 0.05 nm (about 0.045 nm). In 
the images of graphene — a single sheet of graphite — not only the atoms, but also the chemical bonds could be 
observed (see top picture). A movie has been recorded inside the microscope showing hopping of individual carbon 
atoms around a hole punched in a graphene sheet. Similar pictures, resolving carbon atoms and bonds 

between them, have been independently produced for pentacene — a planar organic molecule consisting of five 
carbon rings — using a very different microscopy technique, atomic force microscopy (AFM). In AFM, the 

atoms are probed not by electrons, but by a sharp vibrating tip. 

References 

[1] "Berkeley Scientists Produce First Live Action Movie of Individual Carbon Atoms in Action" (http://newscenter.lbl.gov/press-releases/ 

2009/03/26/atoms-in-action/). March 26, 2009. . 
[2] "The TEM project timeline" (http://ncem.lbl.gov/TEAM-project/files/when_where.html). . 
[3] H. H. Rose (2008). "Optics of high-performance electron Microscopes" (free download review on electron optics). Science and Technology of 

Advanced Materials 9: 014107. doi: 10. 1088/003 1-8949/9/1/014107. 
[4] N. Tanaka (2008). "Present status and future prospects of spherical aberration corrected TEM/STEM for study of nanomaterials" (free 

download review). Set Technol. Adv. Mater. 9: 014111. doi:10.1088/1468-6996/9/l/014111. 
[5] C. Kisielowski et al. (2008). "Detection of Single Atoms and Buried Defects in Three Dimensions by Aberration-Corrected Electron 

Microscope with 0.5-A Information Limit" (http://ncem.lbl.gov/team/TEAM pubs/MAM14-5_Kisielowski_e t_al-Author.pdf) 

(free-download pdf). Microscopy Microanalysis 14: 469-477. doi:10.1017/S1431927608080902. . 
[6] R. Erni et al. (2009). "Atomic-Resolution Imaging with a Sub-50-pm Electron Probe". Physical Review Letters 102: 096101. 

doi: 10.1 103/PhysRevLett.l02.096101. 
[7] C. O. Girit et al. (27 March 2009). "Graphene at the Edge: Stability and Dynamics". Science 323: 1705. doi:10.1126/science.H66999. 
[8] J. C. Meyer et al. (2008). "Direct Imaging of Lattice Atoms and Topological Defects in Graphene Membranes". Nano Lett. 8: 3582. 

doi:10.1021/nl801386m. 
[9] "Single molecule's stunning image" (http://news.bbc.co.Uk/2/hi/science/nature/8225491.stm). 2009-08-28. . Retrieved 2009-08-28. 
[10] L. Gross (2009). "The Chemical Structure of a Molecule Resolved by Atomic Force Microscopy". Science 325: 1110. 

doi: 10.1 126/science.ll76210. 

External links 

• TEAM Project main site (http://ncem.lbl.gov/team/TEAMpage/TEAMpage.html) 



ISIS 166 



ISIS 



Isis was a goddess in Egyptian mythology. 
Isis may also refer to: 

Aircraft 

• Integrated Standby Instrument System, used on the Airbus A320 family of airliners 

• Integrated Sensor is Structure, project to develop an airship for intelligence uses 

Automobiles 

• Morris Isis 

• Toyota Isis 

Comics 

• Isis (Bluewater Comics) 

• Isis (DC Comics) 

• Isis (Marvel Comics) 

Computing 

Image and Scanner Interface Specification, interface for image scanning technologies 

IS-IS, network routing protocol 

ISIS/Draw, chemistry modeling program 

Integrated Software for Imagers and Spectrometers, software used by the United States Geological Survey 

ISIS Modelling Software, river modelling software 

ISIS (operating system), operating system used on the Intel 8085 processor 

ISIS programming language, variant of JOSS 

Integrated Scientific Information System, software program including the MDL Chime plug-in 

Infinitely Scalable Information Storage, Avid Technology storage method known as Avid Unity ISIS, used for 

broadcasting 

ISIS, a networking tool, part of Proteus (design software) 

ISIS Papyrus, Swiss-based commercial enterprise software vendor 

International Species Information System, non-profit software developer for zoos and aquariums 

CDS/ISIS, non-numerical information storage and retrieval software developed by UNESCO 



ISIS 167 

Geography 

• The Isis, segment of the River Thames 

• Isis Highway, Australian highway 

• Shire of Isis, Queensland, Australia 

Music 

Isis (band), American post-metal band 

Isis (Brisbane band), rock band 

Isis (horn-rock band), 1970s all-female band 

Isis (Lully), opera by Jean-Baptiste Lully and Philippe Quinault 

"Isis" (song), by Bob Dylan 

"Isis," song from the album Voyage to Isis by Delta-S 

"Isis," song from the EP Is Is by The Yeah Yeah Yeahs 

Isyss, R&B group 

Organizations 

Isis Innovation, British technology transfer company 

ISIS neutron source 

Institute for Science and International Security 

Institute for the Scientific Investigation of Sexuality, former name of the Family Research Institute 

Institute for the Study of Interdisciplinary Sciences 

International Species Information System, an organization which maintains a database on zoo animal populations 

The Institute of Science in Society, an organization providing scientific information on ecological sustainability 

ISIS (mobile payment system), a joint venture between AT&T, Verizon Wireless and T-Mobile, in the mobile 

payments industry 

Outer space 

• Isis (lunar crater) 

• ISIS (satellite) 

• 42 Isis, an asteroid 

Publications 

• Isis (journal), academic journal 

• Isis magazine, student magazine at Oxford University 



ISIS 168 

Ships 

• HMS Isis, several British Royal Navy ships 

• USC&GS Isis, a survey ship in service in the United States Coast and Geodetic Survey from 1915 to 1917 and 
from 1919 to 1920 

• USS Isis (1901), a United States Navy patrol vessel in commission from 1917 to 1919 

Television 

The Secrets of Isis, 1970s television series 

Isis (Stargate) 

Isis {Battlestar Galactica) 

Isis Tsunami, contestant on America's Next Top Model 

International Secret Intelligence Service, fictional spy agency in Archer (TV series) 

Isis Eaglet, fictional character in Magical Chronicle Lyrical Nanoha Force 

Isis (Smallville) 

Project Isis (Chuck) 

Other uses 

Maria Isis (Maria- Jesus), daughter of Agustin de Iturbide. 

Hurricane Isis (disambiguation), the name of several tropical cyclones 

International Studies of Infarct Survival, a set of clinical trials 

Isis Adventure, puzzle game 

Isis (coral), a soft coral genus 

ISIS Drive, a bicycle bottom bracket interface 



ISIS neutron source 



169 



ISIS neutron source 



v* A i 



ISISW 



r*. m ' 



ISIS Logo 



ISIS is a pulsed neutron and muon source. It 
is situated at the Rutherford Appleton 
Laboratory on the Harwell Science and 
Innovation Campus in Oxfordshire, United 
Kingdom and is part of the Science and 
Technology Facilities Council. It uses the 
techniques muon spectroscopy and neutron 
scattering to probe the structure and 
dynamics of condensed matter on a 
microscopic scale ranging from the 
subatomic to the macromolecular. 

Hundreds of experiments are performed 
annually at ISIS by visiting researchers from 
around the world, in diverse science areas 
including physics, chemistry, materials 
engineering, earth sciences, biology and 
archaeology. 

Neutrons and muons 

Neutrons are uncharged constituents of 
atoms and penetrate materials well, 
deflecting only from the nuclei of atoms. 
The statistical accumulation of deflected 
neutrons at different positions beyond the 
sample can be used to find the structure of a material, and the loss or gain of energy by neutrons can reveal the 
dynamic behaviour of parts of a sample, for example diffusive processes in solids. At ISIS the neutrons are created 
by accelerating 'bunches' of protons in a synchrotron, then colliding these with a heavy tantalum metal target, under a 
constant cooling load to dissipate the heat from the 160 kW proton beam. The impacts cause neutrons to spall off the 
tantalum atoms, and the neutrons are channelled through guides, or beamlines, to about 20 instruments, individually 
optimised for the study of different types of matter. The target station and most of the instruments are set in a large 
hall. Neutrons are a dangerous form of radiation, so the target and beamlines are heavily shielded with concrete. 




ISIS produces muons by colliding a fraction of the proton beam with a graphite target, producing pions which decay 
rapidly into muons, delivered in a spin-polarised beam to sample stations. 



ISIS neutron source 



170 




Another view of the ISIS experimental hall for Target Station 1 



Science at ISIS 

ISIS is administered and operated by the 

Science and Technology Facilities Council 

(previously CCLRC). Experimental time is 

open to academic users from funding 

countries and is applied for through a 

twice-yearly 'call for proposals'. Research 

allocation, or 'beam-time', is allotted to 

applicants via a peer-review process. Users 

and their parent institutions do not pay for 

the running costs of the facility, which are as much as £11,000 per instrument per day. Their transport and living 

costs are also refunded whilst carrying out the experiment. Most users stay in Ridgeway House, a hotel near the site, 

or at Cosener's House, an STFC-run conference centre in Abingdon. Over 600 experiments by 1600 users are 

completed every year. 

A large number of support staff operate the facility, aid users, and carry out research, the control room is staffed 24 
hours a day, every day of the year. Instrument scientists oversee the running of each instrument and liaise with users, 
and other divisions provide sample environment, data analysis and computing expertise, maintain the accelerator, 
and run education programmes. 

Among the important and pioneering work carried out was the discovery of the structure of high-temperature 
superconductors and the solid phase of buckminster-fullerene. 

Construction for a second target station started in 2003, and the first neutrons were delivered to the target on 
December 14, 2007 . It will use low-energy neutrons to study soft condensed matter, biological systems, advanced 
composites and nanomaterials. To supply the extra protons for this, the accelerator is being upgraded. 



History and background of ISIS 

The source was approved in 1977 for the RAL site on the Harwell campus and recycled components from earlier UK 
science programmes including the accelerator hall which had previously been occupied by the Nimrod accelerator. 
The first beam was produced in 1984, and the facility was formally opened by the then Prime Minister Margaret 



Thatcher in October 1985 



[2] 



The name ISIS is not an acronym: it refers to the Ancient Egyptian goddess and the local name for the River 
Thames. The name was selected for the official opening of the facility in 1985, prior to this it was known as the SNS, 
or Spallation Neutron Source. The name was considered appropriate as Isis was a goddess who could restore life to 
the dead, and ISIS made use of equipment previously constructed for the Nimrod and Nina accelerators . 



ISIS neutron source 



171 



External links 



[4] 



ISIS facility 

ISIS Second Target Station 

The Science and Technology Facilities Council 



[5] 



[6] 



References 

[1] ISIS Second Target Station Project (http://ts-2.isis.rl.ac.uk/) 

[2] Linacs at the Rutherford Appleton Laboratory (http://epubs.cclrc.ac.uk/bitstream/692/linacplahistory.pdf) 

[3] Explanation of the name of ISIS (http://www.isis.rl.ac.uk/aboutIsis/index.htm) 

[4] http://www.isis.stfc.ac.uk/ 

[5] http://www.isis.stfc.ac.uk/about-isis/target-station-2/ 

[6] http://www.stfc.ac.uk 

Geographical coordinates: 51°34'18"N 1°19'12"W 



Sudbury Neutrino Observatory 



The Sudbury Neutrino Observatory (SNO) is a neutrino observatory 
located 6,800 feet (about 2 km) underground in Vale Inco's Creighton 
Mine in Sudbury, Ontario, Canada. The detector was designed to detect 
solar neutrinos through their interactions with a large tank of heavy 
water. The detector turned on in May 1999, and was turned off on 28 
November 2006. While new data is no longer being taken the SNO 
collaboration will continue to analyze the data taken during that period 
for the next several years. The underground laboratory has been 
enlarged and continues to operate other experiments at SNOLAB. The 
SNO equipment itself is currently being refurbished for use in the 
SNO+ experiment. 

Experimental motivation 

The first measurements of the number of solar neutrinos reaching the 

earth were taken in the 1960s, and all experiments prior to SNO 

observed a third to a half fewer neutrinos than were predicted by the 

Standard Solar Model. As several experiments confirmed this deficit 

the effect became known as the solar neutrino problem. Over several 

decades many ideas were put forward to try to explain the effect, one 

of which was the hypothesis of neutrino oscillations. All of the solar neutrino detectors prior to SNO had been 

sensitive primarily or exclusively to electron neutrinos and yielded little to no information on muon neutrinos and tau 

neutrinos. 

In 1984, Herb Chen of the University of California at Irvine first pointed out the advantages of using heavy water as 
a detector for solar neutrinos. Unlike previous detectors, using heavy water would make the detector sensitive to two 
reactions, one sensitive to all neutrino flavours, which would allow a detector to measure neutrino oscillations 
directly. The Creighton Mine in Sudbury, among the deepest in the world and accordingly low background radiation, 
was quickly identified as an ideal place for Chen's proposed experiment to be built. 




Artist's concept of SNO's detector. (Courtesy of 
SNO) 



Sudbury Neutrino Observatory 



172 



The SNO collaboration held its first meeting in 1984. At the time it competed with TRIUMF's KAON Factory 
proposal for federal funding, and the wide variety of universities backing SNO quickly led to it being selected for 
development. The official go-ahead was given in 1990. 

The experiment observed the light produced by relativistic electrons in the water created by neutrino interactions. As 
relativistic electrons travel through a medium, they lose energy producing a cone of blue light through the Cerenkov 
effect, and it is this light that is directly detected. 



Detector description 

The SNO detector target consisted of 1000 tonnes (1102 short tons) of 
heavy water contained in a 6-metre (20 ft) radius acrylic vessel. The 
detector cavity outside the vessel was filled with normal water to 
provide both buoyancy for the vessel and radioactive shielding. The 
heavy water was viewed by approximately 9,600 photomultiplier tubes 
(PMTs) mounted on a geodesic sphere at a radius of about 
850 centimetres (335 in). The cavity housing the detector is the largest 
man-made underground cavity in the world, requiring a variety of 
high-performance rock bolting techniques to prevent rock bursts. 

The observatory is located at the end of a 1.5-kilometre (0.9 mi) long 
drift, whimsically named the "SNO drift", isolating it from other 
mining operations. Along the drift are a number of operations and 
equipment rooms, all held in a clean room setting. Most of the facility 
is Class 3000 (fewer than 3,000 particles of 1 urn or larger per 1 m of 
air) but the final cavity containing the detector is Class 1000 



[l] 




Charged current interaction 

In the charged current interaction, a neutrino converts the neutron in a deuteron to a proton. The neutrino is absorbed 
in the reaction and an electron is produced. Solar neutrinos have energies smaller than the mass of muons and tau 
leptons, so only electron neutrinos can participate in this reaction. The emitted electron carries off most of the 
neutrino's energy, on the order of 5—15 MeV, and is detectable. The proton which is produced does not have enough 
energy to be detected easily. The electrons produced in this reaction are emitted in all directions, but there is a slight 
tendency for them to point back in the direction from which the neutrino came. 



Neutral current interaction 

In the neutral current interaction, a neutrino dissociates the deuteron, breaking it into its constituent neutron and 
proton. The neutrino continues on with slightly less energy, and all three neutrino flavours are equally likely to 
participate in this interaction. Heavy water has a small cross section for neutrons, and when neutrons capture on a 
deuterium nucleus a gamma ray (photon) with roughly 6 MeV of energy is produced. The direction of the gamma 
ray is completely uncorrelated with the direction of the neutrino. Some of the neutrons wander past the acrylic vessel 
into the light water, and since light water has a very large cross section for neutron capture these neutrons are 
captured very quickly. A gamma ray with roughly 2 MeV of energy is produced in this reaction, but because this is 
below the detector's energy threshold they are not observable. 



Sudbury Neutrino Observatory 173 

Electron elastic scattering 

In the elastic scattering interaction, a neutrino collides with an atomic electron and imparts some of its energy to the 
electron. All three neutrinos can participate in this interaction through the exchange of the neutral Z boson, and 
electron neutrinos can also participate with the exchange of a charged W boson. For this reason this interaction is 
dominated by electron neutrinos, and this is the channel through which the Super-Kamiokande (Super-K) detector 
can observe solar neutrinos. This interaction is the relativistic equivalent of billiards, and for this reason the electrons 
produced usually point in the direction that the neutrino was travelling (away from the sun). Because this interaction 
takes place on atomic electrons it occurs with the same rate in both the heavy and light water. 

Experimental results and impact 

On 18 June 2001, the first scientific results of SNO were published, bringing the first clear evidence that 

neutrinos oscillate (i.e. that they can transmute into one another), as they travel in the sun. This oscillation in turn 
implies that neutrinos have non-zero masses. The total flux of all neutrino flavours measured by SNO agrees well 
with the theoretical prediction. Further measurements carried out by SNO have since confirmed and improved the 
precision of the original result. 

Although Super-K had beaten SNO to the punch, having published evidence for neutrino oscillation as early as 1998, 
the Super-K results were not conclusive and did not specifically deal with solar neutrinos. SNO's results were the 
first to directly demonstrate oscillations in solar neutrinos. The results of the experiment had a major impact on the 
field, as evidenced by the fact that two of the SNO papers have been cited over 1,500 times, and two others have 
been cited over 750 times. In 2007, the Franklin Institute awarded the director of SNO Art McDonald with the 
Benjamin Franklin Medal in Physics. 

Other possible analyses 

The SNO detector would have been capable of detecting a supernova within our galaxy if one had occurred while the 
detector was online. As neutrinos emitted by a supernova are released earlier than the photons, it is possible to alert 
the astronomical community before the supernova is visible. SNO was a founding member of the Supernova Early 
Warning System (SNEWS) with Super-Kamiokande and the Large Volume Detector. No such supernovas have yet 
been detected. 

The SNO experiment was also able to observe atmospheric neutrinos produced by cosmic ray interactions in the 
atmosphere. Due to the limited size of the SNO detector in comparison with Super-K the low cosmic ray neutrino 
signal is not statistically significant at neutrino energies below 1 GeV. 

Participating institutions 

Large particle physics experiments require large collaborations. With approximately 100 collaborators, SNO was a 
rather small group compared to collider experiments. The participating institutions have included: 

Canada 

Carleton University 

Laurentian University 

Queen's University — designed and built many calibration sources and the device for deploying sources 

TRIUMF 

University of British Columbia 

University of Guelph 



Sudbury Neutrino Observatory 174 

Although no longer a collaborating institution, Chalk River Laboratories led the construction of the acrylic vessel 
that holds the heavy water, and Atomic Energy of Canada Limited was the source of the heavy water. 

United Kingdom 

• University of Oxford — developed much of the experiment's Monte Carlo analysis program (SNOMAN), and 
maintained the program 

United States of America 

• LBNL — Led the construction of the geodesic structure that holds the PMTs 

• LANL 

• University of Pennsylvania — designed and built the front end electronics and trigger 

• University of Washington — designed and built proportional counter tubes for detection of neutrons in the third 
phase of the experiment 

• Brookhaven National Laboratory 

• University of Texas at Austin 

• Massachusetts Institute of Technology 

Honours and awards 

• Asteroid 14724 SNO is named in honour of SNO. 

• In November 2006, the entire SNO team was awarded the inaugural John C. Polanyi Award for "a recent 
outstanding advance in any field of the natural sciences or engineering" conducted in Canada. 

See also 

• SNOLAB — A permanent underground physics laboratory being built around SNO 

• SNO+ - The successor of SNO 

References 

[1] "The Sudbury Neutrino Observatory — Canada's eye on the universe" (http://cerncourier.com/cws/article/cern/28553). CERN Courier. 

CERN. 4 December 2001. . Retrieved 2008-06-04. 
[2] Ahmad, QR; et at (2001). "Measurement of the Rate of v + d — > p + p + e~ Interactions Produced by B Solar Neutrinos at the Sudbury 

Neutrino Observatory". Physical Review Letters 87 (7): 071301. doi:10.1 103/PhysRevLett.87.071301. 
[3] "Sudbury Neutrino Observatory First Scientific Results" (http://www.sno.phy.queensu.ca/sno/first_results/). 3 July 2001. . Retrieved 

2008-06-04. 
[4] "SPIRES HEP Results" (http://www-library.desy.de/cgi-bin/spiface/find/blu/hep/wwwcite?rawcmd=FIND+COLLABORATION+ 

SNO+and+topcite+500+). SPIRES. SLAC. . Retrieved 2009-10-06. 
[5] "Arthur B. McDonald, Ph.D." (http://www.fi. edu/winners/2007/mcdonald_arthur.faw?winner_id=441 1). Franklin Laureate Database. 

Franklin Institute. . Retrieved 2008-06-04. 
[6] "Past Winners — The Sudbury Neutrino Observatory" (http://www.nserc.gc.ca/award_e. asp ?nav=polanyi&lbi=past). NSERC. 3 March 

2008. . Retrieved 2008-06-04. 



Sudbury Neutrino Observatory 175 

External links 

• SNO's official site (http://www.sno.phy.queensu.ca/) 

• Joshua Klein's Introduction to SNO, Solar Neutrinos, and Penn at SNO (http://www.hep.upenn.edu/SNO/ 
intro.html) 

• " Experiment Cave (http://www.pbs.org/kcet/wiredscience/story/49-experiment_cave.html)". WIRED 
Science. PBS. 2007-10-24. No. 104. 

• " The Ghost Particle (http://www.pbs.org/wgbh/nova/neutrino/)". Written and Directed by David Sington. 
Nova. PBS. 2006-02-21. No. 3306 (607), season 34. 

Geographical coordinates: 46°28'00"N 81°10'22"W 



ATLAS experiment 



176 



ATLAS experiment 




ATLAS 



CMS 
LHCb 



ALICE 
TOTEM 



LHCf 
MoEDAL 



LHC experiments 

A Toroidal LHC Apparatus 



Compact Muon Solenoid 
LHC-beauty 



A Large Ion Collider Experiment 

Total Cross Section, Elastic Scattering and Diffraction Dissociation 



LHC-forward 

Monopole and Exotics Detector At the LHC 



p and Pb 



(not marked) 
PS 



SPS 



LHC preaccelerators 

Linear accelerators for protons (Linac 2) and Lead (Linac 3) 



Proton Synchrotron Booster 
Proton Synchrotron 



Super Proton Synchrotron 



Geographical coordinates: 46°14'8"N 6°3'19"E 

ATLAS (A Toroidal LHC Apparatus) is one of the six particle detector 
experiments (ALICE, ATLAS, CMS, TOTEM, LHCb, and LHCf) constructed at 
the Large Hadron Collider (LHC), a new particle accelerator at the European 
Organization for Nuclear Research (CERN) in Switzerland. ATLAS is 44 metres 
long and 25 metres in diameter, weighing about 7,000 tonnes. The project is led by 
Fabiola Gianotti and involves roughly 2,000 scientists and engineers at 165 
institutions in 35 countries. The construction was originally scheduled to be 
completed in June 2007, but was ready and detected its first beam events on 10 
September 2008. The experiment is designed to observe phenomena that involve 
highly massive particles which were not observable using earlier lower-energy 
accelerators and might shed light on new theories of particle physics beyond the 
Standard Model. 

The ATLAS collaboration, the group of physicists building the detector, was 

ATLAS logo. formed in 1992 when the proposed EAGLE (Experiment for Accurate Gamma, 

Lepton and Energy Measurements) and ASCOT (Apparatus with Super 

Conducting Toroids) collaborations merged their efforts into building a single, general-purpose particle detector for 

the Large Hadron Collider. The design was a combination of those two previous designs, as well as the detector 
research and development that had been done for the Superconducting Supercollider. The ATLAS experiment was 




ATLAS experiment 



177 



proposed in its current form in 1994, and officially funded by the CERN member countries beginning in 1995. 
Additional countries, universities, and laboratories joined in subsequent years, and further institutions and physicists 
continue to join the collaboration even today. The work of construction began at individual institutions, with detector 
components shipped to CERN and assembled in the ATLAS experimental pit beginning in 2003. 

ATLAS is designed as a general-purpose detector. When the proton beams produced by the Large Hadron Collider 
interact in the center of the detector, a variety of different particles with a broad range of energies may be produced. 
Rather than focusing on a particular physical process, ATLAS is designed to measure the broadest possible range of 
signals. This is intended to ensure that, whatever form any new physical processes or particles might take, ATLAS 
will be able to detect them and measure their properties. Experiments at earlier colliders, such as the Tevatron and 
Large Electron-Positron Collider, were designed based on a similar philosophy. However, the unique challenges of 
the Large Hadron Collider — its unprecedented energy and extremely high rate of collisions — require ATLAS to be 
larger and more complex than any detector ever built. 



Background 




The first cyclotron, an early type of particle accelerator, was built by 

Ernest O. Lawrence in 1931, with a radius of just a few centimetres 

and a particle energy of 1 megaelectronvolt (MeV). Since then, 

accelerators have grown enormously in the quest to produce new 

particles of greater and greater mass. As accelerators have grown, so 

too has the list of known particles that they might be used to 

investigate. The most comprehensive model of particle interactions 

available today is known as the Standard Model of Particle Physics. 

With the important exception of the Higgs boson, all of the particles 

predicted by the model have been observed. While the standard model 

predicts that quarks, electrons, and neutrinos should exist, it does not 

explain why the masses of the particles are so very different. Due to 

this violation of "naturalness" most particle physicists believe it is 

possible that the Standard Model will break down at energies beyond 

the current energy frontier of about one teraelectronvolt (TeV) (set at the Tevatron). If such 

beyond-the-Standard-Model physics is observed it is hoped that a new model, which is identical to the Standard 

Model at energies thus far probed, can be developed to describe particle physics at higher energies. Most of the 

currently proposed theories predict new higher-mass particles, some of which are hoped to be light enough to be 

observed by ATLAS. At 27 kilometres in circumference, the Large Hadron Collider (LHC) will collide two beams of 

protons together, each proton carrying about 7 TeV of energy — enough energy to produce particles with masses up 

to roughly ten times more massive than any particles currently known — assuming of course that such particles 

exist. With an energy seven million times that of the first accelerator the LHC represents a "new generation" of 

particle accelerators. 



ATLAS experiment detector under construction 

in October 2004 in its experimental pit; the 

current status of construction can be seen on the 
[41 
CERN website. Note the people in the 

background, for comparison. 



Particles that are produced in accelerators must also be observed, and this is the task of particle detectors. While 
interesting phenomena may occur when protons collide it is not enough to just produce them. Particle detectors must 
be built to detect particles, their masses, momentum, energies, charges, and nuclear spins. In order to identify all 
particles produced at the interaction point where the particle beams collide, particle detectors are usually designed 
with a similarity to an onion. The layers are made up of detectors of different types, each of which is adept at 
observing specific types of particles. The different features that particles leave in each layer of the detector allow for 
effective particle identification and accurate measurements of energy and momentum. (The role of each layer in the 
detector is discussed below.) As the energy of the particles produced by the accelerator increases, the detectors 
attached to it must grow to effectively measure and stop higher-energy particles. ATLAS is the largest detector ever 



ATLAS experiment 



178 



built at a particle collider as of 2008 



[l] 



Physics Program 

ATLAS is intended to investigate many different types of physics that 
might become detectable in the energetic collisions of the LHC. Some 
of these are confirmations or improved measurements of the Standard 
Model, while many others are searches for new physical theories. 

One of the most important goals of ATLAS is to investigate a missing 
piece of the Standard Model, the Higgs boson. The Higgs 
mechanism, which includes the Higgs boson, is invoked to give masses 
to elementary particles, giving rise to the differences between the weak 
force and electromagnetism by giving the W and Z bosons masses 
while leaving the photon massless. If the Higgs boson is not discovered 
by ATLAS, it is expected that another mechanism of electroweak 
symmetry breaking that explains the same phenomena, such as 
technicolour, will be discovered. The Standard Model is simply not 
mathematically consistent at the energies of the LHC without such a 
mechanism. The Higgs boson would be detected by the particles it 
decays into; the easiest to observe are two photons, two bottom quarks, 
or four leptons. Sometimes these decays can only be definitively 
identified as originating with the Higgs boson when they are associated 
with additional particles; for an example of this, see the diagram at 
right. 




A schematic, called a Feynman diagram, of two 

virtual gluons from colliding LHC protons 

interacting to produce a hypothetical Higgs 

boson, a top quark, and an antitop quark. These in 

turn decay into a specific combination of quarks 

and leptons that is very unlikely to be duplicated 

by other processes. Collecting sufficient evidence 

of signals like this one may eventually allow 

ATLAS collaboration members to discover the 

Higgs boson. 



[5] 



The asymmetry between the behavior of matter and antimatter, known as CP violation, will also be investigated. 
Current CP-violation experiments, such as BaBar and Belle, have not yet detected sufficient CP violation in the 
Standard Model to explain the lack of detectable antimatter in the universe. It is possible that new models of physics 
will introduce additional CP violation, shedding light on this problem; these models might either be detected directly 
by the production of new particles, or indirectly by measurements made of the properties of B-mesons. (LHCb, an 
LHC experiment dedicated to B-mesons, is likely to be better suited to the latter) 



[6] 



The top quark, discovered at Fermilab in 1995, has thus far had its properties measured only approximately. With 
much greater energy and greater collision rates, LHC will produce a tremendous number of top quarks, allowing 
ATLAS to make much more precise measurements of its mass and interactions with other particles. These 
measurements will provide indirect information on the details of the Standard Model, perhaps revealing 
inconsistencies that point to new physics. Similar precision measurements will be made of other known particles; for 
example, ATLAS may eventually measure the mass of the W boson twice as accurately as has previously been 
achieved. 

Perhaps the most exciting lines of investigation are those searching directly for new models of physics. One theory 
that is the subject of much current research is broken supersymmetry. The theory is popular because it could 
potentially solve a number of problems in theoretical physics and is present in almost all models of string theory. 
Models of supersymmetry involve new, highly massive particles; in many cases these decay into high-energy quarks 
and stable heavy particles that are very unlikely to interact with ordinary matter. The stable particles would escape 
the detector, leaving as a signal one or more high-energy quark jets and a large amount of "missing" momentum. 
Other hypothetical massive particles, like those in Kaluza-Klein theory, might leave a similar signature, but its 
discovery would certainly indicate that there was some kind of physics beyond the Standard Model. 



ATLAS experiment 



179 



One remote possibility (if the universe contains large extra dimensions) is that microscopic black holes might be 

tot 

produced by the LHC. These would decay immediately by means of Hawking radiation, producing all particles in 

191 
the Standard Model in equal numbers and leaving an unequivocal signature in the ATLAS detector. In fact, if this 

occurs, the primary studies of Higgs bosons and top quarks would be conducted on those produced by the black 

holes. 



Components 



The ATLAS detector consists of a series of ever-larger concentric cylinders around the interaction point where the 
proton beams from the LHC collide. It can be divided into four major parts: the Inner Detector, the calorimeters, the 
muon spectrometer and the magnet systems. Each of these is in turn made of multiple layers. The detectors are 
complementary: the Inner Detector tracks particles precisely, the calorimeters measure the energy of easily stopped 
particles, and the muon system makes additional measurements of highly penetrating muons. The two magnet 
systems bend charged particles in the Inner Detector and the muon spectrometer, allowing their momenta to be 
measured. 

The only established stable particles that cannot be detected directly are neutrinos; their presence is inferred by 
noticing a momentum imbalance among detected particles. For this to work, the detector must be "hermetic", and 
detect all non-neutrinos produced, with no blind spots. Maintaining detector performance in the high radiation areas 
immediately surrounding the proton beams is a significant engineering challenge. 



Inner detector 

The Inner Detector begins a few centimetres from the proton beam 
axis, extends to a radius of 1.2 metres, and is seven metres in length 
along the beam pipe. Its basic function is to track charged particles by 
detecting their interaction with material at discrete points, revealing 
detailed information about the type of particle and its momentum. 
The magnetic field surrounding the entire inner detector causes 
charged particles to curve; the direction of the curve reveals a particle's 
charge and the degree of curvature reveals its momentum. The starting 
points of the tracks yield useful information for identifying particles; 
for example, if a group of tracks seem to originate from a point other 
than the original proton— proton collision, this may be a sign that the 
particles came from the decay of a bottom quark (see B-tagging). The 
Inner Detector has three parts, which are explained below. 




The ATLAS TRT central section, the outermost 

part of the Inner Detector, as of September 2005, 

assembled on the surface and taking data from 

cosmic rays. 



The Pixel Detector, the innermost part of the detector, contains three layers and three disks on each end-cap, with a 
total of 1744 modules, each measuring two centimetres by six centimetres. The detecting material is 250 |am thick 
silicon. Each module contains 16 readout chips and other electronic components. The smallest unit that can be read 
out is a pixel (each 50 by 400 micrometres); there are roughly 47,000 pixels per module. The minute pixel size is 
designed for extremely precise tracking very close to the interaction point. In total, the Pixel Detector will have over 
80 million readout channels, which is about 50% of the total readout channels; such a large count created a design 
and engineering challenge. Another challenge was the radiation the Pixel Detector will be exposed to because of its 
proximity to the interaction point, requiring that all components be radiation hardened in order to continue operating 
after significant exposures. 

The Semi-Conductor Tracker (SCT) is the middle component of the inner detector. It is similar in concept and 
function to the Pixel Detector but with long, narrow strips rather than small pixels, making coverage of a larger area 
practical. Each strip measures 80 micrometres by 12.6 centimetres. The SCT is the most critical part of the inner 



ATLAS experiment 



180 



detector for basic tracking in the plane perpendicular to the beam, since it measures particles over a much larger area 
than the Pixel Detector, with more sampled points and roughly equal (albeit one dimensional) accuracy. It is 
composed of four double layers of silicon strips, and has 6.2 million readout channels and a total area of 61 square 
meters. 

The Transition radiation tracker (TRT), the outermost component of the inner detector, is a combination of a straw 
tracker and a transition radiation detector. The detecting elements are drift tubes (straws), each four millimetres in 
diameter and up to 144 centimetres long. The uncertainty of track position measurements (position resolution) is 
about 200 micrometres, not as precise as those for the other two detectors, a necessary sacrifice for reducing the cost 
of covering a larger volume and having transition radiation detection capability. Each straw is filled with gas that 
becomes ionized when a charged particle passes through. The straws are held at about -1500V, driving the negative 
ions to a fine wire down the centre of each straw, producing a current pulse (signal) in the wire. The wires with 
signals create a pattern of 'hit' straws that allow the path of the particle to be determined. Between the straws, 
materials with widely varying indices of refraction cause ultra-relativistic charged particles to produce transition 
radiation and leave much stronger signals in some straws. Xenon gas is used to increase the number of straws with 
strong signals. Since the amount of transition radiation is greatest for highly relativistic particles (those with a speed 
very near the speed of light), and particles of a particular energy have a higher speed the lighter they are, particle 
paths with many very strong signals can be identified as the lightest charged particles, electrons. The TRT has about 
298,000 straws in total. 







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Calorimeters 

The calorimeters are situated outside the solenoidal magnet that 
surrounds the inner detector. Their purpose is to measure the energy 
from particles by absorbing it. There are two basic calorimeter 
systems: an inner electromagnetic calorimeter and an outer hadronic 

1121 

calorimeter. Both are sampling calorimeters; that is, they absorb 
energy in high-density metal and periodically sample the shape of the 
resulting particle shower, inferring the energy of the original particle 
from this measurement. 

The electromagnetic (EM) calorimeter absorbs energy from particles 
that interact electromagnetically, which include charged particles and 
photons. It has high precision, both in the amount of energy absorbed 
and in the precise location of the energy deposited. The angle between 
the particle's trajectory and the detector's beam axis (or more precisely 
the pseudorapidity) and its angle within the perpendicular plane are 
both measured to within roughly 0.025 radians. The energy-absorbing 
materials are lead and stainless steel, with liquid argon as the sampling 
material, and a cryostat is required around the EM calorimeter to keep 
it sufficiently cool. 

The hadron calorimeter absorbs energy from particles that pass through 
the EM calorimeter, but do interact via the strong force; these particles 
are primarily hadrons. It is less precise, both in energy magnitude and 
in the localization (within about 0.1 radians only). The 
energy-absorbing material is steel, with scintillating tiles that sample 
the energy deposited. Many of the features of the calorimeter are chosen for their cost-effectiveness; the instrument 




One of the sections of the extensions of the 

hadronic calorimeter, waiting to be inserted in 

late February 2006 



ATLAS experiment 



181 



is large and comprises a huge amount of construction material: the main part of the calorimeter — the tile 
calorimeter — is eight metres in diameter and covers 12 metres along the beam axis. The far-forward sections of the 
hadronic calorimeter are contained within the EM calorimeter's cryostat, and use liquid argon as it does. 



Muon spectrometer 

The muon spectrometer is an extremely large tracking system, extending from a radius of 4.25 m around the 
calorimeters out to the full radius of the detector (11 m). Its tremendous size is required to accurately measure the 
momentum of muons, which penetrate other elements of the detector; the effort is vital because one or more muons 
are a key element of a number of interesting physical processes, and because the total energy of particles in an event 
could not be measured accurately if they were ignored. It functions similarly to the inner detector, with muons 
curving so that their momentum can be measured, albeit with a different magnetic field configuration, lower spatial 
precision, and a much larger volume. It also serves the function of simply identifying muons — very few particles of 
other types are expected to pass through the calorimeters and subsequently leave signals in the muon spectrometer. It 
has roughly one million readout channels, and its layers of detectors have a total area of 12,000 square meters. 



Magnet system 

The ATLAS detector uses two large superconducting magnet systems 
to bend charged particles so that their momenta can be measured. This 
bending is due to the Lorentz force, which is proportional to velocity. 
Since all particles produced in the LHC's proton collisions will be 
traveling at very close to the speed of light, the force on particles of 
different momenta is equal. (In the theory of relativity, momentum is 
not proportional to velocity at such speeds.) Thus high-momentum 
particles will curve very little, while low-momentum particles will 
curve significantly; the amount of curvature can be quantified and the 
particle momentum can be determined from this value. 




The ends of four of eight ATLAS toroid magnets, 
seen from the surface, about 90 metres above, in 
September 2005. 



The inner solenoid produces a two tesla magnetic field surrounding the 

ri3i 

Inner Detector. This high magnetic field allows even very energetic 

particles to curve enough for their momentum to be determined, and its nearly uniform direction and strength allow 
measurements to be made very precisely. Particles with momenta below roughly 400 MeV will be curved so strongly 
that they will loop repeatedly in the field and most likely not be measured; however, this energy is very small 
compared to the several TeV of energy released in each proton collision. 

The outer toroidal magnetic field is produced by eight very large air-core superconducting barrel loops and two 
end-caps, all situated outside the calorimeters and within the muon system. This magnetic field is 26 metres long 
and 20 metres in diameter, and it stores 1.6 gigajoules of energy. Its magnetic field is not uniform, because a 
solenoid magnet of sufficient size would be prohibitively expensive to build. Fortunately, measurements need to be 
much less precise to measure momentum accurately in the large volume of the muon system. 



ATLAS experiment 



182 



Forward detectors 

The ATLAS detector will be complemented with a set of detectors in 
the very forward region. These detectors will be located in the LHC 
tunnel far away from the interaction point. The basic idea is to measure 
elastic scattering at very small angles in order to get a handle on the 
absolute luminosity at the interaction point of ATLAS. 




Part of the ATLAS, as it looked February 2007. 



Data systems and analysis 

The detector generates unmanageably large amounts of raw data, about 25 megabytes per event (raw; zero 
suppression reduces this to 1.6 MB)times 23 events per beam crossing, times 40 million beam crossings per second 

ri4i 

in the center of the detector, for a total of 23 petabyte/second of raw data. The trigger system uses simple 
information to identify, in real time, the most interesting events to retain for detailed analysis. There are three trigger 
levels, the first based in electronics on the detector and the other two primarily run on a large computer cluster near 
the detector. After the first-level trigger, about 100,000 events per second have been selected. After the third-level 
trigger, a few hundred events remain to be stored for further analysis. This amount of data will require over 
100 megabytes of disk space per second — at least a petabyte each year. 

Offline event reconstruction will be performed on all permanently stored events, turning the pattern of signals from 
the detector into physics objects, such as jets, photons, and leptons. Grid computing will be extensively used for 
event reconstruction, allowing the parallel use of university and laboratory computer networks throughout the world 
for the CPU-intensive task of reducing large quantities of raw data into a form suitable for physics analysis. The 
software for these tasks has been under development for many years, and will continue to be refined once the 
experiment is running. 

Individuals and groups within the collaboration will write their own code to perform further analysis of these objects, 
searching in the pattern of detected particles for particular physical models or hypothetical particles. These studies 
are already being developed and tested on detailed simulations of particles and their interactions with the detector. 
Such simulations give physicists a good sense of which new particles can be detected and how long it will take to 
confirm them with sufficient statistical certainty. 



ATLAS experiment 183 

See also 

LHC 

Notes 

[I] CERN (2006-11-20). "World's largest superconducting magnet switches on" (http://press.web.cern.ch/Press/PressReleases/ 
Releases2006/PR17.06E.html). Press release. . Retrieved 2007-03-03. 

[2] "First beam and first events in ATLAS" (http://www.atlas.ch/news/2008/first-beam-and-event.html). Atlas. ch. . Retrieved 2008-09-13. 

[3] "ATLAS Collaboration records" (http://library.cern.ch/archives/isad/isaatlas.html). CERN Archive. . Retrieved 2007-02-25. 

[4] "UX15 Installation; WEB cameras" (http://atlaseye-webpub.web.cern.ch/atlaseye-webpub/web-sites/pages/UX15_webcams.htm). 

ATLAS Control Room, cern.ch. . Retrieved September 15, 2010. 
[5] "Introduction and Overview" (http://atlas.web.cern.ch/Atlas/TP/NEW/HTML/tp9new/node4. 

html#SECTION00400000000000000000). ATLAS Technical Proposal. CERN. 1994. . 
[6] N. V. Krasnikov, V. A. Matveev (September 1997). "Physics at LHC" (http://arxiv.org/abs/hep-ph/9703204). Physics of Particles and 

Nuclei 28 (5): 441^70. doi:10.1134/1.953049. . 
[7] "Top-Quark Physics" (http://atlas.web.cern.ch/ Atlas/TP/NEW/HTML/tp9new/node416.html#SECTION0024100000000000000000). 

ATLAS Technical Proposal. CERN. 1994. . 
[8] CM. Harris, M.J. Palmer, M.A. Parker, P. Richardson, A. Sabetfakhri and B.R. Webber (2005). "Exploring higher dimensional black holes at 

the Large Hadron Collider". Journal of High Energy Physics 5: 053. doi: 10. 1088/1 126-6708/2005/05/053. 
[9] J. Tanaka, T. Yamamura, S. Asai, J. Kanzaki (2005). "Study of Black Holes with the ATLAS detector at the LHC" (http://www. 

springerlink.com/content/x067g845688470r4/). The European Physical Journal C 41 (s2): 19-33. doi: 10.1 140/epjcd/s2005-02-008-x. . 
[10] "Overall detector concept" (http://atlas.web.cern.ch/ Atlas/TP/NEW/HTML/tp9new/node6.html#SECTION00420000000000000000). 

ATLAS Technical Proposal. CERN. 1994. . 

[II] "Inner detector" (http://atlas.web.cern.en/Atlas/TP/NEW/HTML/tp9new/nodelO.html#SECTION00433000000000000000). ATLAS 
Technical Proposal. CERN. 1994. . 

[12] "Calorimetry" (http://atlas.web.cern.en/Atlas/TP/NEW/HTML/tp9new/node9.html#SECTION00432000000000000000). ATLAS 

Technical Proposal. CERN. 1994. . 
[13] "Magnet system" (http://atlas.web.cern.ch/ Atlas/TP/NEW/HTML/tp9new/node8.html#SECTION0043 1000000000000000). ATLAS 

Technical Proposal. CERN. 1994. . 
[14] . http://atlas.ch/detector.html.See also 32:30 for information on the various trigger levels. 
[15] "The sensitive giant" (http://www.eurekalert.org/features/doe/2004-03/dnal-tsg032604.php). United States Department of Energy 

Research News. March 2004. . 

References 

• ATLAS Technical Proposal. (http://atlas.web.cern.ch/Atlas/TP/tp.html) CERN: The Atlas Experiment. 
Retrieved on 2007-04-10 

• ATLAS Detector and Physics Performance Technical Design Report. (http://atlas.web.cern.ch/Atlas/ 
GROUPS/PHYSICS/TDR/access.html) CERN: The Atlas Experiment. Retrieved on 2007-04-10 

• N. V. Krasnikov, V. A. Matveev (September 1997). "Physics at LHC" (http://arxiv.org/abs/hep-ph/9703204). 
Physics of Particles and Nuclei 28 (5): 441-470. doi: 10. 1134/1.953049. 

External links 

• Official ATLAS Public Webpage (http://atlas.ch) at CERN (The "award winning ATLAS movie" is a very good 
general introduction! ) 

• Official ATLAS Collaboration Webpage (http://atlas.web.cern.ch/Atlas/internal/Welcome.html) at CERN 
(Lots of technical and logistical information) 

• ATLAS Cavern Webcams (http://atlaseye-webpub.web.cern.ch/atlaseye-webpub/web-sites/pages/ 
UX 1 5_webcams . htm) 

• Time lapse video of the assembly (http://www.youtube. com/watch ?v=kVrUR_SOykk) 

• ATLAS section from US/LHC Website (http://www.uslhc.us/What_is_the_LHC/Experiments/ATLAS) 

• PhysicsWorld article on LHC and experiments (http://physicsweb.Org/articles/world/13/5/9/l) 



ATLAS experiment 184 

• New York Times article on LHC and experiments (http://www.nytimes.eom/2000/l l/21/science/21HIGG. 
html?ex= 1 1 30040000&en=5282f5 1 cfO 1 9f 1 b7&ei=5070&ex= 1 08200 1 600&en=39ccf65ca6047eb2&ei=5070) 

• United States Department of Energy article on ATLAS (http://www.eurekalert.org/features/doe/2004-03/ 
dnal-tsg032604.php) 

• The Large Hadron Collider ATLAS Experiment Virtual Reality (VR) photography panoramas (http://www. 
petermccready . com/portfolio/0509 1901. html) 

• Large Hadron Collider Project Director Dr Lyn Evans CBE on the engineering behind the ATLAS experiment, 
Ingenia magazine, June 2008 (http://www.ingenia.org.uk/ingenia/articles.aspx?Index=489) 

• Atlas Experiment News and social networking (http://www.AtlasExperiment.net) 

• The ATLAS Collaboration, G Aad et al. (2008-08-14). "The ATLAS Experiment at the CERN Large Hadron 
Collider" (http://www.iop. org/EJ/journal/-page=extra.lhc/jinst). Journal of Instrumentation 3 (S08003): 
S08003. doi:10.1088/1748-0221/3/08/S08003. Retrieved 2008-08-26. (Full design documentation) 

• Press release from October 2008 by EB Industries regarding the ATLAS project (http://ebindustries.com/ 
ATLAS article.pdf) 



185 



Techniques 



Microscopy 



Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the 
unaided eye (objects that are not within the resolution range of the normal eye). There are three well-known branches 
of microscopy, optical, electron, and scanning probe microscopy. 

Optical and electron microscopy involve the diffraction, reflection, or refraction of electromagnetic 
radiation/electron beams interacting with the specimen, and the subsequent collection of this scattered radiation or 
another signal in order to create an image. This process may be carried out by wide-field irradiation of the sample 
(for example standard light microscopy and transmission electron microscopy) or by scanning of a fine beam over 
the sample (for example confocal laser scanning microscopy and scanning electron microscopy). Scanning probe 
microscopy involves the interaction of a scanning probe with the surface of the object of interest. The development 
of microscopy revolutionized biology and remains an essential technique in the life and physical sciences. 







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Scanning electron microscope image of pollen. 



Microscopy 



186 



Optical microscopy 



Optical or light microscopy involves passing visible light transmitted 
through or reflected from the sample through a single or multiple 
lenses to allow a magnified view of the sample. The resulting image 
can be detected directly by the eye, imaged on a photographic plate or 
captured digitally. The single lens with its attachments, or the system 
of lenses and imaging equipment, along with the appropriate lighting 
equipment, sample stage and support, makes up the basic light 
microscope. The most recent development is the digital microscope, 
which uses a CCD camera to focus on the exhibit of interest. The 
image is shown on a computer screen since the camera is attached to it 
via a USB port, so eye-pieces are unnecessary. 

Limitations 

Limitations of standard optical microscopy (bright field microscopy) 
lie in three areas; 

• The technique can only image dark or strongly refracting objects 
effectively. 

• Diffraction limits resolution to approximately 0.2 micrometre (see: 
microscope). 

• Out of focus light from points outside the focal plane reduces image clarity. 

Live cells in particular generally lack sufficient contrast to be studied successfully, internal structures of the cell are 
colourless and transparent. The most common way to increase contrast is to stain the different structures with 
selective dyes, but this involves killing and fixing the sample. Staining may also introduce artifacts, apparent 
structural details that are caused by the processing of the specimen and are thus not a legitimate feature of the 
specimen. 

These limitations have all been overcome to some extent by specific microscopy techniques that can non-invasively 
increase the contrast of the image. In general, these techniques make use of differences in the refractive index of cell 
structures. It is comparable to looking through a glass window: you (bright field microscopy) don't see the glass but 
merely the dirt on the glass. There is however a difference as glass is a denser material, and this creates a difference 
in phase of the light passing through. The human eye is not sensitive to this difference in phase but clever optical 
solutions have been thought out to change this difference in phase into a difference in amplitude (light intensity). 




Stereo microscope 



Microscopy 



187 



Techniques 

In order to improve specimen contrast or highlight certain structures in a sample special techniques must be used. A 
huge selection of microscopy techniques are available to increase contrast or label a sample. 

Four examples of transilumination techniques used to generate contrast in a sample of [[tissue 

paper]]. 1.559 urn/pixel. 




Bright field illumination, sample 

contrast comes from absorbance 

of light in the sample. 



Cross-polarized light 

illumination, sample contrast 

comes from rotation of polarized 

light through the sample. 



Dark field illumination, sample 

contrast comes from light 

scattered by the sample. 



Phase contrast illumination, 
sample contrast comes from 
interference of different path 
lengths of light through the 
sample. 



Bright field 

Bright field microscopy is the simplest of all the light microscopy techniques. Sample illumination is via transmitted 
white light, i.e. illuminated from below and observed from above. Limitations include low contrast of most 
biological samples and low apparent resolution due to the blur of out of focus material. The simplicity of the 
technique and the minimal sample preparation required are significant advantages. 

Oblique illumination 

The use of oblique (from the side) illumination gives the image a 3-dimensional appearance and can highlight 
otherwise invisible features. A more recent technique based on this method is Hoffmann's modulation contrast, a 
system found on inverted microscopes for use in cell culture. Oblique illumination suffers from the same limitations 
as bright field microscopy (low contrast of many biological samples; low apparent resolution due to out of focus 
objects), but may highlight otherwise invisible structures. 



Dark field 



[2] 



Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark field 
illumination uses a carefully aligned light source to minimize the quantity of directly-transmitted (unscattered) light 
entering the image plane, collecting only the light scattered by the sample. Darkfield can dramatically improve 
image contrast — especially of transparent objects — while requiring little equipment setup or sample preparation. 
However, the technique does suffer from low light intensity in final image of many biological samples, and 
continues to be affected by low apparent resolution. 

Rheinberg illumination is a special variant of dark field illumination in which transparent, colored filters are inserted 
just before the condenser so that light rays at high aperture are differently colored than those at low aperture (i.e. the 
background to the specimen may be blue while the object appears self-luminous yellow). Other color combinations 



are possible but their effectiveness is quite variable 



[3] 



Microscopy 



188 



Dispersion staining 

Dispersion staining is an optical technique that results in a colored image of a colorless object. This is an optical 
staining technique and requires no stains or dyes to produce a color effect. There are five different microscope 
configurations used in the broader technique of dispersion staining. They include brightfield Becke" line, oblique, 
darkfield, phase contrast, and objective stop dispersion staining. 



Phase contrast 

In electron microscopy: Phase-contrast 
imaging 

More sophisticated techniques will show 
proportional differences in optical density . Phase 
contrast is a widely used technique that shows 
differences in refractive index as difference in 
contrast. It was developed by the Dutch physicist 
Frits Zernike in the 1930s (for which he was awarded 
the Nobel Prize in 1953). The nucleus in a cell for 
example will show up darkly against the surrounding 
cytoplasm. Contrast is excellent; however it is not for 
use with thick objects. Frequently, a halo is formed 
even around small objects, which obscures detail. 
The system consists of a circular annulus in the 
condenser, which produces a cone of light. This cone 
is superimposed on a similar sized ring within the 
phase-objective. Every objective has a different size 
ring, so for every objective another condenser setting 
has to be chosen. The ring in the objective has 
special optical properties: it first of all reduces the 
direct light in intensity, but more importantly, it 
creates an artificial phase difference of about a 
quarter wavelength. As the physical properties of this 
direct light have changed, interference with the 
diffracted light occurs, resulting in the phase contrast 
image. 




Phase-contrast image of uncalcified matrix (top) and calcified matrix 
(bottom). 



Differential interference contrast 

Superior and much more expensive is the use of interference contrast. Differences in optical density will show up 
as differences in relief. A nucleus within a cell will actually show up as a globule in the most often used differential 
interference contrast system according to Georges Nomarski. However, it has to be kept in mind that this is an 
optical effect, and the relief does not necessarily resemble the true shape! Contrast is very good and the condenser 
aperture can be used fully open, thereby reducing the depth of field and maximizing resolution. 

The system consists of a special prism (Nomarski prism, Wollaston prism) in the condenser that splits light in an 
ordinary and an extraordinary beam. The spatial difference between the two beams is minimal (less than the 
maximum resolution of the objective). After passage through the specimen, the beams are reunited by a similar prism 
in the objective. 



Microscopy 189 

In a homogeneous specimen, there is no difference between the two beams, and no contrast is being generated. 
However, near a refractive boundary (say a nucleus within the cytoplasm), the difference between the ordinary and 
the extraordinary beam will generate a relief in the image. Differential interference contrast requires a polarized light 
source to function; two polarizing filters have to be fitted in the light path, one below the condenser (the polarizer), 
and the other above the objective (the analyzer). 

Note: In cases where the optical design of a microscope produces an appreciable lateral separation of the two beams 
we have the case of classical interference microscopy, which does not result in relief images, but can nevertheless be 
used for the quantitative determination of mass-thicknesses of microscopic objects. 

Interference reflection microscopy 

An additional technique using interference is interference reflection microscopy (also known as reflected 
interference contrast, or RIC). It is used to examine the adhesion of cells to a glass surface, using polarized light of a 
narrow range of wavelengths to be reflected whenever there is an interface between two substances with different 
refractive indices. Whenever a cell is attached to the glass surface, reflected light from the glass and from the 
attached cell will interfere, while if there is no cell attached to the glass, there will be no interference. 

Interference reflection microscopy can be obtained by using the same elements used by DIC, but without the prisms. 
Also, the light that is being detected is reflected and not transmitted as it is when DIC is employed. 

Fluorescence 

When certain compounds are illuminated with high energy light, they then emit light of a different, lower frequency. 
This effect is known as fluorescence. Often specimens show their own characteristic autofluorescence image, based 
on their chemical makeup. 

This method is of critical importance in the modern life sciences, as it can be extremely sensitive, allowing the 
detection of single molecules. Many different fluorescent dyes can be used to stain different structures or chemical 
compounds. One particularly powerful method is the combination of antibodies coupled to a fluorochrome as in 
immunostaining. Examples of commonly used fluorochromes are fluorescein or rhodamine. The antibodies can be 
made tailored specifically for a chemical compound. For example, one strategy often in use is the artificial 
production of proteins, based on the genetic code (DNA). These proteins can then be used to immunize rabbits, 
which then form antibodies which bind to the protein. The antibodies are then coupled chemically to a fluorochrome 
and then used to trace the proteins in the cells under study. 

Highly-efficient fluorescent proteins such as the green fluorescent protein (GFP) have been developed using the 
molecular biology technique of gene fusion, a process that links the expression of the fluorescent compound to that 
of the target protein. Piston DW, Patterson GH, Lippincott-Schwartz J, Claxton NS, Davidson MW (2007). "Nikon 
MicroscopyU: Introduction to Fluorescent Proteins" . Nikon MicroscopyU . Retrieved 2007-08-22. This combined 
fluorescent protein is, in general, non-toxic to the organism and rarely interferes with the function of the protein 
under study. Genetically modified cells or organisms directly express the fluorescently-tagged proteins, which 
enables the study of the function of the original protein in vivo. 

Since fluorescence emission differs in wavelength (color) from the excitation light, an ideal fluorescent image shows 
only the structure of interest that was labeled with the fluorescent dye. This high specificity led to the widespread use 
of fluorescence light microscopy in biomedical research. Different fluorescent dyes can be used to stain different 
biological structures, which can then be detected simultaneously, while still being specific due to the individual color 
of the dye. 

To block the excitation light from reaching the observer or the detector, filter sets of high quality are needed. These 
typically consist of an excitation filter selecting the range of excitation wavelengths, a dichroic mirror, and an 
emission filter blocking the excitation light. Most fluorescence microscopes are operated in the Epi-illumination 
mode (illumination and detection from one side of the sample) to further decrease the amount of excitation light 



Microscopy 190 

entering the detector. 

See also total internal reflection fluorescence microscope. 

Confocal 

Confocal microscopy generates the image in a completely different way to normal "wide-field" microscopes. Using a 
scanning point of light instead of full sample illumination confocal microscopy gives slightly higher resolution, and 
significant improvements in optical sectioning by blocking the influence of out-of-focus light that would otherwise 
degrade the image. Confocal microscopy is, therefore, commonly used where 3D structure is important. 

Deconvolution 

Fluorescence microscopy is extremely powerful due to its ability to show specifically labeled structures within a 
complex environment and also because of its inherent ability to provide three-dimensional information of biological 
structures. However, this information is blurred by the fact that, upon illumination, all fluorescently labeled 
structures emit light no matter whether they are in focus or not. This means that an image of a certain structure is 
always blurred by the contribution of light from structures that are out of focus. This phenomenon becomes apparent 
as a loss of contrast especially when using objectives with a high resolving power, typically oil immersion objectives 
with a high numerical aperture. 

However, this phenomenon is not caused by random processes such as light scattering but can be relatively well 
defined by the optical properties of the image formation in the microscope imaging system. If one considers a small 
fluorescent light source (essentially a bright spot), light coming from this spot spreads out the further out of focus 
one is. Under ideal conditions, this produces a sort of "hourglass" shape of this point source in the third (axial) 
dimension. This shape is called the point spread function (PSF) of the microscope imaging system. Since any 
fluorescence image is made up of a large number of such small fluorescent light sources, the image is said to be 
"convolved by the point spread function". 

Knowing this point spread function means that it is possible to reverse this process to a certain extent by 
computer-based methods commonly known as deconvolution microscopy. There are various algorithms available 
for 2D or 3D deconvolution. They can be roughly classified in nonrestorative and restorative methods. While the 
nonrestorative methods can improve contrast by removing out-of-focus light from focal planes, only the restorative 
methods can actually reassign light to its proper place of origin. This can be an advantage over other types of 3D 
microscopy such as confocal microscopy, because light is not thrown away but reused. For 3D deconvolution, one 
typically provides a series of images derived from different focal planes (called a Z-stack) plus the knowledge of the 
PSF, which can be derived either experimentally or theoretically from knowing all contributing parameters of the 
microscope. 

Sub-diffraction techniques 

It is well known that there is a spatial limit to which light can focus: approximately half of the wavelength of the 
light one is using. But this is not a true barrier, because this diffraction limit is only true in the far-field and 
localization precision can be increased with many photons and careful analysis; and like the sound barrier, the 
diffraction barrier is breakable. This section explores some approaches to imaging objects smaller than -250 nm. In 
1978, the first theoretical ideas had been developed to break this barrier using a 4Pi microscope as a confocal laser 
scanning fluorescence microscope where the light is focused ideally from all sides to a common focus that is used to 
scan the object by 'point-by-point' excitation combined with 'point-by-point' detection . Most of the following 
information was gathered (with permission) from a chemistry blog's review of sub-diffraction microscopy techniques 
Part I and Part II . For a review, see also reference 



Microscopy 



191 



Vertico SMI - SPDMphymod Superresolution Microscopy 

Localization Microscopy/Spatially Structured Illumination 

Around 1995, Christoph Cremer commenced with the development of a light microscopic process, which achieved a 
substantially improved size resolution of cellular nanostructures stained with a fluorescent marker. This time he 
employed the principle of wide field microscopy combined with structured laser illumination (spatially modulated 
illumination, SMI . In addition, this technology is no longer subjected to the speed limitations of the focusing 
microscopy so that it becomes possible to undertake 3D analyses of whole cells within short observation times (at 
the moment around a few seconds). 

Also since around 1995, Christoph Cremer developed and realized new fluorescence-based wide-field microscopy 
approaches that had as their goal the improvement of the effective optical resolution (in terms of the smallest 
detectable distance between two localized objects) down to a fraction of the conventional resolution (spectral 
precision distance/position determination microscopy, SPDM). 

ri2i 

Combining SPDM and SMI, known as Vertico-SMI microscopy Christoph Cremer can currently achieve a 

ri3i 

resolution of approx. 10 nm in 2D and 40 nm in 3D in wide field images of whole living cells . The Vertico SMI 
is currently the fastest optical 3D nanoscope for the three-dimensional structural analysis of whole cells world-wide. 

The Vertico SMI works with high recording speed and processes a complete 3D stack in 40 seconds (2000 frames: 
50 frames/s), the very fast image processing based on specific proprietary algorithms makes the image available after 
2min/3min (l-/2-color). The specific wide-field technique captures very large areas up to 5000 |jm2. 

SPDMphymod: Super Resolution Microscopy Images of standard GFP, RFP, YFP fluorescent 

dyes 




Single YFP molecule 

detection in a human 

cancer cell. Typical 

distance measurements 

in the 15 nm range (5 nm 

standard deviation) 




Co-localisation microscopy (2CLM) with GFP and RFP fusion 

proteins (nucleus of a bone cancer cell) 120.000 localized molecules in 

a widefield area(470 um2) 



Microscopy 192 

Use of standard dyes like normal GFP 

In 2008, Cremer's lab discovered that super resolution microscopy was also possible for many standard fluorescent 
dyes like GFP, Alexa dyes and fluorescein molecules, provided certain photo-physical conditions are present. Using 

his specific localization microscopy called SPDMPhymod, it is possible to detect and count two different fluorescent 

ri4i 
molecule types (this technology is referred to as 2CLM, 2 Color Localization Microscopy) 

Near-field scanning 

Near-field scanning is also called NSOM. Probably the most conceptual way to break the diffraction barrier is to use 
a light source and/or a detector that is itself nanometer in scale. Diffraction as we know it is truly a far-field effect: 
The light from an aperture is the Fourier transform of the aperture in the far-field. But, in the near-field, all of this 
is not necessarily the case. Near-field scanning optical microscopy (NSOM) forces light through the tiny tip of a 
pulled fiber — and the aperture can be on the order of tens of nanometers. When the tip is brought to nanometers 
away from a molecule, the resolution is limited not by diffraction but by the size of the tip aperture (because only 
that one molecule will see the light coming out of the tip). An image can be built by a raster scan of the tip over the 
surface to create an image. 

The main down-side to NSOM is the limited number of photons you can force out a tiny tip, and the minuscule 
collection efficiency (if one is trying to collect fluorescence in the near-field). Other techniques such as ANSOM 
(see below) try to avoid this drawback. 

Local enhancement / ANSOM / optical nano-antennas 

Instead of forcing photons down a tiny tip, some techniques create a local bright spot in an otherwise 
diffraction-limited spot. ANSOM is apertureless NSOM: it uses a tip very close to a fluorophore to enhance the local 

ri7i 

electric field the fluorophore sees. Basically, the ANSOM tip is like a lightning rod which creates a hot spot of 
light. 

Bowtie nanoantennas have been used to greatly and reproducibly enhance the electric field in the nanometer gap 
between the tips two gold triangles. Again, the point is to enhance a very small region of a diffraction-limited spot, 
thus improving the mismatch between light and nanoscale objects — and breaking the diffraction barrier. 

Stimulated emission depletion 

Stefan Hell at the Max Planck Institute for Biophysical Chemistry - Gottingen (Germany) developed STED 
microscopy (stimulated emission depletion), which uses two laser pulses. The first pulse is a diffraction-limited spot 
that is tuned to the absorption wavelength, so excites any fluorophores in that region; an immediate second pulse is 
red-shifted to the emission wavelength and stimulates emission back to the ground state before, thereby depleting the 
excited state of any fluorophores in this depletion pulse. The trick is that the depletion pulse goes through a phase 
modulator that makes the pulse illuminate the sample in the shape of a donut, so the outer part of the diffraction 
limited spot is depleted and the small center can still fluoresce. By saturating the depletion pulse, the center of the 
donut gets smaller and smaller until they can get resolution of tens of nanometers. 

This technique also requires a raster scan like NSOM and standard confocal laser scanning microscopy. 

Fitting the point-spread function 

Fitting the point-spread function (also called PSF). The methods above (and below) use experimental techniques to 
circumvent the diffraction barrier, but one can also use crafty analysis to increase the ability to know where a 
nanoscale object is located. The image of a point source on a charge-coupled device camera is called a point-spread 
function (PSF), which is limited by diffraction to be no less than approximately half the wavelength of the light. But 
it is possible to simply fit that PSF with a Gaussian to locate the center of the PSF — and thus the location of the 
fluorophore. The precision by which this technique can locate the center depends on the number of photons collected 



Microscopy 



193 



T211 
(as well as the CCD pixel size and other factors). This concept was first used to achieve resolution beyond the 

diffraction limit with single molecules by Van Oijen et al. in 1998 (Chem. Phys. Lett. V.292, pl83). Subsequently at 

T221 
room temperature, groups like the Selvin lab and many others have employed this analysis to localize single 

fluorophores to a few nanometers. This, of course, requires careful measurements and collecting many photons. 

PALM, STORM 

What fitting a PSF is to localization, photo-activated localization microscopy (PALM) is to "resolution" — this term 

T231 
is here used loosely to mean measuring the distance between objects, not true optical resolution. Eric Betzig and 

T241 1251 

colleagues developed PALM; Xiaowei Zhuang at Harvard used a similar technique and calls it STORM: 

stochastic optical reconstruction microscopy. Sam Hess at University of Maine developed the technique 

simultaneously . The basic premise of both techniques is to fill the imaging area with many dark fluorophores that 

can be photoactivated into a fluorescing state by a flash of light. Because photoactivation is stochastic, only a few, 

well-separated molecules "turn on." Then Gaussians are fit to their PSFs to high precision (see section above). After 

the few bright dots photobleach, another flash of the photoactivating light activates random fluorophores again and 

the PSFs are fit of these different well-spaced objects. This process is repeated many times, building up an image 

molecule-by-molecule; and, because the molecules were localized at different times, the "resolution" of the final 

image can be much higher than that limited by diffraction. 

The major problem with these techniques is that to get these beautiful pictures, it takes on the order of hours to 
collect the data. This is not the technique to study dynamics (fitting the PSF is better for that). 



Structured illumination 

There is also the wide-field structured-illumination (SI) approach to 

[OQ] [291 

breaking the diffraction limit of light . SI — or patterned 

illumination — relies on both specific microscopy protocols and 
extensive software analysis post-exposure. But, because SI is a 
wide-field technique, it is usually able to capture images at a higher 
rate than confocal-based schemes like STED (This is only a 
generalization, because SI is not actually superfast). The main concept 
of SI is to illuminate a sample with patterned light and increase the 
resolution by measuring the fringes in the Moire pattern (from the 
interference of the illumination pattern and the sample). 
"Otherwise-unobservable sample information can be deduced from the 



fringes and computationally restored. 



.,[30] 



SI enhances spatial resolution by collecting information from 
frequency space outside the observable region. This process is done in 
reciprocal space: The Fourier transform (FT) of an SI image contains 
superimposed additional information from different areas of reciprocal 
space; with several frames with the illumination shifted by some phase, 




Comparison of the resolution obtained by 
confocal laser scanning microscopy (top) and 3D 

structured illumination microscopy 
(3D-SIM-Microscopy, bottom). Shown are details 
of a nuclear envelope. Nuclear pores (anti-NPC) 

red, nuclear envelope (anti-Lamin) green, 
chromatin (DAPI-staining) blue. Scale bars: lum. 



it is possible to computationally separate and reconstruct the FT image, 

which has much more resolution information. The reverse FT returns the reconstructed image to a super-resolution 

image. 



But this enhances the resolution only by a factor of 2 (because the SI pattern cannot be focused to anything smaller 
than half the wavelength of the excitation light). To further increase the resolution, one can introduce nonlinearities, 
which show up as higher-order harmonics in the FT. In reference , Gustafsson uses saturation of the fluorescent 
sample as the nonlinear effect. A sinusoidal saturating excitation beam produces the distorted fluorescence intensity 
pattern in the emission. This nonpolynomial nonlinearity yields a series of higher-order harmonics in the FT. 



Microscopy 



194 



Each higher-order harmonic in the FT allows another set of images that can be used to reconstruct a larger area in 
reciprocal space, and thus a higher resolution. In this case, Gustafsson achieves less than 50-nm resolving power, 
more than five times that of the microscope in its normal configuration. 

The main problems with SI are that, in this incarnation, saturating excitation powers cause more photodamage and 
lower fluorophore photostability, and sample drift must be kept to below the resolving distance. The former 
limitation might be solved by using a different nonlinearity (such as stimulated emission depletion or reversible 
photoactivation, both of which are used in other sub-diffraction imaging schemes); the latter limits live-cell imaging 
and may require faster frame rates or the use of some fiduciary markers for drift subtraction. Nevertheless, SI is 
certainly a strong contender for further application in the field of super-resolution microscopy. 

Images of [[cell nucleuslcell nuclei]] and [[Mitosislmitotic]] stages recorded with 3D-SIM 

Microscopy. 





Comparison 
confocal 

microscopy - 
3D-SIM 



Cell nucleus in prophase from 
various angles 




Two 

mouse cell 

nuclei in 

prophase. 




mouse cell in 
telophase 



Extensions 

Most modern instruments provide simple solutions for micro-photography and image recording electronically. 
However such capabilities are not always present and the more experienced microscopist will, in many cases, still 
prefer a hand drawn image rather than a photograph. This is because a microscopist with knowledge of the subject 
can accurately convert a three dimensional image into a precise two dimensional drawing . In a photograph or other 
image capture system however, only one thin plane is ever in good focus. 

The creation of careful and accurate micrographs requires a microscopical technique using a monocular eyepiece. It 
is essential that both eyes are open and that the eye that is not observing down the microscope is instead concentrated 
on a sheet of paper on the bench besides the microscope. With practice, and without moving the head or eyes, it is 
possible to accurately record the observed details by tracing round the observed shapes by simultaneously "seeing" 
the pencil point in the microscopical image. 

Practicing this technique also establishes good general microscopical technique. It is always less tiring to observe 
with the microscope focused so that the image is seen at infinity and with both eyes open at all times. 



Microscopy 195 

X-ray 

As resolution depends on the wavelength of the light. Electron microscopy has been developed since the 1930s that 
use electron beams instead of light. Because of the much smaller wavelength of the electron beam, resolution is far 
higher. 

Though less common, X-ray microscopy has also been developed since the late 1940s. The resolution of X-ray 
microscopy lies between that of light microscopy and electron microscopy. 

Electron microscopy 

For light microscopy, the wavelength of the light limits the resolution to around 0.2 micrometers. In order to gain 
higher resolution, the use of an electron beam with a far smaller wavelength is used in electron microscopes. 

• Transmission electron microscopy (TEM) is quite similar to the compound light microscope, by sending an 
electron beam through a very thin slice of the specimen. The resolution limit in 2005 was around 0.05 nanometer 
and has not increased appreciably since that time. 

• Scanning electron microscopy (SEM) visualizes details on the surfaces of cells and particles and gives a very nice 
3D view. It gives results much like those of the stereo light microscope, and, akin to that, its most useful 
magnification is in the lower range than that of the transmission electron microscope. 

Atomic de Broglie 



The atomic de Broglie microscope is an imaging system which is expected to provide resolution at the nanometer 

T311 T321 

scale using neutral He atoms as probe particles. . Such a device could provide the resolution at nanometer 

scale and be absolutely non-destructive, but it is not developed as well as optical or electron microscopes. 



Scanning probe microscopy 

This is a sub-diffraction technique. Examples of scanning probe microscopes are the atomic force microscope 
(AFM), the Scanning tunneling microscope and the photonic force microscope. All such methods imply a solid 
probe tip in the vicinity (near field) of an object, which is supposed to be almost flat. 

Ultrasonic force 

Ultrasonic Force Microscopy (UFM) has been developed in order to improve the details and image contrast on "flat" 
areas of interest where the AFM images are limited in contrast. The combination of AFM-UFM allows a near field 
acoustic microscopic image to be generated. The AFM tip is used to detect the ultrasonic waves and overcomes the 
limitation of wavelength that occurs in acoustic microscopy. By using the elastic changes under the AFM tip, an 
image of much greater detail than the AFM topography can be generated. 

Ultrasonic force microscopy allows the local mapping of elasticity in atomic force microscopy by the application of 
ultrasonic vibration to the cantilever or sample. In an attempt to analyze the results of ultrasonic force microscopy in 
a quantitative fashion, a force-distance curve measurement is done with ultrasonic vibration applied to the cantilever 
base, and the results are compared with a model of the cantilever dynamics and tip-sample interaction based on the 
finite-difference technique. 



Microscopy 196 

Infrared microscopy 

The term infrared microscope covers two main types of diffraction-limited microscopy. The first provides optical 
visualization plus IR spectroscopic data collection. The second (more recent and more advanced) technique employs 
focal plane array detection for infrared chemical imaging, where the image contrast is determined by the response of 
individual sample regions to particular IR wavelengths selected by the user. 

T331 
IR versions of sub-diffraction microscopy (see above) exist also. These include IR NSOM and photothermal 

microspectroscopy. 

Digital holographic microscopy 

In digital holographic microscopy (DHM), interfering wave fronts 
from a coherent (monochromatic) light-source are recorded on a 
sensor. The image is digitally reconstructed by a computer from the 
recorded hologram. Besides the ordinary bright field image, a phase 
shift image is created as well. 

DHM can operate both in reflection and transmission mode. In 
reflection mode, the phase shift image provides a relative distance 
measurement and thus represents a topography map of the reflecting 
surface. In transmission mode, the phase shift image provides a 
label-free quantitative measurement of the optical thickness of the 
specimen. Phase shift images of biological cells are very similar to 
images of stained cells and have successfully been analyzed by high 
content analysis software. 




Human cells imaged by DHM phase shift (left) 
and phase contrast microscopy (right). 



A unique feature of DHM is the ability to adjust focus after the image is recorded, since all focus planes are recorded 
simultaneously by the hologram. This feature makes it possible to image moving particles in a volume or to rapidly 
scan a surface. Another attractive feature is DHM's ability to use low cost optics by correcting optical aberrations by 
software. 

Digital Pathology (virtual microscopy) 

Digital Pathology is an image-based information environment enabled by computer technology that allows for the 
management of information generated from a digital slide. Digital pathology is enabled in part by virtual 
microscopy, which is the practice of converting glass slides into digital slides that can be viewed, managed, and 
analyzed. 

Amateur microscopy 

Amateur Microscopy is the investigation and observation of biological and non-biological specimens for recreational 
purposes. Collectors of minerals, insects, seashells, and plants may use microscopes as tools to uncover features that 
help them classify their collected items. Other amateurs may be interested in observing the life found in pond water 
and of other samples. Microscopes may also prove useful for the water quality assessment for people that keep a 
home aquarium. Photographic documentation and drawing of the microscopic images are additional tasks that 
augment the spectrum of tasks of the amateur. There are even competitions for photomicrograph art. Participants of 
this pastime either may use commercially prepared microscopic slides or may engage in the task of specimen 
preparation. 

While microscopy is a central tool in the documentation of biological specimens, it is, in general, insufficient to 
justify the description of a new species based on microscopic investigations alone. Often genetic and biochemical 



Microscopy 



197 



tests are necessary to confirm the discovery of a new species. A laboratory and access to academic literature is a 
necessity, which is specialized and, in general, not available to amateurs. There is, however, one huge advantage that 
amateurs have above professionals: time to explore their surroundings. Often, advanced amateurs team up with 
professionals to validate their findings and (possibly) describe new species. 

In the late 1800s, amateur microscopy became a popular hobby in the United States and Europe. Several 
'professional amateurs' were being paid for their sampling trips and microscopic explorations by philanthropists, to 
keep them amused on the Sunday afternoon (e.g., the diatom specialist A. Grunow, being paid by (among others) a 
Belgian industrialist). Professor John Phin published "Practical Hints on the Selection and Use of the Microscope 
(Second Edition, 1878)," and was also the editor of the "American Journal of Microscopy." 

In 1995, a loose group of amateur microscopists, drawn from several organizations in the UK and USA, founded a 
site for microscopy based on the knowledge and input of amateur (perhaps better referred to as 'enthusiast') 
microscopists. This was the first attempt to establish 'amateur' microscopy as a serious subject in the then-emerging 
new media of the Internet. Today, it remains as a powerful established international resource for all ages, to input 
their findings and share information. It is a nonprofit-making web presence dedicated to the pursuit of science and 
understanding of the small-scale world: [34] 

Examples of amateur microscopy images: 




f t 






"house bee" Mouth 100X 



Rice Stem cs 400X 



Rabbit Testis 100X 



Fern Porothallium 400X 



See also 

• Acronyms in microscopy 

• Digital microscope 

• Digital Pathology 

• Interferometric microscopy 

• Kohler illumination 

• Timeline of microscope technology 

• Two-photon excitation microscopy 



References 

[1] Abramowitz M, Davidson MW (2007). "Introduction to Microscopy" (http://micro.magnet.fsu.edu/primer/anatomy/introduction.html). 

Molecular Expressions. . Retrieved 2007-08-22. 
[2] Abramowitz M, Davidson MW (2003-08-01). "Darkfield Illumination" (http://micro.magnet.fsu.edu/primer/techniques/darkfield.html). 

Retrieved 2008-10-21. 
[3] Abramowitz M, Davidson MW (2003-08-01). "Rheinberg Illumination" (http://micro.magnet.fsu.edu/primer/techniques/rheinberg. 

html). . Retrieved 2008-10-21. 
[4] http://www.microscopyu.com/articles/livecellimaging/fpintro.html 
[5] Nasse M. J., Woehl J. C. (2010). "Realistic modeling of the illumination point spread function in confocal scanning optical microscopy". 

Opt. Soc. Am. A 27 (2): 295-302. doi:10.1364/IOSAA.27.000295. 
[6] Wallace W, Schaefer LH, Swedlow IR (2001). "A workingperson's guide to deconvolution in light microscopy". BioTechniques 31 (5): 

1076-8, 1080, 1082 passim. PMID 11730015. 
[7] Considerations on a laser-scanning-microscope with high resolution and depth of field: C. Cremer and T. Cremer in M1CROSCOPICA 

ACTA VOL. 81 NUMBER 1 September.pp. 31—44 (1978) 



Microscopy 198 

[8] http://blog.everydayscientist.com/?p=184 

[9] http://blog.everydayscientist.com/?p=354 

[10] WEM News and Views (http://dx.doi.org/10.1038/nmethl006-781) 

[11] Nano- structure analysis using Spatially Modulated Illumination microscopy: D. Baddeley, C. Batram, Y. Weiland, C. Cremer, UJ. Birk in 

NATURE PROTOCOLS, Vol 2, pp. 2640-2646 (2007) 
[12] High-precision structural analysis of subnuclear complexes in fixed and live cells via Spatially Modulated Illumination (SMI) microscopy: J. 

Reymann, D. Baddeley, P. Lemmer, W. Stadter, T. Jegou, K. Rippe, C. Cremer, U. Birk in CHROMOSOME RESEARCH, Vol. 16, pp. 367 

-382 (2008) 
[13] SPDM — Light Microscopy with Single Molecule Resolution at the Nanoscale: P. Lemmer, M.Gunkel, D. Baddeley, R. Kaufmann, A. Urich, 

Y. Weiland, J.Reymann, P. Milller, M. Hausmann, C. Cremer in APPLIED PHYSICS B, Vol 93, pp. 1-12 (2008). 
[14] Manuel Gunkel, Fabian Erdel, Karsten Rippe, Paul Lemmer, Rainer Kaufmann, Christoph Hormann, Roman Amberger and Christoph 

Cremer: Dual color localization microscopy of cellular nanostructures. In: Biotechnology Journal, 2009, 4, 927-938. ISSN 1860-6768 
[15] "Fresnel Diffraction Applet" (http://www.falstad.com/diffraction/) (Java applet). . Retrieved 2007-08-22. 
[16] Cummings JR, Fellers TJ, Davidson MW (2007). "Specialized Microscopy Techniques - Near-Field Scanning Optical Microscopy" (http:// 

www.olympusmicro.com/primer/techniques/nearfield/nearfieldintro.html). Olympus Microscopy Resource Center. . Retrieved 

2007-08-22. 
[17] Sanchez EJ, Novotny L, Xie XS (1999). "Near-Field Fluorescence Microscopy Based on Two-Photon Excitation with Metal Tips" (http:// 

link.aps.org/abstract/PRL/v82/p4014). Phys Rev Lett 82: 4014-7. doi:10.1 103/PhysRevLett.82.4014. . 
[18] Schuck PJ, Fromm DP, Sundaramurthy A, Kino GS, Moerner WE (2005). "Improving the Mismatch between Light and Nanoscale Objects 

with Gold Bowtie Nanoantennas" (http://link.aps.org/abstract/PRL/v94/e017402). Phys Rev Lett 94 (1): 017402. 

doi:10.1103/PhysRevLett.94.017402. PMID 15698131. . 
[19] Muhlschlegel P, Eisler H-J, Martin OJF, Hecht B, Pohl DW (2005). "Resonant optical antennas" (http://www.sciencemag.org/cgi/ 

content/abstract/308/5728/1607). Science 308: 1607. doi:10.1126/science.H11886. . 
[20] STED (http://dx.doi.org/10.1038/nbt895) 

[21] Webb paper (http://www.biophysj.Org/cgi/content/abstract/82/5/2775) 
[22] http://www.physics.uiuc.edu/people/Selvin/index.htm 
[23] http://www.hhmi.org/news/betzig.html 
[24] PALM (http://dx.doi.org/10. 1 126/science. 1 127344) 
[25] http://zhuang.harvard.edu/ 
[26] STORM (http://dx.doi.org/10.1038/nmeth929) 
[27] http ://dx. doi. org/ 10.1 529/biophy sj . 1 06. 09 1 1 1 6 
[28] Bailey, B.; Farkas, D. L.; Taylor, D. L.; Lanni, F. Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation 

(http://dx.doi.org/10.1038/366044a0). Nature 1993, 366, 44-48. 
[29] Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy (http://dx.doi.org/ 

10.1046/j.l365-2818.2000.00710.x). J. ofMicrosc. 2000, 198(2), 82-87. 
[30] Gustafsson, M. G. L. Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution 

(http://dx.doi.org/10.1073/pnas.0406877102). PNAS 2005, 702(37), 13081-13086. 
[31] D.Kouznetsov; H. Oberst, K. Shimizu, A. Neumann, Y. Kuznetsova, J.-F. Bisson, K. Ueda, S. R. J. Brueck (2006). "Ridged atomic mirrors 

and atomic nanoscope" (http://stacks.iop.org/0953-4075/39/1605). JOPB 39: 1605-1623. doi: 10. 1088/0953-4075/39/7/005. . 
[32] Atom Optics and Helium Atom Microscopy. Cambridge University, http://www-sp.phy.cam.ac.uk/research/mirror.php3 
[33] H M Pollock and D A Smith, The use of near-field probes for vibrational spectroscopy and photothermal imaging, in Handbook of 

vibrational spectroscopy, J.M. Chalmers and P.R. Griffiths (eds), John Wiley & Sons Ltd, Vol. 2, pp. 1472 - 1492 (2002) 
[34] http ://www. microscopy-uk.org. uk 

Further reading 

• Advanced Light Microscopy vol. 1 Principles and Basic Properties by Maksymilian Pluta, Elsevier (1988) 

• Advanced Light Microscopy vol. 2 Specialised Methods by Maksymilian Pluta, Elsevier (1989) 

• Introduction to Light Microscopy by S. Bradbury, B. Bracegirdle, BIOS Scientific Publishers (1998) 

• Video Microscopy by Shinya Inoue, Plenum Press (1986) 

• (http://www.kip.uni-heidelberg.de/AG_Cremer/pdf-files/Cremer_Micros_Acta_1978.pdf) 1978: Theoretical 
basis of super resolution 4Pi microscopy & design of a confocal laser scanning fluorescence microscope 

• Portraits of life, one molecule at a time (http://pubs.acs.org/subscribe/journals/ancham/79/i05/pdf/ 
0307feature_willis.pdf), a feature article on sub-diffraction microscopy from the March 1, 2007 issue of 
Analytical Chemistry (http://pubs3. acs.org/acs/journals/toc. page?incoden=ancham&indecade=0& 
involume=79&inissue=5) 



Microscopy 199 

External links 
General 

• (http://www.imaging-git.com/news/world-s-fastest-optical-microscope-chosen-best-business-idea) Bwcon 
award: world's fastest superresolution microscope as best business idea 

GFP Superresolution (PDF file; 330 kB) (http://www.tlb.de/uploads/media/GFP_Superresolution_01.pdf) 
Olympus Microscopy Resource Center (http://www.olympusmicro.com/) ( website critique (http://www. 
genengnews.com/bestofweb/list. aspx?iid=93)) 
Nikon MicroscopyU (http://www.microscopyu.com) 

Andor Microscopy Techniques (http://www.andor.com/microscopy_systems/techniques/) - Various 
techniques used in microscopy. 

Carl Zeiss "Microscopy from the very beginning" (http://www.zeiss.de/C1256B5E0047FF3F7Open), a step by 
step tutorial into the basics of microscopy. 

Microscopy in Detail (http://www.biologie.uni-hamburg.de/b-online/e03/03.htm) - A resource with many 
illustrations elaborating the most common microscopy techniques 

Microscopy Information (http://www.microbehunter.com) - Microscopy information and techniques for 
teachers, educators and enthusiasts. 

WITec SNOM System (http://witec.de/en/products/snom/alpha300s/) - NSOM/SNOM and Hybrid 
Microscopy techniques in combination with AFM, RAMAN, Confocal, Dark-field, DIC & Fluorescence 
Microscopy techniques. 

Manawatu Microscopy (http://confocal-manawatu.pbworks.com/) - first known collaboration environment for 
Microscopy and Image Analysis. 

Audio microscope glossary (http://www.histology-world.com/microscope/audiomicroscope/ 
audiomicroscope. htm) 

PSF Lab (http://onemolecule.chem.uwm.edu/software), freeware (for academic use) permitting the calculation 
of the Point Spread Function in stratified media including polarization effects based on a rigorous vector-based 
model. 

Techniques 

• Ratio-metric Imaging Applications For Microscopes (http://www.pti-nj.com/EasyRatio/ 
EasyRatioPro-Applications.html) Examples of Ratiometric Imaging Work on a Microscope 

• Interactive Fluorescence Dye and Filter Database (https://www. micro-shop. zeiss.com/?s=2525647761b33& 
l=en&p=us&f=f) Carl Zeiss Interactive Fluorescence Dye and Filter Database. 

• Images formed by simple microscopes (http://www.brianjford.com/wav-spf.htm) - examples of observations 
with single-lens microscopes. 

Organizations 

• Royal Microscopical Society (http://www.rms.org.uk/) (RMS) 

• Microscopy Society of America (http://www.microscopy.org/) (MSA) 

• European Microscopy Society (http://www.eurmicsoc.org/) (EMS) 

• Non-membership International online organisation (http://www.microscopy-uk.org.uk/) (Mic-UK) 



X-ray crystallography 



200 



X-ray crystallography 



X-ray crystallography is a method of determining the arrangement of 
atoms within a crystal, in which a beam of X-rays strikes a crystal and 
diffracts into many specific directions. From the angles and intensities 
of these diffracted beams, a crystallographer can produce a 
three-dimensional picture of the density of electrons within the crystal. 
From this electron density, the mean positions of the atoms in the 
crystal can be determined, as well as their chemical bonds, their 
disorder and various other information. 



•Vr. ~< 



) «*V."«vr 



. m 






1 ■>*.'>*""«• 



X-ray crystallography can locate every atom in a 

zeolite, an aluminosilicate with many important 

applications, such as water purification. 



Since many materials can form crystals — such as salts, metals, 

minerals, semiconductors, as well as various inorganic, organic and 

biological molecules — X-ray crystallography has been fundamental in 

the development of many scientific fields. In its first decades of use, 

this method determined the size of atoms, the lengths and types of 

chemical bonds, and the atomic-scale differences among various 

materials, especially minerals and alloys. The method also revealed the 

structure and functioning of many biological molecules, including 

vitamins, drugs, proteins and nucleic acids such as DNA. X-ray crystallography is still the chief method for 

characterizing the atomic structure of new materials and in discerning materials that appear similar by other 

experiments. X-ray crystal structures can also account for unusual electronic or elastic properties of a material, shed 

light on chemical interactions and processes, or serve as the basis for designing pharmaceuticals against diseases. 

In an X-ray diffraction measurement, a crystal is mounted on a goniometer and gradually rotated while being 
bombarded with X-rays, producing a diffraction pattern of regularly spaced spots known as reflections. The 
two-dimensional images taken at different rotations are converted into a three-dimensional model of the density of 
electrons within the crystal using the mathematical method of Fourier transforms, combined with chemical data 
known for the sample. Poor resolution (fuzziness) or even errors may result if the crystals are too small, or not 
uniform enough in their internal makeup. 

X-ray crystallography is related to several other methods for determining atomic structures. Similar diffraction 
patterns can be produced by scattering electrons or neutrons, which are likewise interpreted as a Fourier transform. If 
single crystals of sufficient size cannot be obtained, various other X-ray methods can be applied to obtain less 
detailed information; such methods include fiber diffraction, powder diffraction and small-angle X-ray scattering 
(SAXS). If the material under investigation is only available in the form of nanocrystalline powders or suffers from 
poor crystallinity, the methods of electron crystallography can be applied for determining the atomic structure. 

For all above mentioned X-ray diffraction methods, the scattering is elastic; the scattered X-rays have the same 
wavelength as the incoming X-ray. By contrast, inelastic X-ray scattering methods are useful in studying excitations 
of the sample, rather than the distribution of its atoms. 



X-ray crystallography 



201 



History 



Early scientific history of crystals and X-rays 

Crystals have long been admired for their regularity and symmetry, but 
they were not investigated scientifically until the 17th century. 
Johannes Kepler hypothesized in his work Strena seu de Nive 
Sexangula (1611) that the hexagonal symmetry of snowflake crystals 
was due to a regular packing of spherical water particles 



[l] 



\74 mjj &dftru$pr&wi [ohdoTHi 

I an! 






*vnejkprafi, &&b we infrtjfe : c 



Drawing of square (Figure A, above) and 

hexagonal (Figure B, below) packing from 

Kepler's work, Strena seu de Nive Sexangula. 




As shown by X-ray crystallography, the 

hexagonal symmetry of snowflakes results from 

the tetrahedral arrangement of hydrogen bonds 

about each water molecule. The water molecules 

are arranged similarly to the silicon atoms in the 

tridymite polymorph of SiO The resulting 

crystal structure has hexagonal symmetry when 

viewed along a principal axis. 



Crystal symmetry was first investigated experimentally by Nicolas 
Steno (1669), who showed that the angles between the faces are the 
same in every exemplar of a particular type of crystal, and by Rene 
Just Haiiy (1784), who discovered that every face of a crystal can be 
described by simple stacking patterns of blocks of the same shape and 
size. Hence, William Hallowes Miller in 1839 was able to give each 
face a unique label of three small integers, the Miller indices which are 
still used today for identifying crystal faces. Hauy's study led to the 
correct idea that crystals are a regular three-dimensional array (a 
Bravais lattice) of atoms and molecules; a single unit cell is repeated 
indefinitely along three principal directions that are not necessarily 
perpendicular. In the 19th century, a complete catalog of the possible 
symmetries of a crystal was worked out by Johann Hessel, Auguste 
Bravais, Yevgraf Fyodorov, Arthur Schonflies and (belatedly) 
William Barlow. From the available data and physical reasoning, 
Barlow proposed several crystal structures in the 1880s that were 
validated later by X-ray crystallography; however, the available data 
were too scarce in the 1880s to accept his models as conclusive. 



X-ray crystallography 



202 




Hydrogen 
bonds 



X-rays were discovered by Wilhelm Conrad Rontgen in 1895, just as 

the studies of crystal symmetry were being concluded. Physicists were 

initially uncertain of the nature of X-rays, although it was soon 

suspected (correctly) that they were waves of electromagnetic 

radiation, in other words, another form of light. At that time, the wave 

model of light — specifically, the Maxwell theory of electromagnetic 

radiation — was well accepted among scientists, and experiments by 

Charles Glover Barkla showed that X-rays exhibited phenomena 

associated with electromagnetic waves, including transverse 

polarization and spectral lines akin to those observed in the visible 

wavelengths. Single-slit experiments in the laboratory of Arnold 

Sommerfeld suggested the wavelength of X-rays was about 1 

Angstrom. However, X-rays are composed of photons, and thus are not 

only waves of electromagnetic radiation but also exhibit particle-like 

properties. The photon concept was introduced by Albert Einstein in 

1905, but it was not broadly accepted until 1922, when Arthur 

Compton confirmed it by the scattering of X-rays from electrons. 

Therefore, these particle-like properties of X-rays, such as their ionization of gases, caused William Henry Bragg to 

argue in 1907 that X-rays were not electromagnetic radiation. Nevertheless, Bragg's view was not 

broadly accepted and the observation of X-ray diffraction in 1912 confirmed for most scientists that X-rays were 

a form of electromagnetic radiation. 



X-ray crystallography shows the arrangement of 

water molecules in ice, revealing the hydrogen 

bonds that hold the solid together. Few other 

methods can determine the structure of matter 

with such sub-atomic precision (resolution). 



X-ray analysis of crystals 




The incoming beam (coming from upper left) 

causes each scatterer to re-radiate a small portion 

of its intensity as a spherical wave. If scatterers 

are arranged symmetrically with a separation d, 

these spherical waves will be in sync (add 

constructively) only in directions where their 

path-length difference 2d sin 8 equals an integer 

multiple of the wavelength X. In that case, part of 

the incoming beam is deflected by an angle 28, 

producing a reflection spot in the diffraction 

pattern. 



Crystals are regular arrays of atoms, and X-rays can be considered 
waves of electromagnetic radiation. Atoms scatter X-ray waves, 
primarily through the atoms' electrons. Just as an ocean wave striking a 
lighthouse produces secondary circular waves emanating from the 
lighthouse, so an X-ray striking an electron produces secondary 
spherical waves emanating from the electron. This phenomenon is 
known as elastic scattering, and the electron (or lighthouse) is known 
as the scatterer. A regular array of scatterers produces a regular array 
of spherical waves. Although these waves cancel one another out in 
most directions through destructive interference, they add 
constructively in a few specific directions, determined by Bragg's law: 



2d sin 9 = nX 

Here d is the spacing between diffracting planes, Q is the incident angle, n is any integer, and "k is the wavelength of 
the beam. These specific directions appear as spots on the diffraction pattern called reflections. Thus, X-ray 
diffraction results from an electromagnetic wave (the X-ray) impinging on a regular array of scatterers (the repeating 
arrangement of atoms within the crystal). 



X-ray crystallography 203 

X-rays are used to produce the diffraction pattern because their wavelength X is typically the same order of 
magnitude (1-100 Angstroms) as the spacing d between planes in the crystal. In principle, any wave impinging on a 
regular array of scatterers produces diffraction, as predicted first by Francesco Maria Grimaldi in 1665. To produce 
significant diffraction, the spacing between the scatterers and the wavelength of the impinging wave should be 
similar in size. For illustration, the diffraction of sunlight through a bird's feather was first reported by James 
Gregory in the later 17th century. The first artificial diffraction gratings for visible light were constructed by David 
Rittenhouse in 1787, and Joseph von Fraunhofer in 1821. However, visible light has too long a wavelength 
(typically, 5500 Angstroms) to observe diffraction from crystals. Prior to the first X-ray diffraction experiments, the 
spacings between lattice planes in a crystal were not known with certainty. 

The idea that crystals could be used as a diffraction grating for X-rays arose in 1912 in a conversation between Paul 
Peter Ewald and Max von Laue in the English Garden in Munich. Ewald had proposed a resonator model of crystals 
for his thesis, but this model could not be validated using visible light, since the wavelength was much larger than 
the spacing between the resonators. Von Laue realized that electromagnetic radiation of a shorter wavelength was 
needed to observe such small spacings, and suggested that X-rays might have a wavelength comparable to the 
unit-cell spacing in crystals. Von Laue worked with two technicians, Walter Friedrich and his assistant Paul 
Knipping, to shine a beam of X-rays through a copper sulfate crystal and record its diffraction on a photographic 
plate. After being developed, the plate showed a large number of well-defined spots arranged in a pattern of 
intersecting circles around the spot produced by the central beam. Von Laue developed a law that connects 

the scattering angles and the size and orientation of the unit-cell spacings in the crystal, for which he was awarded 
the Nobel Prize in Physics in 1914. [18] 

As described in the mathematical derivation below, the X-ray scattering is determined by the density of electrons 
within the crystal. Since the energy of an X-ray is much greater than that of a valence electron, the scattering may be 
modeled as Thomson scattering, the interaction of an electromagnetic ray with a free electron. This model is 
generally adopted to describe the polarization of the scattered radiation. The intensity of Thomson scattering declines 
as 1/ra 2 with the mass m of the charged particle that is scattering the radiation; hence, the atomic nuclei, which are 
thousands of times heavier than an electron, contribute negligibly to the scattered X-rays. 



X-ray crystallography 



204 



Development from 1912 to 1920 

After Von Laue's pioneering research, the field developed rapidly, 
most notably by physicists William Lawrence Bragg and his father 
William Henry Bragg. In 1912-1913, the younger Bragg developed 
Bragg's law, which connects the observed scattering with reflections 
from evenly spaced planes within the crystal. The Braggs, 

father and son, shared the 1915 Nobel Prize in Physics for their work 
in crystallography. The earliest structures were generally simple and 
marked by one-dimensional symmetry. However, as computational and 
experimental methods improved over the next decades, it became 
feasible to deduce reliable atomic positions for more complicated two- 
and three-dimensional arrangements of atoms in the unit-cell. 

The potential of X-ray crystallography for determining the structure of 
molecules and minerals — then only known vaguely from chemical 
and hydrodynamic experiments — was realized immediately. The 
earliest structures were simple inorganic crystals and minerals, but 
even these revealed fundamental laws of physics and chemistry. The 
first atomic-resolution structure to be "solved" (i.e. determined) in 

[221 [231 [241 

1914 was that of table salt. The distribution of electrons in 

the table-salt structure showed that crystals are not necessarily 

composed of covalently bonded molecules, and proved the existence of 

[251 
ionic compounds. The structure of diamond was solved in the same 

year, proving the tetrahedral arrangement of its chemical bonds 

and showing that the length of C— C single bond was 1.52 Angstroms. 

no] 

Other early structures included copper, calcium fluoride (CaF , also 

[291 
known as fluorite), calcite (CaCO ) and pyrite (FeS ) in 1914; 

spinel (MgAl O ) in 1915; the rutile and anatase forms of 

[321 

titanium dioxide (TiO ) in 1916; pyrochroite Mn(OH) and, by 
extension, brucite Mg(OH) 2 in 1919;. [33] [34] Also in 1919 sodium 
nitrate (NaNO ) and cesium dichloroiodide (CsICl ) were determined 
by Ralph Walter Graystone Wyckoff, and the wurtzite (hexagonal 




Although diamonds (top left) and graphite (top 

right) are identical in chemical composition — 

being both pure carbon — X-ray crystallography 

revealed the arrangement of their atoms (bottom) 

accounts for their different properties. In 

diamond, the carbon atoms are arranged 

tetrahedrally and held together by single covalent 

bonds, making it strong in all directions. By 

contrast, graphite is composed of stacked sheets. 

Within the sheet, the bonding is covalent and has 

hexagonal symmetry, but there are no covalent 

bonds between the sheets, making graphite easy 

to cleave into flakes. 



ZnS) structure became known in 1920 



[35] 



The structure of graphite was solved in 1916 by the related method of powder diffraction, which was 

T3R1 

developed by Peter Debye and Paul Scherrer and, independently, by Albert Hull in 1917. The structure of 

graphite was determined from single-crystal diffraction in 1924 by two groups independently. Hull also used 

T411 [421 

the powder method to determine the structures of various metals, such as iron and magnesium. 



Contributions to chemistry and material science 

X-ray crystallography has led to a better understanding of chemical bonds and non-covalent interactions. The initial 
studies revealed the typical radii of atoms, and confirmed many theoretical models of chemical bonding, such as the 
tetrahedral bonding of carbon in the diamond structure, the octahedral bonding of metals observed in ammonium 

[431 [291 

hexachloroplatinate (IV), and the resonance observed in the planar carbonate group and in aromatic 

[441 [451 

molecules. Kathleen Lonsdale's 1928 structure of hexamethylbenzene established the hexagonal symmetry of 



X-ray crystallography 205 

benzene and showed a clear difference in bond length between the aliphatic C— C bonds and aromatic C— C bonds; 
this finding led to the idea of resonance between chemical bonds, which had profound consequences for the 
development of chemistry. Her conclusions were anticipated by William Henry Bragg, who published models of 

[441 [47] 

naphthalene and anthracene in 1921 based on other molecules, an early form of molecular replacement. 

Also in the 1920s, Victor Moritz Goldschmidt and later Linus Pauling developed rules for eliminating chemically 
unlikely structures and for determining the relative sizes of atoms. These rules led to the structure of brookite (1928) 
and an understanding of the relative stability of the rutile, brookite and anatase forms of titanium dioxide. 

The distance between two bonded atoms is a sensitive measure of the bond strength and its bond order; thus, X-ray 
crystallographic studies have led to the discovery of even more exotic types of bonding in inorganic chemistry, such 

as metal-metal double bonds, metal-metal quadruple bonds, and three-center, two-electron 

T541 
bonds. X-ray crystallography — or, strictly speaking, an inelastic Compton scattering experiment — has also 

provided evidence for the partly covalent character of hydrogen bonds. In the field of organometallic chemistry, 

the X-ray structure of ferrocene initiated scientific studies of sandwich compounds, while that of Zeise's salt 

stimulated research into "back bonding" and metal-pi complexes. Finally, X-ray crystallography had a 

pioneering role in the development of supramolecular chemistry, particularly in clarifying the structures of the crown 

ethers and the principles of host-guest chemistry. 

In material sciences, many complicated inorganic and organometallic systems have been analyzed using 
single-crystal methods, such as fullerenes, metalloporphyrins, and other complicated compounds. Single-crystal 
diffraction is also used in the pharmaceutical industry, due to recent problems with polymorphs. The major factors 
affecting the quality of single-crystal structures are the crystal's size and regularity; recrystallization is a commonly 
used technique to improve these factors in small-molecule crystals. The Cambridge Structural Database contains 
over 500,000 structures; over 99% of these structures were determined by X-ray diffraction. 

Mineralogy and metallurgy 

Since the 1920s, X-ray diffraction has been the principal method for determining the arrangement of atoms in 
minerals and metals. The application of X-ray crystallography to mineralogy began with the structure of garnet, 
which was determined in 1924 by Menzer. A systematic X-ray crystallographic study of the silicates was undertaken 
in the 1920s. This study showed that, as the Si/O ratio is altered, the silicate crystals exhibit significant changes in 
their atomic arrangements. Machatschki extended these insights to minerals in which aluminium substitutes for the 
silicon atoms of the silicates. The first application of X-ray crystallography to metallurgy likewise occurred in the 
mid-1920s. Most notably, Linus Pauling's structure of the alloy Mg Sn led to his theory of 

the stability and structure of complex ionic crystals. 



X-ray crystallography 



206 



Early organic and small biological molecules 

The first structure of an organic compound, hexamethylenetetramine, 
was solved in 1923. This was followed by several studies of 
long-chain fatty acids, which are an important component of biological 
membranes. [71] [72] [73] [74] [75] [76] [77] [78] [79] In the 1930s, the 
structures of much larger molecules with two-dimensional complexity 
began to be solved. A significant advance was the structure of 
phthalocyanine, a large planar molecule that is closely related to 
porphyrin molecules important in biology, such as heme, corrin and 
chlorophyll. 

X-ray crystallography of biological molecules took off with Dorothy 
Crowfoot Hodgkin, who solved the structures of cholesterol (1937), 
vitamin B12 (1945) and penicillin (1954), for which she was awarded 
the Nobel Prize in Chemistry in 1964. In 1969, she succeeded in 
solving the structure of insulin, on which she worked for over thirty years. 




The three-dimensional structure of penicillin, for 

which Dorothy Crowfoot Hodgkin was awarded 

the Nobel Prize in Chemistry in 1964. The green, 

white, red, yellow and blue spheres represent 

atoms of carbon, hydrogen, oxygen, sulfur and 

nitrogen, respectively. 

[81] 




Ribbon diagram of the structure of myoglobin, 
showing colored alpha helices. Such proteins are 
long, linear molecules with thousands of atoms; 

yet the relative position of each atom has been 
determined with sub-atomic resolution by X-ray 
crystallography. Since it is difficult to visualize 
all the atoms at once, the ribbon shows the rough 
path of the protein polymer from its N-terminus 
(blue) to its C-terminus (red). 

such as ion channels and receptors. 



Biological macromolecular crystallography 

Crystal structures of proteins (which are irregular and hundreds of 
times larger than cholesterol) began to be solved in the late 1950s, 
beginning with the structure of sperm whale myoglobin by Max Perutz 
and Sir John Cowdery Kendrew, for which they were awarded the 

TR21 

Nobel Prize in Chemistry in 1962. Since that success, over 48970 
X-ray crystal structures of proteins, nucleic acids and other biological 
molecules have been determined. For comparison, the nearest 
competing method in terms of structures analyzed is nuclear magnetic 
resonance (NMR) spectroscopy, which has resolved 7806 chemical 
structures. Moreover, crystallography can solve structures of 
arbitrarily large molecules, whereas solution-state NMR is restricted to 
relatively small ones (less than 70 kDa). X-ray crystallography is now 
used routinely by scientists to determine how a pharmaceutical drug 

roc] 

interacts with its protein target and what changes might improve it. 
However, intrinsic membrane proteins remain challenging to 
crystallize because they require detergents or other means to solubilize 
them in isolation, and such detergents often interfere with 
crystallization. Such membrane proteins are a large component of the 
genome and include many proteins of great physiological importance, 



Relationship to other scattering techniques 



Elastic vs. inelastic scattering 

X-ray crystallography is a form of elastic scattering; the outgoing X-rays have the same energy, and thus same 
wavelength, as the incoming X-rays, only with altered direction. By contrast, inelastic scattering occurs when energy 
is transferred from the incoming X-ray to the crystal, e.g., by exciting an inner-shell electron to a higher energy 



X-ray crystallography 207 

level. Such inelastic scattering reduces the energy (or increases the wavelength) of the outgoing beam. Inelastic 
scattering is useful for probing such excitations of matter, but not in determining the distribution of scatterers within 
the matter, which is the goal of X-ray crystallography. 

X-rays range in wavelength from 10 to 0.01 nanometers; a typical wavelength used for crystallography is 1 A 
(0.1 nm), which is on the scale of covalent chemical bonds and the radius of a single atom. Longer-wavelength 
photons (such as ultraviolet radiation) would not have sufficient resolution to determine the atomic positions. At the 
other extreme, shorter-wavelength photons such as gamma rays are difficult to produce in large numbers, difficult to 
focus, and interact too strongly with matter, producing particle-antiparticle pairs. Therefore, X-rays are the 
"sweetspot" for wavelength when determining atomic-resolution structures from the scattering of electromagnetic 
radiation. 

Other X-ray techniques 

Other forms of elastic X-ray scattering include powder diffraction, SAXS and several types of X-ray fiber 
diffraction, which was used by Rosalind Franklin in determining the double-helix structure of DNA. In general, 
single-crystal X-ray diffraction offers more structural information than these other techniques; however, it requires a 
sufficiently large and regular crystal, which is not always available. 

These scattering methods generally use monochromatic X-rays, which are restricted to a single wavelength with 
minor deviations. A broad spectrum of X-rays (that is, a blend of X-rays with different wavelengths) can also be 
used to carry out X-ray diffraction, a technique known as the Laue method. This is the method used in the original 
discovery of X-ray diffraction. Laue scattering provides much structural information with only a short exposure to 
the X-ray beam, and is therefore used in structural studies of very rapid events (Time resolved crystallography). 
However, it is not as well-suited as monochromatic scattering for determining the full atomic structure of a crystal 
and therefore works better with crystals with relatively simple atomic arrangements. 

The Laue back reflection mode records X-rays scattered backwards from a broad spectrum source. This is useful if 
the sample is too thick for X-rays to transmit through it. The diffracting planes in the crystal are determined by 
knowing that the normal to the diffracting plane bisects the angle between the incident beam and the diffracted beam. 

roo] 

A Greninger chart can be used to interpret the back reflection Laue photograph. 

Electron and neutron diffraction 

Other particles, such as electrons and neutrons, may be used to produce a diffraction pattern. Although electron, 
neutron, and X-ray scattering are based on different physical processes, the resulting diffraction patterns are analyzed 
using the same coherent diffraction imaging techniques. 

As derived below, the electron density within the crystal and the diffraction patterns are related by a simple 
mathematical method, the Fourier transform, which allows the density to be calculated relatively easily from the 
patterns. However, this works only if the scattering is weak, i.e., if the scattered beams are much less intense than the 
incoming beam. Weakly scattered beams pass through the remainder of the crystal without undergoing a second 
scattering event. Such re-scattered waves are called "secondary scattering" and hinder the analysis. Any sufficiently 
thick crystal will produce secondary scattering, but since X-rays interact relatively weakly with the electrons, this is 
generally not a significant concern. By contrast, electron beams may produce strong secondary scattering even for 
relatively thin crystals (>100 nm). Since this thickness corresponds to the diameter of many viruses, a promising 
direction is the electron diffraction of isolated macromolecular assemblies, such as viral capsids and molecular 
machines, which may be carried out with a cryo-electron microscope. Moreover the strong interaction of electrons 
with matter (about 1000 times stronger than for X-rays) allows determination of the atomic structure of extremely 
small volumes. The field of applications for electron crystallography ranges from bio molecules like membrane 
proteins over organic thin films to the complex structures of (nanocrystalline) intermetallic compounds and zeolites. 



X-ray crystallography 



208 



Neutron diffraction is an excellent method for structure determination, although it has been difficult to obtain 
intense, monochromatic beams of neutrons in sufficient quantities. Traditionally, nuclear reactors have been used, 
although the new Spallation Neutron Source holds much promise in the near future. Being uncharged, neutrons 
scatter much more readily from the atomic nuclei rather than from the electrons. Therefore, neutron scattering is very 
useful for observing the positions of light atoms with few electrons, especially hydrogen, which is essentially 
invisible in the X-ray diffraction. Neutron scattering also has the remarkable property that the solvent can be made 
invisible by adjusting the ratio of normal water, HO, and heavy water, DO. 

Methods 



Overview of single-crystal X-ray diffraction 

The oldest and most precise method of X-ray crystallography is 
single-crystal X-ray diffraction, in which a beam of X-rays strikes a 
single crystal, producing scattered beams. When they land on a piece 
of film or other detector, these beams make a diffraction pattern of 
spots; the strengths and angles of these beams are recorded as the 
crystal is gradually rotated. Each spot is called a reflection, since it 
corresponds to the reflection of the X-rays from one set of evenly 
spaced planes within the crystal. For single crystals of sufficient purity 
and regularity, X-ray diffraction data can determine the mean chemical 
bond lengths and angles to within a few thousandths of an Angstrom 
and to within a few tenths of a degree, respectively. The atoms in a 
crystal are not static, but oscillate about their mean positions, usually 
by less than a few tenths of an Angstrom. X-ray crystallography allows 
measuring the size of these oscillations. 

Procedure 

The technique of single-crystal X-ray crystallography has three basic 
steps. The first — and often most difficult — step is to obtain an 
adequate crystal of the material under study. The crystal should be 
sufficiently large (typically larger than 0. 1 mm in all dimensions), pure 
in composition and regular in structure, with no significant internal imperfections such as cracks or twinning. 

In the second step, the crystal is placed in an intense beam of X-rays, usually of a single wavelength {monochromatic 
X-rays), producing the regular pattern of reflections. As the crystal is gradually rotated, previous reflections 
disappear and new ones appear; the intensity of every spot is recorded at every orientation of the crystal. Multiple 
data sets may have to be collected, with each set covering slightly more than half a full rotation of the crystal and 
typically containing tens of thousands of reflections. 

In the third step, these data are combined computationally with complementary chemical information to produce and 
refine a model of the arrangement of atoms within the crystal. The final, refined model of the atomic arrangement — 
now called a crystal structure — is usually stored in a public database. 



'*&■ crystal 


x-rays H 






diffraction 
pattern 




phases 1 


m 

C 
V 

E 

V 

c 
c 
y 


f|fcg||lpL_ electron 
^^^^^^^H density map 




fitting 1 




^» fil'jJnjOSi atomic 


Workflow for solving the structure of a molecule 


by X-ray crystallography. 



X-ray crystallography 



209 



Limitations 

As the crystal's repeating unit, its unit cell, becomes larger and more complex, the atomic-level picture provided by 
X-ray crystallography becomes less well-resolved (more "fuzzy") for a given number of observed reflections. Two 
limiting cases of X-ray crystallography — "small-molecule" and "macromolecular" crystallography — are often 
discerned. Small-molecule crystallography typically involves crystals with fewer than 100 atoms in their asymmetric 
unit; such crystal structures are usually so well resolved that the atoms can be discerned as isolated "blobs" of 
electron density. By contrast, macromolecular crystallography often involves tens of thousands of atoms in the unit 
cell. Such crystal structures are generally less well-resolved (more "smeared out"); the atoms and chemical bonds 
appear as tubes of electron density, rather than as isolated atoms. In general, small molecules are also easier to 
crystallize than macromolecules; however, X-ray crystallography has proven possible even for viruses with hundreds 
of thousands of atoms. 



Crystallization 




A protein crystal seen under a microscope. 

Crystals used in X-ray crystallography may be 

smaller than a millimeter across. 



Although crystallography can be used to characterize the disorder in an 
impure or irregular crystal, crystallography generally requires a pure 
crystal of high regularity to solve the structure of a complicated 
arrangement of atoms. Pure, regular crystals can sometimes be 
obtained from natural or synthetic materials, such as samples of metals, 
minerals or other macroscopic materials. The regularity of such 
crystals can sometimes be improved with macromolecular crystal 



annealing 



[90] [91] [92] 



and other methods. However, in many cases, 



obtaining a diffraction-quality crystal is the chief barrier to solving its 



atomic -resolution structure 



[93] 



Small-molecule and macromolecular crystallography differ in the 
range of possible techniques used to produce diffraction-quality 
crystals. Small molecules generally have few degrees of conformational freedom, and may be crystallized by a wide 
range of methods, such as chemical vapor deposition and recrystallization. By contrast, macromolecules generally 
have many degrees of freedom and their crystallization must be carried out to maintain a stable structure. For 
example, proteins and larger RNA molecules cannot be crystallized if their tertiary structure has been unfolded; 
therefore, the range of crystallization conditions is restricted to solution conditions in which such molecules remain 
folded. 



X-ray crystallography 



210 



Protein crystals are almost always grown in solution. The most common 

approach is to lower the solubility of its component molecules very gradually; if 

this is done too quickly, the molecules will precipitate from solution, forming a 

useless dust or amorphous gel on the bottom of the container. Crystal growth in 

solution is characterized by two steps: nucleation of a microscopic crystallite 

(possibly having only 100 molecules), followed by growth of that crystallite, 

[941 
ideally to a diffraction-quality crystal. The solution conditions that favor the 

first step (nucleation) are not always the same conditions that favor the second 

step (subsequent growth). The crystallographer's goal is to identify solution 

conditions that favor the development of a single, large crystal, since larger 

crystals offer improved resolution of the molecule. Consequently, the solution 

conditions should disfavor the first step (nucleation) but favor the second 

(growth), so that only one large crystal forms per droplet. If nucleation is favored 

too much, a shower of small crystallites will form in the droplet, rather than one 

large crystal; if favored too little, no crystal will form whatsoever. 

It is extremely difficult to predict good conditions for nucleation or growth of 

well-ordered crystals. In practice, favorable conditions are identified by 

screening; a very large batch of the molecules is prepared, and a wide variety of 

crystallization solutions are tested. Hundreds, even thousands, of solution 

conditions are generally tried before finding the successful one. The various 

conditions can use one or more physical mechanisms to lower the solubility of 

the molecule; for example, some may change the pH, some contain salts of the 

Hofmeister series or chemicals that lower the dielectric constant of the solution, 

and still others contain large polymers such as polyethylene glycol that drive the 

molecule out of solution by entropic effects. It is also common to try several temperatures for encouraging 

crystallization, or to gradually lower the temperature so that the solution becomes supersaturated. These methods 

require large amounts of the target molecule, as they use high concentration of the molecule(s) to be crystallized. 

Due to the difficulty in obtaining such large quantities (milligrams) of crystallization grade protein, robots have been 

developed that are capable of accurately dispensing crystallization trial drops that are in the order of 100 nanoliters 

in volume. This means that 10-fold less protein is used per-experiment when compared to crystallization trials setup 

by hand (in the order of 1 microliter) 




Three methods of preparing crystals, 

A: Hanging drop. B: Sitting drop. C: 

Microdialysis 



[97] 



Several factors are known to inhibit or mar crystallization. The growing crystals are generally held at a constant 
temperature and protected from shocks or vibrations that might disturb their crystallization. Impurities in the 
molecules or in the crystallization solutions are often inimical to crystallization. Conformational flexibility in the 
molecule also tends to make crystallization less likely, due to entropy. Ironically, molecules that tend to 
self-assemble into regular helices are often unwilling to assemble into crystals. Crystals can be marred by twinning, 
which can occur when a unit cell can pack equally favorably in multiple orientations; although recent advances in 
computational methods may allow solving the structure of some twinned crystals. Having failed to crystallize a 
target molecule, a crystallographer may try again with a slightly modified version of the molecule; even small 
changes in molecular properties can lead to large differences in crystallization behavior. 



X-ray crystallography 



211 



Data collection 



Mounting the crystal 

The crystal is mounted for measurements so that it may be held in the X-ray beam and rotated. There are several 

methods of mounting. Although crystals were once loaded into glass capillaries with the crystallization solution (the 

mother liquor), a modern approach is to scoop the crystal up in a tiny loop, made of nylon or plastic and attached to a 

solid rod, that is then flash-frozen with liquid nitrogen. This freezing reduces the radiation damage of the X-rays, 

as well as the noise in the Bragg peaks due to thermal motion (the Debye-Waller effect). However, untreated crystals 

[99] 
often crack if flash-frozen; therefore, they are generally pre-soaked in a cryoprotectant solution before freezing. 

Unfortunately, this pre-soak may itself cause the crystal to crack, ruining it for crystallography. Generally, successful 

cryo-conditions are identified by trial and error. 

The capillary or loop is mounted on a goniometer, which allows it to be positioned accurately within the X-ray beam 
and rotated. Since both the crystal and the beam are often very small, the crystal must be centered within the beam to 
within -25 micrometers accuracy, which is aided by a camera focused on the crystal. The most common type of 
goniometer is the "kappa goniometer", which offers three angles of rotation: the to angle, which rotates about an axis 
perpendicular to the beam; the k angle, about an axis at -50° to the ro axis; and, finally, the ep angle about the 
loop/capillary axis. When the k angle is zero, the w and cp axes are aligned. The k rotation allows for convenient 
mounting of the crystal, since the arm in which the crystal is mounted may be swung out towards the 
crystallographer. The oscillations carried out during data collection (mentioned below) involve the to axis only. An 
older type of goniometer is the four-circle goniometer, and its relatives such as the six-circle goniometer. 



X-ray sources 

The mounted crystal is then irradiated with a beam of monochromatic X-rays. The brightest and most useful X-ray 
sources are synchrotrons; their much higher luminosity allows for better resolution. They also make it convenient to 
tune the wavelength of the radiation, which is useful for multi-wavelength anomalous dispersion (MAD) phasing, 
described below. Synchrotrons are generally national facilities, each with several dedicated beamlines where data is 
collected around the clock, seven days a week. 

Smaller, X-ray generators are often used in laboratories to check the 
quality of crystals before bringing them to a synchrotron and 
sometimes to solve a crystal structure. In such systems, electrons are 
boiled off of a cathode and accelerated through a strong electric 
potential of -50 kV; having reached a high speed, the electrons collide 
with a metal plate, emitting bremsstrahlung and some strong spectral 
lines corresponding to the excitation of inner-shell electrons of the 
metal. The most common metal used is copper, which can be kept cool 
easily, due to its high thermal conductivity, and which produces strong 
K and K lines. The K line is sometimes suppressed with a thin 

a p p 

(-10 |am) nickel foil. The simplest and cheapest variety of sealed X-ray 

tube has a stationary anode (the Crookes tube) and produces -2 kW of X-ray radiation. The more expensive variety 

has a rotating-anode type source that produces - 14 kW of X-ray radiation. 

X-rays are generally filtered (by use of X-Ray Filters) to a single wavelength (made monochromatic) and collimated 
to a single direction before they are allowed to strike the crystal. The filtering not only simplifies the data analysis, 
but also removes radiation that degrades the crystal without contributing useful information. Collimation is done 
either with a collimator (basically, a long tube) or with a clever arrangement of gently curved mirrors. Mirror 
systems are preferred for small crystals (under 0.3 mm) or with large unit cells (over 150 A) 




X-ray crystallography 



212 




Recording the reflections 

When a crystal is mounted and exposed to an intense beam of X-rays, 
it scatters the X-rays into a pattern of spots or reflections that can be 
observed on a screen behind the crystal. A similar pattern may be seen 
by shining a laser pointer at a compact disc. The relative intensities of 
these spots provide the information to determine the arrangement of 
molecules within the crystal in atomic detail. The intensities of these 
reflections may be recorded with photographic film, an area detector or 
with a charge-coupled device (CCD) image sensor. The peaks at small 
angles correspond to low-resolution data, whereas those at high angles 
represent high-resolution data; thus, an upper limit on the eventual 
resolution of the structure can be determined from the first few images. 
Some measures of diffraction quality can be determined at this point, 
such as the mosaicity of the crystal and its overall disorder, as observed 
in the peak widths. Some pathologies of the crystal that would render it 
unfit for solving the structure can also be diagnosed quickly at this 
point. 

One image of spots is insufficient to reconstruct the whole crystal; it represents only a small slice of the full Fourier 
transform. To collect all the necessary information, the crystal must be rotated step-by-step through 180°, with an 
image recorded at every step; actually, slightly more than 180° is required to cover reciprocal space, due to the 
curvature of the Ewald sphere. However, if the crystal has a higher symmetry, a smaller angular range such as 90° or 
45° may be recorded. The rotation axis should be changed at least once, to avoid developing a "blind spot" in 
reciprocal space close to the rotation axis. It is customary to rock the crystal slightly (by 0.5-2°) to catch a broader 
region of reciprocal space. 

Multiple data sets may be necessary for certain phasing methods. For example, MAD phasing requires that the 
scattering be recorded at least three (and usually four, for redundancy) wavelengths of the incoming X-ray radiation. 
A single crystal may degrade too much during the collection of one data set, owing to radiation damage; in such 



An X-ray diffraction pattern of a crystallized 

enzyme. The pattern of spots (called reflections) 

can be used to determine the structure of the 

enzyme. 



cases, data sets on multiple crystals must be taken 



[100] 



Data analysis 

Crystal symmetry, unit cell, and image scaling 

The recorded series of two-dimensional diffraction patterns, each corresponding to a different crystal orientation, is 
converted into a three-dimensional model of the electron density; the conversion uses the mathematical technique of 
Fourier transforms, which is explained below. Each spot corresponds to a different type of variation in the electron 
density; the crystallographer must determine which variation corresponds to which spot {indexing), the relative 
strengths of the spots in different images {merging and scaling) and how the variations should be combined to yield 
the total electron density {phasing). 

Data processing begins with indexing the reflections. This means identifying the dimensions of the unit cell and 
which image peak corresponds to which position in reciprocal space. A byproduct of indexing is to determine the 
symmetry of the crystal, i.e., its space group. Some space groups can be eliminated from the beginning. For 
example, reflection symmetries cannot be observed in chiral molecules; thus, only 65 space groups of 243 possible 
are allowed for protein molecules which are almost always chiral. Indexing is generally accomplished using an 
autoindexing routine. Having assigned symmetry, the data is then integrated. This converts the hundreds of 
images containing the thousands of reflections into a single file, consisting of (at the very least) records of the Miller 
index of each reflection, and an intensity for each reflection (at this state the file often also includes error estimates 



X-ray crystallography 213 

and measures of partiality (what part of a given reflection was recorded on that image)). 

A full data set may consist of hundreds of separate images taken at different orientations of the crystal. The first step 
is to merge and scale these various images, that is, to identify which peaks appear in two or more images (merging) 
and to scale the relative images so that they have a consistent intensity scale. Optimizing the intensity scale is critical 
because the relative intensity of the peaks is the key information from which the structure is determined. The 
repetitive technique of crystallographic data collection and the often high symmetry of crystalline materials cause the 
diffractometer to record many symmetry-equivalent reflections multiple times. This allows calculating the 
symmetry-related R-factor, a reliability index based upon how similar are the measured intensities of 
symmetry-equivalent reflections, thus assessing the quality of the data. 

Initial phasing 

The data collected from a diffraction experiment is a reciprocal space representation of the crystal lattice. The 
position of each diffraction 'spot' is governed by the size and shape of the unit cell, and the inherent symmetry within 
the crystal. The intensity of each diffraction 'spot' is recorded, and this intensity is proportional to the square of the 
structure factor amplitude. The structure factor is a complex number containing information relating to both the 
amplitude and phase of a wave. In order to obtain an interpretable electron density map, both amplitude and phase 
must be known (an electron density map allows a crystallographer to build a starting model of the molecule). The 
phase cannot be directly recorded during a diffraction experiment: this is known as the phase problem. Initial phase 
estimates can be obtained in a variety of ways: 

• Ab initio phasing or direct methods - This is usually the method of choice for small molecules (<1000 
non-hydrogen atoms), and has been used successfully to solve the phase problems for small proteins. If the 
resolution of the data is better than 1.4 A (140 pm), direct methods can be used to obtain phase information, by 
exploiting known phase relationships between certain groups of reflections. 

• Molecular replacement - if a related structure is known, it can be used as a search model in molecular 
replacement to determine the orientation and position of the molecules within the unit cell. The phases obtained 
this way can be used to generate electron density maps. 

• Anomalous X-ray scattering (MAD or SAD phasing) - the X-ray wavelength may be scanned past an absorption 
edge of an atom, which changes the scattering in a known way. By recording full sets of reflections at three 
different wavelengths (far below, far above and in the middle of the absorption edge) one can solve for the 
substructure of the anomalously diffracting atoms and thence the structure of the whole molecule. The most 
popular method of incorporating anomalous scattering atoms into proteins is to express the protein in a 
methionine auxotroph (a host incapable of synthesizing methionine) in a media rich in seleno-methionine, which 
contains selenium atoms. A MAD experiment can then be conducted around the absorption edge, which should 
then yield the position of any methionine residues within the protein, providing initial phases. 

• Heavy atom methods (multiple isomorphous replacement) - If electron-dense metal atoms can be introduced into 
the crystal, direct methods or Patterson-space methods can be used to determine their location and to obtain initial 
phases. Such heavy atoms can be introduced either by soaking the crystal in a heavy atom-containing solution, or 
by co-crystallization (growing the crystals in the presence of a heavy atom). As in MAD phasing, the changes in 
the scattering amplitudes can be interpreted to yield the phases. Although this is the original method by which 
protein crystal structures were solved, it has largely been superseded by MAD phasing with 
selenomethionine. 



X-ray crystallography 



214 



Model building and phase refinement 

Having obtained initial phases, an initial model can be built. This 
model can be used to refine the phases, leading to an improved model, 
and so on. Given a model of some atomic positions, these positions and 
their respective Debye-Waller factors (or B-factors, accounting for the 
thermal motion of the atom) can be refined to fit the observed 
diffraction data, ideally yielding a better set of phases. A new model 
can then be fit to the new electron density map and a further round of 
refinement is carried out. This continues until the correlation between 
the diffraction data and the model is maximized. The agreement is 
measured by an 7?-factor defined as 




A protein crystal structure at 2.7 A resolution. 

The mesh encloses the region in which the 
electron density exceeds a given threshold. The 

straight segments represent chemical bonds 

between the non-hydrogen atoms of an arginine 

(upper left), a tyrosine (lower left), a disulfide 

bond (upper right, in yellow), and some peptide 

groups (running left-right in the middle). The two 

curved green tubes represent spline fits to the 

polypeptide backbone. 



R 



E 



all reflections 



\F - F c 



E 



all reflections 



\F n 



A similar quality criterion is R , which is calculated from a subset (-10%) of reflections that were not included in 
the structure refinement. Both R factors depend on the resolution of the data. As a rule of thumb, R should be 
approximately the resolution in Angstroms divided by 10; thus, a data-set with 2 A resolution should yield a final 
R ~ 0.2. Chemical bonding features such as stereochemistry, hydrogen bonding and distribution of bond lengths 
and angles are complementary measures of the model quality. Phase bias is a serious problem in such iterative model 
building. Omit maps are a common technique used to check for this. 

It may not be possible to observe every atom of the crystallized molecule - it must be remembered that the resulting 
electron density is an average of all the molecules within the crystal. In some cases, there is too much residual 
disorder in those atoms, and the resulting electron density for atoms existing in many conformations is smeared to 
such an extent that it is no longer detectable in the electron density map. Weakly scattering atoms such as hydrogen 
are routinely invisible. It is also possible for a single atom to appear multiple times in an electron density map, e.g., 
if a protein sidechain has multiple (<4) allowed conformations. In still other cases, the crystallographer may detect 
that the covalent structure deduced for the molecule was incorrect, or changed. For example, proteins may be cleaved 
or undergo post-translational modifications that were not detected prior to the crystallization. 



X-ray crystallography 215 

Deposition of the structure 

Once the model of a molecule's structure has been finalized, it is often deposited in a crystallographic database such 
as the Cambridge Structural Database (for small molecules), the Inorganic Crystal Structure Database (ICSD) (for 
inorganic compounds) or the Protein Data Bank (for protein structures). Many structures obtained in private 
commercial ventures to crystallize medicinally relevant proteins, are not deposited in public crystallographic 
databases. 

Diffraction theory 

The main goal of X-ray crystallography is to determine the density of electrons fir) throughout the crystal, where r 
represents the three-dimensional position vector within the crystal. To do this, X-ray scattering is used to collect data 
about its Fourier transform F(q), which is inverted mathematically to obtain the density defined in real space, using 
the formula 

where the integral is taken over all values of q. The three-dimensional real vector q represents a point in reciprocal 
space, that is, to a particular oscillation in the electron density as one moves in the direction in which q points. The 
length of q corresponds to 2 7T divided by the wavelength of the oscillation. The corresponding formula for a 
Fourier transform will be used below 



F(cL) = Jdrf(r)e 



jq-r 



where the integral is summed over all possible values of the position vector r within the crystal. 

The Fourier transform F(q) is generally a complex number, and therefore has a magnitude LF(q)l and a phase qp(q) 
related by the equation 

F(q) = |F(q)|e^ q ) 
The intensities of the reflections observed in X-ray diffraction give us the magnitudes LF(q)l but not the phases q>(q). 
To obtain the phases, full sets of reflections are collected with known alterations to the scattering, either by 
modulating the wavelength past a certain absorption edge or by adding strongly scattering (i.e., electron-dense) metal 
atoms such as mercury. Combining the magnitudes and phases yields the full Fourier transform F(q), which may be 
inverted to obtain the electron density fir). 

Crystals are often idealized as being perfectly periodic. In that ideal case, the atoms are positioned on a perfect 
lattice, the electron density is perfectly periodic, and the Fourier transform F(q) is zero except when q belongs to the 
reciprocal lattice (the so-called Bragg peaks). In reality, however, crystals are not perfectly periodic; atoms vibrate 
about their mean position, and there may be disorder of various types, such as mosaicity, dislocations, various point 
defects, and heterogeneity in the conformation of crystallized molecules. Therefore, the Bragg peaks have a finite 
width and there may be significant diffuse scattering, a continuum of scattered X-rays that fall between the Bragg 
peaks. 

Intuitive understanding by Bragg's law 

An intuitive understanding of X-ray diffraction can be obtained from the Bragg model of diffraction. In this model, a 
given reflection is associated with a set of evenly spaced sheets running through the crystal, usually passing through 
the centers of the atoms of the crystal lattice. The orientation of a particular set of sheets is identified by its three 
Miller indices (h, lc, I), and let their spacing be noted by d. William Lawrence Bragg proposed a model in which the 
incoming X-rays are scattered specularly (mirror-like) from each plane; from that assumption, X-rays scattered from 
adjacent planes will combine constructively (constructive interference) when the angle 6 between the plane and the 
X-ray results in a path-length difference that is an integer multiple n of the X-ray wavelength X. 



X-ray crystallography 216 

2d sin 9 = nX 
A reflection is said to be indexed when its Miller indices (or, more correctly, its reciprocal lattice vector components) 
have been identified from the known wavelength and the scattering angle 26. Such indexing gives the unit-cell 
parameters, the lengths and angles of the unit-cell, as well as its space group. Since Bragg's law does not interpret the 
relative intensities of the reflections, however, it is generally inadequate to solve for the arrangement of atoms within 
the unit-cell; for that, a Fourier transform method must be carried out. 

Scattering as a Fourier transform 

The incoming X-ray beam has a polarization and should be represented as a vector wave; however, for simplicity, let 
it be represented here as a scalar wave. We also ignore the complication of the time dependence of the wave and just 
focus on the wave's spatial dependence. Plane waves can be represented by a wave vector k. , and so the strength of 
the incoming wave at time t=0 is given by 

Ae ikm ' r 
At position r within the sample, let there be a density of scatterers fir); these scatterers should produce a scattered 
spherical wave of amplitude proportional to the local amplitude of the incoming wave times the number of scatterers 
in a small volume dV about r 

amplitude of scattered wave = Ae l T Sf(r)dV 
where S is the proportionality constant. 

Let's consider the fraction of scattered waves that leave with an outgoing wave-vector of k and strike the screen at 

fe fe out 

r . Since no energy is lost (elastic, not inelastic scattering), the wavelengths are the same as are the magnitudes 

screen 

of the wave-vectors Ik. I=lk I. From the time that the photon is scattered at r until it is absorbed at r , the 

in out screen 

photon undergoes a change in phase 

The net radiation arriving at r is the sum of all the scattered waves throughout the crystal 

screen 

which may be written as a Fourier transform 

ASe ik out -r^ een / dr/(r)e- iq - r = ASe ikmtmI " xm F(q) 
where q = k - k. . The measured intensity of the reflection will be square of this amplitude 

^ 2 5 2 |F(q)| 2 

Friedel and Bijvoet mates 

For every reflection corresponding to a point q in the reciprocal space, there is another reflection of the same 
intensity at the opposite point -q. This opposite reflection is known as the Friedel mate of the original reflection. 
This symmetry results from the mathematical fact that the density of electrons fir) at a position r is always a real 
number. As noted above, fir) is the inverse transform of its Fourier transform F(q); however, such an inverse 
transform is a complex number in general. To ensure that/(r) is real, the Fourier transform F(q) must be such that 
the Friedel mates F(-q) and F(q) are complex conjugates of one another. Thus, F(-q) has the same magnitude as 
F(q) but they have the opposite phase, i.e., q>(q) = -q>(q) 

F(-q) = |F(-q)|e^- q ) = F*{q) = |F(q)| e -^ q ) 

2 

The equality of their magnitudes ensures that the Friedel mates have the same intensity \F\ . This symmetry allows 
one to measure the full Fourier transform from only half the reciprocal space, e.g., by rotating the crystal slightly 
more than a 180°, instead of a full turn. In crystals with significant symmetry, even more reflections may have the 
same intensity (Bijvoet mates); in such cases, even less of the reciprocal space may need to be measured, e.g., 
slightly more than 90°. 



X-ray crystallography 217 

The Friedel-mate constraint can be derived from the definition of the inverse Fourier transform 



/w = /(^ F(qy, ' = / 



w - j !V |F(q)|Wr 



Since Euler's formula states that e = cos(x) + i sin(x), the inverse Fourier transform can be separated into a sum of a 
purely real part and a purely imaginary part 

The function fir) is real if and only if the second integral / . is zero for all values of r. In turn, this is true if and only 
if the above constraint is satisfied 

since / . = — / . implies that / . =0. 

sin sin sin 

Ewald's sphere 

Each X-ray diffraction image represents only a slice, a spherical slice of reciprocal space, as may be seen by the 

Ewald sphere construction. Both k and k. have the same length, due to the elastic scattering, since the wavelength 
F out in fe fe & 

has not changed. Therefore, they may be represented as two radial vectors in a sphere in reciprocal space, which 
shows the values of q that are sampled in a given diffraction image. Since there is a slight spread in the incoming 
wavelengths of the incoming X-ray beam, the values oflF(q)lcan be measured only for q vectors located between the 
two spheres corresponding to those radii. Therefore, to obtain a full set of Fourier transform data, it is necessary to 
rotate the crystal through slightly more than 180°, or sometimes less if sufficient symmetry is present. A full 360° 
rotation is not needed because of a symmetry intrinsic to the Fourier transforms of real functions (such as the 
electron density), but "slightly more" than 180° is needed to cover all of reciprocal space within a given resolution 
because of the curvature of the Ewald sphere. In practice, the crystal is rocked by a small amount (0.25-1°) to 
incorporate reflections near the boundaries of the spherical Ewald shells. 

Patterson function 

A well-known result of Fourier transforms is the autocorrelation theorem, which states that the autocorrelation c(r) 
of a function fir) 



c(r) = J dx/(x)/(x + r) = J J^C{$e<* 

has a Fourier transform C(q) that is the squared magnitude of F(q) 



Therefore, the autocorrelation function c(r) of the electron density (also known as the Patterson function ) can 
be computed directly from the reflection intensities, without computing the phases. In principle, this could be used to 
determine the crystal structure directly; however, it is difficult to realize in practice. The autocorrelation function 
corresponds to the distribution of vectors between atoms in the crystal; thus, a crystal of N atoms in its unit cell may 
have N(N-l) peaks in its Patterson function. Given the inevitable errors in measuring the intensities, and the 
mathematical difficulties of reconstructing atomic positions from the interatomic vectors, this technique is rarely 
used to solve structures, except for the simplest crystals. 

Advantages of a crystal 

In principle, an atomic structure could be determined from applying X-ray scattering to non-crystalline samples, 
even to a single molecule. However, crystals offer a much stronger signal due to their periodicity. A crystalline 
sample is by definition periodic; a crystal is composed of many unit cells repeated indefinitely in three independent 
directions. Such periodic systems have a Fourier transform that is concentrated at periodically repeating points in 
reciprocal space known as Bragg peaks; the Bragg peaks correspond to the reflection spots observed in the 
diffraction image. Since the amplitude at these reflections grows linearly with the number N of scatterers, the 
observed intensity of these spots should grow quadratically, like N 2 . In other words, using a crystal concentrates the 



X-ray crystallography 218 

weak scattering of the individual unit cells into a much more powerful, coherent reflection that can be observed 
above the noise. This is an example of constructive interference. 

In a liquid, powder or amorphous sample, molecules within that sample are in random orientations. Such samples 
have a continuous Fourier spectrum that uniformly spreads its amplitude thereby reducing the measured signal 
intensity, as is observed in SAXS. More importantly, the orientational information is lost. Although theoretically 
possible, it is experimentally difficult to obtain atomic -resolution structures of complicated, asymmetric molecules 
from such rotationally averaged data. An intermediate case is fiber diffraction in which the subunits are arranged 
periodically in at least one dimension. 

See also 

• Bragg diffraction 

• Bravais lattice 

• Crystallographic database 

• Crystallographic point groups 

• Difference density map 

• Electron crystallography 

• Electron diffraction 

• Neutron diffraction 

• Ptychography 

• Powder diffraction 

• Scherrer Equation 

• Small angle X-ray scattering (SAXS) 

• Structure determination 

• Wide angle X-ray scattering (WAXS) 

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Martin TW, Derewenda ZS (1999). "The name is Bond — H bond". Nature Structural Biology 6 (5): 403. doi: 10.1038/8195. 
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Further reading 

International Tables for Crystallography 

• Theo Hahn, ed (2002). International Tables for Crystallography. Volume A, Space-group Symmetry (5 ed.). 
Dordrecht: Kluwer Academic Publishers, for the International Union of Crystallography. ISBN 0792365909. 

• Michael G. Rossmann and Eddy Arnold, ed (2001). International Tables for Crystallography. Volume F, 
Crystallography of biological molecules. Dordrecht: Kluwer Academic Publishers, for the International Union of 
Crystallography. ISBN 0792368576. 

• Theo Hahn, ed (1996). International Tables for Crystallography. Brief Teaching Edition of Volume A, 
Space-group Symmetry (4 ed.). Dordrecht: Kluwer Academic Publishers, for the International Union of 
Crystallography. ISBN 0792342526. 



X-ray crystallography 222 

Bound collections of articles 

• Charles W. Carter and Robert M. Sweet., ed (1997). Macromolecular Crystallography, Part A (Methods in 
Enzymology, v. 276). San Diego: Academic Press. ISBN 0121821773. 

• Charles W. Carter Jr., Robert M. Sweet., ed (1997). Macromolecular Crystallography, Part B (Methods in 
Enzymology, v. 277). San Diego: Academic Press. ISBN 0121821781. 

• A. Ducruix and R. Giege, ed (1999). Crystallization of Nucleic Acids and Proteins: A Practical Approach (2 ed.). 
Oxford: Oxford University Press. ISBN 0199636788. 

Textbooks 

Blow D (2002). Outline of Crystallography for Biologists. Oxford: Oxford University Press. ISBN 0198510519. 

Burns G., Glazer A M (1990). Space Groups for Scientists and Engineers (2nd ed.). Boston: Academic Press, Inc. 

ISBN 0121457613. 

Clegg W (1998). Crystal Structure Determination (Oxford Chemistry Primer). Oxford: Oxford University Press. 

ISBN 0198559011. 

Cullity B.D. (1978). Elements ofX-Ray Diffraction (2nd ed.). Reading, Massachusetts: Addison-Wesley 

Publishing Company. ISBN 0534553966. 

Drenth J (1999). Principles of Protein X-Ray Crystallography. New York: Springer- Verlag. ISBN 0387985875. 

Giacovazzo C et al. (1992). Fundamentals of Crystallography . Oxford: Oxford University Press. 

ISBN 0198555784. 

Glusker JP, Lewis M, Rossi M (1994). Crystal Structure Analysis for Chemists and Biologists. New York: VCH 

Publishers. ISBN 0471185434. 

Massa W (2004). Crystal Structure Determination. Berlin: Springer. ISBN 3540206442. 

McPherson A (1999). Crystallization of Biological Macromolecules. Cold Spring Harbor, NY: Cold Spring 

Harbor Laboratory Press. ISBN 0879696176. 

McPherson A (2003). Introduction to Macromolecular Crystallography . John Wiley & Sons. ISBN 0471251224. 

McRee DE (1993). Practical Protein Crystallography. San Diego: Academic Press. ISBN 0124860508. 

OKeeffe M, Hyde B G (1996). Crystal Structures; I. Patterns and Symmetry. Washington, DC: Mineralogical 

Society of America, Monograph Series. ISBN 0939950405. 

Rhodes G (2000). Crystallography Made Crystal Clear. San Diego: Academic Press. ISBN 0125870728., PDF 

copy of select chapters (http://www.chem.uwec.edu/Chem406_F06/Pages/lecture_notes/lect07/ 

Crystallography_Rhodes.pdf) 

Rupp B (2009). Biomolecular Crystallography : Principles, Practice and Application to Structural Biology. New 

York: Garland Science. ISBN 0815340818. 

Zachariasen WH (1945). Theory of X-ray Diffraction in Crystals. New York: Dover Publications. 

LCCN 67-26967. 



X-ray crystallography 223 

Applied computational data analysis 

• Young, R.A., ed (1993). The Rietveld Method. Oxford: Oxford University Press & International Union of 
Crystallography. ISBN 0198555776. 

Historical 

• Bijvoet JM, Burgers WG, Hagg G, eds. (1969). Early Papers on Diffraction of X-rays by Crystals (Volume I). 
Utrecht: published for the International Union of Crystallography by A. Oosthoek's Uitgeversmaatschappij N.V.. 

• Bijvoet JM, Burgers WG, Hagg G, eds. (1972). Early Papers on Diffraction of X-rays by Crystals (Volume II). 
Utrecht: published for the International Union of Crystallography by A. Oosthoek's Uitgeversmaatschappij N.V.. 

• Bragg W L, Phillips D C and Lipson H (1992). The Development of X-ray Analysis. New York: Dover. 
ISBN 0486673162. 

• Ewald PP, editor, and numerous crystallographers (1962). Fifty Years of X-ray Diffraction. Utrecht: published for 
the International Union of Crystallography by A. Oosthoek's Uitgeversmaatschappij N.V.. 

• Ewald, P. P., editor 50 Years ofX-Ray Diffraction (http://www.iucr.org/iucr-top/publ/ 
50YearsOfXrayDiffraction/) (Reprinted in pdf format for the IUCr XVIII Congress, Glasgow, Scotland, 
International Union of Crystallography). 

• Friedrich W (1922). "Die Geschichte der Auffindung der Rontgenstrahlinterferenzen". Die Naturwissenschaften 
10: 363. doi:10.1007/BF01565289. 

• Lonsdale K (1949). Crystals and X-rays. New York: D. van Nostrand. 

External links 
Tutorials 

• Crystallography for beginners (http://www.xtal.iqfr.csic.es/Cristalografia/index-en.html) 

• Simple, non technical introduction (http://stein.bioch.dundee.ac.uk/~charlie/index.php?section=l) 

• "Small Molecule Crystalization" (http://acaschool.iit.edu/lectures04/JLiangXtal.pdf) (PDF) at Illinois 
Institute of Technology website 

• International Union of Crystallography (http://iucr.org) 

• Crystallography 101 (http://www.ruppweb.org/Xray/101index.html) 

• Interactive structure factor tutorial (http://www.ysbl.york.ac.uk/~cowtan/sfapplet/sfintro.html), 
demonstrating properties of the diffraction pattern of a 2D crystal. 

• Picturebook of Fourier Transforms (http://www.ysbl.york.ac.uk/~cowtan/fourier/fourier.html), illustrating 
the relationship between crystal and diffraction pattern in 2D. 

• Lecture notes on X-ray crystallography and structure determination (http://www.chem.uwec.edu/ 
Chem406_F06/Pages/lectnotes.html#lecture7) 

• Online lecture on Modern X-ray Scattering Methods for Nanoscale Materials Analysis (http://nanohub.org/ 
resources/5580) by Richard J. Matyi 



X-ray crystallography 224 

Primary databases 

Crystallography Open Database (http://www.crystallography.net/) (COD) 

Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) (PDB) 

Nucleic Acid Databank (http://ndbserver.rutgers.edu/) (NDB) 

Cambridge Structural Database (http://www.ccdc.cam.ac.uk/products/csd/) (CSD) 

Inorganic Crystal Structure Database (http://www.fiz-karlsruhe.de/icsd.html) (ICSD) 

Biological Macromolecule Crystallization Database (http://xpdb.nist.gov:8060/BMCD4/) (BMCD) 

Derivative databases 

PDB sum (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/) 

Proteopeida - the collaborative, 3D encyclopedia of proteins and other molecules (http://www.proteopedia.org) 

RN AB ase (http ://w w w .rnabase . org/) 

HIC-Up database of PDB ligands (http://xray.bmc.uu.se/hicup/) 

Structural Classification of Proteins database 

CATH Protein Structure Classification 

List of transmembrane proteins with known 3D structure (http://blanco.biomol.uci.edu/ 

Membrane_Proteins_xtal . html) 

• Orientations of Proteins in Membranes database 

Structural validation 

• WHAT-IF structural validation suite (http://swift.cmbi.kun.n1/WIWWWI//) 

• Biotech structural validation suite (http://biotech.ebi.ac.uk/) (formerly ProCheck) 

• MolProbity structural validation suite (http://molprobity.biochem.duke.edu/) 

• ProSA-web (https://prosa.services.came.sbg.ac.at/prosa.php) 

• NQ-Flipper (https://flipper.services.came.sbg.ac.at/) (check for unfavorable rotamers of Asn and Gin 
residues) 

• DALI server (http://www.ebi.ac.uk/dali/) (identifies proteins similar to a given protein) 



X-ray scattering techniques 



225 



X-ray scattering techniques 



X-ray scattering techniques are a family of 
non-destructive analytical techniques which reveal 
information about the crystallographic structure, 
chemical composition, and physical properties of 
materials and thin films. These techniques are based on 
observing the scattered intensity of an X-ray beam 
hitting a sample as a function of incident and scattered 
angle, polarization, and wavelength or energy. 

X-ray diffraction techniques 

X-ray diffraction yields the atomic structure of 
materials and is based on the elastic scattering of 
X-rays from the electron clouds of the individual atoms 
in the system. The most comprehensive description of 
scattering from crystals is given by the dynamical 
theory of diffraction 




[l] 



This is an X-ray diffraction pattern formed when X-rays are focused 

on a crystalline material, in this case a protein. Each dot, called a 

reflection, forms from the coherent interference of scattered X-rays 

passing through the crystal. 



• Single-crystal X-ray diffraction is a technique used 
to solve the complete structure of crystalline 
materials, ranging from simple inorganic solids to 
complex macromolecules, such as proteins. 

• Powder diffraction (XRD) is a technique used to characterise the crystallographic structure, crystallite size (grain 
size), and preferred orientation in polycrystalline or powdered solid samples. Powder diffraction is commonly 
used to identify unknown substances, by comparing diffraction data against a database maintained by the 
International Centre for Diffraction Data. It may also be used to characterize heterogeneous solid mixtures to 
determine relative abundance of crystalline compounds and, when coupled with lattice refinement techniques, 
such as Rietveld refinement, can provide structural information on unknown materials. Powder diffraction is also 
a common method for determining strains in crystalline materials. An effect of the finite crystallite sizes is seen as 
a broadening of the peaks in an X-ray diffraction as is explained by the Scherrer Equation. 

• Thin film diffraction and grazing incidence X-ray diffraction may be used to characterize the crystallographic 
structure and preferred orientation of substrate-anchored thin films. 

• High-resolution X-ray diffraction is used to characterize thickness, crystallographic structure, and strain in thin 
epitaxial films. It employs parallel-beam optics. 

• X-ray pole figure analysis enables one to analyze and determine the distribution of crystalline orientations within 
a crystalline thin-film sample. 

• X-ray rocking curve analysis is used to quantify grain size and mosaic spread in crystalline materials. 



X-ray scattering techniques 226 

Scattering techniques 
Elastic scattering 

Materials that do not have long range order may also be studied by scattering methods that rely on elastic scattering 
of monochromatic X-rays. 

• Small angle X-ray scattering (SAXS) probes structure in the nanometer to micrometer range by measuring 

121 
scattering intensity at scattering angles 26 close to 0°. 

• X-ray reflectivity is an analytical technique for determining thickness, roughness, and density of single layer and 
multilayer thin films. 

• Wide angle X-ray scattering (WAXS), a technique concentrating on scattering angles 26 larger than 5°. 

Inelastic scattering 

When the energy and angle of the inelastically scattered X-rays are monitored scattering techniques can be used to 
probe the electronic band structure of materials. 

• Compton scattering 

• Resonant inelastic X-ray scattering (RIXS) 

• X-ray Raman scattering 

• X-ray diffraction pattern 

See also 

Structure determination 

Materials Science 

Metallurgy 

Mineralogy 

X-ray crystallography 

X-ray generator 

References 

[1] Azaroff, L. V.; R. Kaplow, N. Kato, R. J. Weiss, A. J. C. Wilson, R. A. Young (1974). X-ray diffraction. McGraw-Hill. 

[2] Glatter, O.; O. Kratky (1982). Small Angle X-ray Scattering (http://physchem.kfunigraz.ac.at/sm/Software.htm). Academic Press. . 

External links 

• International Union of Crystallography (http://www.iucr.ac.uk/) 

• IUCr Crystallography Online (http://www.iucr.org/cww-top/crystal.index.html) 

• The International Centre for Diffraction Data (ICDD) (http://www.icdd.com/) 

• The British Crystallographic Association (http://crystallography.org.uk/) 

• Introduction to X-ray Diffraction (http://www.mrl.ucsb.edu/mrl/centralfacilities/xray/xray-basics/index. 
html) at University of California, Santa Barbara 



Fourier transform spectroscopy 



227 



Fourier transform spectroscopy 



Fourier transform spectroscopy is a measurement technique whereby spectra are collected based on measurements 
of the coherence of a radiative source, using time-domain or space-domain measurements of the electromagnetic 
radiation or other type of radiation. It can be applied to a variety of types of spectroscopy including optical 
spectroscopy, infrared spectroscopy (FTIR, FT-NIRS), nuclear magnetic resonance (NMR) and magnetic resonance 
spectroscopic imaging (MRSI) , mass spectrometry and electron spin resonance spectroscopy. There are several 
methods for measuring the temporal coherence of the light (see: field-autocorrelation), including the continuous 
wave Michelson or Fourier transform spectrometer and the pulsed Fourier transform spectrograph (which is more 
sensitive and has a much shorter sampling time than conventional spectroscopic techniques, but is only applicable in 
a laboratory environment). 

The term Fourier transform spectroscopy reflects the fact that in all these techniques, a Fourier transform is required 
to turn the raw data into the actual spectrum, and in many of the cases in optics involving interferometers, is based 
on the Wiener— Khinchin theorem. 



Conceptual introduction 



Measuring an emission spectrum 

One of the most basic tasks in spectroscopy is to characterize the 
spectrum of a light source: How much light is emitted at each different 
wavelength. The most straightforward way to measure a spectrum is to 
pass the light through a monochromator, an instrument that blocks all 
of the light except the light at a certain wavelength (the un-blocked 
wavelength is set by a knob on the monochromator). Then the intensity 
of this remaining (single-wavelength) light is measured. The measured 
intensity directly indicates how much light is emitted at that 
wavelength. By varying the monochromator' s wavelength setting, the 
full spectrum can be measured. This simple scheme in fact describes 
how some spectrometers work. 







C; 








" 












r^ 




CN 1 CH 




it 


r^i 



300 400 500 600 700 

Wavelength / nm 

An example of a spectrum: The spectrum of light 
emitted by the blue flame of a butane torch. The 
horizontal axis is the wavelength of light, and the 
vertical axis represents how much light is emitted 
by the torch at that wavelength. 



Fourier transform spectroscopy is a less intuitive way to get the same 

information. Rather than allowing only one wavelength at a time to 

pass through to the detector, this technique lets through a beam 

containing many different wavelengths of light at once, and measures the total beam intensity. Next, the beam is 

modified to contain a different combination of wavelengths, giving a second data point. This process is repeated 

many times. Afterwards, a computer takes all this data and works backwards to infer how much light there is at each 

wavelength. 

To be more specific, between the light source and the detector, there is a certain configuration of mirrors that allows 
some wavelengths to pass through but blocks others (due to wave interference). The beam is modified for each new 
data point by moving one of the mirrors; this changes the set of wavelengths that can pass through. 

As mentioned, computer processing is required to turn the raw data (light intensity for each mirror position) into the 
desired result (light intensity for each wavelength). The processing required turns out to be a common algorithm 
called the Fourier transform (hence the name, "Fourier transform spectroscopy"). The raw data is sometimes called 
an "interferogram". 



Fourier transform spectroscopy 



228 




Measuring an absorption spectrum 

The method of Fourier transform spectroscopy can also be used for 
absorption spectroscopy. The primary example is "FTIR 
Spectroscopy", a common technique in chemistry. 

In general, the goal of absorption spectroscopy is to measure how well 
a sample absorbs or transmits light at each different wavelength. 
Although absorption spectroscopy and emission spectroscopy are 
different in principle, they are closely related in practice; any technique 
for emission spectroscopy can also be used for absorption 
spectroscopy. First, the emission spectrum of a broadband lamp is 
measured (this is called the "background spectrum"). Second, the 
emission spectrum of the same lamp shining through the sample is 
measured (this is called the "sample spectrum"). The sample will 
absorb some of the light, causing the spectra to be different. The ratio 
of the "sample spectrum" to the "background spectrum" is directly related to the sample's absorption spectrum. 

Accordingly, the technique of "Fourier transform spectroscopy" can be used both for measuring emission spectra (for 
example, the emission spectrum of a star), and absorption spectra (for example, the absorption spectrum of a glass of 
liquid). 



10720 10700 10BS0 10600 10B40 

relative Ortskccrrjir i < I 

An "interferogram" from a Fourier transform 

spectrometer. The horizontal axis is the position 

of the mirror, and the vertical axis is the amount 

of light detected. This is the "raw data" which can 

be Fourier transformed into an actual spectrum. 



coherent 
light source 



Continuous wave Michelson or Fourier transform spectrograph 

The Michelson spectrograph is similar to the 

instrument used in the Michelson-Morley experiment. 

Light from the source is split into two beams by a 

half-silvered mirror, one is reflected off a fixed mirror 

and one off a moving mirror which introduces a time 

delay — the Fourier transform spectrometer is just a 

Michelson interferometer with a movable mirror. The 

beams interfere, allowing the temporal coherence of the 

light to be measured at each different time delay 

setting, effectively converting the time domain into a 

spatial coordinate. By making measurements of the 

signal at many discrete positions of the moving mirror, 

the spectrum can be reconstructed using a Fourier 

transform of the temporal coherence of the light. 

Michelson spectrographs are capable of very high 

spectral resolution observations of very bright sources. 

The Michelson or Fourier transform spectrograph was 

popular for infra-red applications at a time when 

infra-red astronomy only had single pixel detectors. 

Imaging Michelson spectrometers are a possibility, but in general have been supplanted by imaging Fabry— Perot 

instruments which are easier to construct. 




The Fourier transform spectrometer is just a Michelson 

interferometer but one of the two fully-reflecting mirrors is movable, 

allowing a variable delay (in the travel-time of the light) to be 

included in one of the beams. 



Fourier transform spectroscopy 229 

Extracting the spectrum 

The intensity as a function of the path length difference in the interferometer pand wavenumber y = 1 /\ is 

Ifa V ) = I(l>) [1 + COs(27TI>p)] , 

where 1(D) is the spectrum to be determined. Note that it is not necessary for 1(D) to be modulated by the sample 
before the interferometer. In fact, most FTIR spectrometers place the sample after the interferometer in the optical 
path. The total intensity at the detector is 

I(p) = I{p,D)dD = I(D)[l + cos(2nDp)]dD. 
Jo Jo 

This is just a Fourier cosine transform. The inverse gives us our desired result in terms of the measured quantity 

("OO 

Up) = 4 / [I(p) - \l{p = 0)] cos(27ri>p)dp. 
Jo 

Pulsed Fourier transform spectrometer 

A pulsed Fourier transform spectrometer does not employ transmittance techniques. In the most general description 
of pulsed FT spectrometry, a sample is exposed to an energizing event which causes a periodic response. The 
frequency of the periodic response, as governed by the field conditions in the spectrometer, is indicative of the 
measured properties of the analyte. 

Examples of pulsed Fourier transform spectrometry 

In magnetic spectroscopy (EPR, NMR), an RF pulse in a strong ambient magnetic field is used as the energizing 
event. This turns the magnetic particles at an angle to the ambient field, resulting in gyration. The gyrating spins then 
induce a periodic current in a detector coil. Each spin exhibits a characteristic frequency of gyration (relative to the 
field strength) which reveals information about the analyte. 

In Fourier transform mass spectrometry, the energizing event is the injection of the charged sample into the strong 
electromagnetic field of a cyclotron. These particles travel in circles, inducing a current in a fixed coil on one point 
in their circle. Each traveling particle exhibits a characteristic cyclotron frequency-field ratio revealing the masses in 
the sample. 

Free induction decay 

Pulsed FT spectrometry gives the advantage of requiring a single, time-dependent measurement which can easily 
deconvolute a set of similar but distinct signals. The resulting composite signal, is called a free induction decay, 
because typically the signal will decay due to inhomogeneities in sample frequency, or simply unrecoverable loss of 
signal due to entropic loss of the property being measured. 

Stationary forms of Fourier transform spectrometers 

In addition to the scanning forms of Fourier transform spectrometers, there are a number of stationary or 
self-scanned forms. While the analysis of the interferometric output is similar to that of the typical scanning 
interferometer, significant differences apply, as shown in the published analyses. Some stationary forms retain the 
Fellgett multiplex advantage, and their use in the spectral region where detector noise limits apply is similar to the 
scanning forms of the FTS. In the photon-noise limited region, the application of stationary interferometers is 
dictated by specific consideration for the spectral region and the application. 



Fourier transform spectroscopy 230 

Fellgett advantage 

One of the most important advantages of Fourier transform spectroscopy was shown by P.B. Fellgett, an early 
advocate of the method. The Fellgett advantage, also known as the multiplex principle, states that when obtaining a 
spectrum when measurement noise is dominated by detector noise, a multiplex spectrometer such as a Fourier 
transform spectrometer will produce a relative improvement in signal-to-noise ratio, compared to an equivalent 
scanning monochromator, of the order of the square root of m, where m is the number of sample points comprising 
the spectrum. 

Converting spectra from time domain to frequency domain 

/•oo 
-oo 
The sum is performed over all contributing frequencies to give a signal S(t) in the time domain. 

/•oo 
S(t)e il/2nt dt 
-oo 

gives non-zero value when S(t) contains a component that matches the oscillating function. 
Remember that 

e lx = cos x + i sin x 

See also 

• Applied spectroscopy 

• Forensic chemistry 

• Forensic polymer engineering 

• Nuclear Magnetic Resonance 

• Infrared spectroscopy 

References 

[1] Antoine Abragam. 1968. Principles of Nuclear Magnetic Resonance., Cambridge University Press: Cambridge, UK. 
[2] Peter Atkins, Julio De Paula. 2006. Physical Chemistry, 8th ed. Oxford University Press: Oxford, UK. 

[3] William H. Smith U.S. Patent 4976542 (http://www. google.com/patents ?vid=4976542) Digital Array Scanned Interferometer, issued Dec. 
11, 1990 

External links 

• Description of how a Fourier transform spectrometer works (http://scienceworld.wolfram.com/physics/ 
FourierTransformSpectrometer.html) 

• The Michelson or Fourier transform spectrograph (http://www.astro.livjm.ac.uk/courses/phys362/notes/) 

• Internet Journal of Vibrational Spectroscopy - How FTIR works (http://www.ijvs.com/volume5/edition5/ 
sectionl .html#Feature) 

• Fourier Transform Spectroscopy Topical Meeting and Tabletop Exhibit (http://www.osa.org/meetings/ 
topicalmeetings/fts/default.aspx) 



Hyperspectral imaging 



231 



Hyperspectral imaging 



Hyperspectral imaging collects and processes information from across the electromagnetic spectrum. Unlike the 
human eye, which just sees visible light, hyperspectral imaging is more like the eyes of the mantis shrimp, which can 
see visible light as well as from the ultraviolet to infrared. Hyperspectral capabilities enable the mantis shrimp to 
recognize different types of coral, prey, or predators, all of which may appear as the same color to the human eye. 

Humans build sensors and processing systems to provide the same type of capability for application in agriculture, 
mineralogy, physics, and surveillance. Hyperspectral sensors look at objects using a vast portion of the 
electromagnetic spectrum. Certain objects leave unique 'fingerprints' across the electromagnetic spectrum. These 
'fingerprints' are known as spectral signatures and enable identification of the materials that make up a scanned 
object. For example, having the spectral signature for oil helps mineralogists find new oil fields. 



Acquisition and Analysis 

Hyperspectral sensors collect information as a set of 'images'. Each 
image represents a range of the electromagnetic spectrum and is also 
known as a spectral band. These 'images' are then combined and form a 
three dimensional hyperspectral cube for processing and analysis. 

Hyperspectral cubes are generated from airborne sensors like the 
NASA's Airborne Visible/Infrared Imaging Spectrometer (AVIRIS), or 
from satellites like NASA's Hyperion. However, for many 
development and validation studies handheld sensors are used. 

The precision of these sensors is typically measured in spectral 

resolution, which is the width of each band of the spectrum that is 

captured. If the scanner picks up on a large number of fairly narrow 

frequency bands, it is possible to identify objects even if said objects 

are only captured in a handful of pixels. However, spatial resolution is 

a factor in addition to spectral resolution. If the pixels are too large, then multiple objects are captured in the same 

pixel and become difficult to identify. If the pixels are too small, then the energy captured by each sensor-cell is low, 

and the decreased signal-to-noise ratio reduces the reliability of measured features. 

MicroMSI, Opticks and Envi are three remote sensing applications that support the processing and analysis of 
hyperspectral data. The acquisition and processing of hyperspectral images is also referred to as imaging 
spectroscopy. 




Example of a hyperspectral cube 



Hyperspectral imaging 



232 



Differences between hyperspectral and multispectral imaging 



Multispectral/ 



Hyperspectral imaging is part of a class of techniques commonly 
referred to as spectral imaging or spectral analysis. Hyperspectral 
imaging is related to multispectral imaging. The distinction between 
hyper- and multi-spectral should not be based on a random or arbitrary 
"number of bands". A distinction that is based on the type of 
measurement may be more appropriate. 

Multispectral deals with several images at discrete and somewhat 
narrow bands. The "discrete and somewhat narrow" is what 
distinguishes multispectral in the visible from color photography. A 
multispectral sensor may have many bands covering the spectrum from 
the visible to the longwave infrared. Multispectral images do not 
produce the "spectrum" of an object. Landsat is an excellent example. 

Hyperspectral deals with imaging narrow spectral bands over a 

contiguous spectral range, and produce the spectra of all pixels in the scene. So a sensor with only 20 bands can also 
be hyperspectral when it covers the range from 500 to 700 nm with 20 10-nm wide bands. (While a sensor with 20 
discrete bands covering the VIS, NIR, SWIR, MWIR, and LWIR would be considered multispectral.) 

Ultraspectral could be reserved for interferometer type imaging sensors with a very fine spectral resolution. These 
sensor often have (but not necessarily) a low spatial resolution of several pixels only, a restriction imposed by the 
high data rate. 




Hyperspectral and Multispectral Differences. 



Applications 

Hyperspectral remote sensing is used in a wide array of real-life applications. Although originally developed for 
mining and geology (the ability of hyperspectral imaging to identify various minerals makes it ideal for the mining 
and oil industries, where it can be used to look for ore and oil ) it has now spread into fields as widespread as 

ecology and surveillance, as well as historical manuscript research such as the imaging of the Archimedes 
Palimpsest. This technology is continually becoming more available to the public, and has been used in a wide 
variety of ways. Organizations such as NASA and the USGS have catalogues of various minerals and their spectral 
signatures, and have posted them online to make them readily available for researchers. 



Agriculture 

Although the costs of acquiring hyperspectral images is typically high, for specific crops and in specific climates 
hyperspectral remote sensing is used more and more for monitoring the development and health of crops. In 
Australia work is under way to use imaging spectrometers to detect grape variety, and develop an early warning 
system for disease outbreaks. Furthermore work is underway to use hyperspectral data to detect the chemical 
composition of plants which can be used to detect the nutrient and water status of wheat in irrigated systems . 

Another important area in agriculture is the detection of animal proteins in compound feeds in order to avoid the 
Bovine spongiform encephalopathy (BSE) or mad-cow disease (MCD). For this, different studies have been done in 
order to propose alternative tools to the reference method (classical microscopy). One of the first alternatives is the 
use of NIR microscopy (Infrared microscopy), which combines the advantages of microscopy and NIR. In 2004, the 
first study relating this problematic with Hyperspectral imaging was published . Hyperspectral libraries are 
constructed, which are representative of the wide diversity of ingredients usually present in the preparation of 
compound feeds. These libraries can be used together with chemometric tools to investigate the limit of detection, 
specificity and reproducibility of the NIR hyperspectral imaging method for the detection and quantification of 
animal ingredient in feed. 



Hyperspectral imaging 233 

Mineralogy 

The original field of development for hyperspectral remote sensing, hyperspectral sensing of minerals is now well 
developed. Many minerals can be identified from images, and their relation to the presence of valuable minerals such 
as gold and diamonds is well understood. Currently the move is towards understanding the relation between oil and 
gas leakages from pipelines and natural wells; their effect on the vegetation and the spectral signatures. Recent work 
includes the PhD dissertations of Werff and Noomen . 

Physics 

Physicists use an electron microscopy technique that involves microanalysis using either Energy dispersive X-ray 
spectroscopy (EDS), Electron energy loss spectroscopy (EELS), Infrared Spectroscopy(IR), Raman Spectroscopy, or 
cathodoluminescence (CL) spectroscopy, in which the entire spectrum measured at each point is recorded. EELS 
hyperspectral imaging is performed in a scanning transmission electron microscope (STEM); EDS and CL mapping 
can be performed in STEM as well, or in a scanning electron microscope or electron probe microanalyzer (EPMA). 
Often, multiple techniques (EDS, EELS, CL) are used simultaneously. 

In a "normal" mapping experiment, an image of the sample will be made that is simply the intensity of a particular 
emission mapped in an XY raster. For example, an EDS map could be made of a steel sample, in which iron x-ray 
intensity is used for the intensity grayscale of the image. Dark areas in the image would indicate not-iron-bearing 
impurities. This could potentially give misleading results; if the steel contained tungsten inclusions, for example, the 
high atomic number of tungsten could result in bremsstrahlung radiation that made the iron-free areas appear to be 
rich in iron. 

By hyperspectral mapping, instead, the entire spectrum at each mapping point is acquired, and a quantitative analysis 
can be performed by computer post-processing of the data, and a quantitative map of iron content produced. This 
would show which areas contained no iron, despite the anomalous x-ray counts caused by bremsstrahlung. Because 
EELS core-loss edges are small signals on top of a large background, hyperspectral imaging allows large 
improvements to the quality of EELS chemical maps. 

Similarly, in CL mapping, small shifts in the peak emission energy could be mapped, which would give information 
regarding slight chemical composition changes or changes in the stress state of a sample. 

Surveillance 

Hyperspectral surveillance is the implementation of hyperspectral scanning technology for surveillance purposes. 
Hyperspectral imaging is particularly useful in military surveillance because of measures that military entities now 
take to avoid airborne surveillance. Airborne surveillance has been in effect since soldiers used tethered balloons to 
spy on troops during the American Civil War, and since that time we have learned not only to hide from the naked 
eye, but to mask our heat signature to blend in to the surroundings and avoid infrared scanning, as well. The idea that 
drives hyperspectral surveillance is that hyperspectral scanning draws information from such a large portion of the 
light spectrum that any given object should have a unique spectral signature in at least a few of the many bands that 
get scanned. 



Hyperspectral imaging 234 

Advantages and disadvantages 

The primary advantages to hyperspectral imaging is that, because an entire spectrum is acquired at each point, the 
operator needs no prior knowledge of the sample, and post-processing allows all available information from the 
dataset to be mined. 

The primary disadvantages are cost and complexity. Fast computers, sensitive detectors, and large data storage 
capacities are needed for analyzing hyperspectral data. Significant data storage capacity is necessary since 
hyperspectral cubes are large multi-dimensional datasets, potentially exceeding hundreds of megabytes. All of these 
factors greatly increase the cost of acquiring and processing hyperspectral data. Also, one of the hurdles that 
researchers have had to face is finding ways to program hyperspectral satellites to sort through data on their own and 
transmit only the most important images, as both transmission and storage of that much data could prove difficult 
and costly. As a relatively new analytical technique, the full potential of hyperspectral imaging has not yet been 
realized. 

See also 

• Airborne Real-time Cueing Hyperspectral Enhanced Reconnaissance 

• Full spectral imaging 

• Multi-spectral image 

• Chemical imaging 

• Remote Sensing 

• Sensor fusion 

• ERD AS IMAGINE 

• Liquid Crystal Tunable Filter 

References 

[1] Schurmer, J.H., (Dec 2003), Air Force Research Laboratories Technology Horizons 

[2] Ellis, J., (Jan 2001) Searching for oil seeps and oil-impacted soil with hyperspectral imagery (http://www.eomonline.com/Common/ 

currentissues/JanOl/ellis.htm), Earth Observation Magazine. 
[3] Smith, R.B. (July 14, 2006), Introduction to hyperspectral imaging with TMIPS (http://www.microimages.com/getstart/pdf/hyprspec. 

pdf), Microimages Tutorial Web site 
[4] Lacar, F.M., et al., Use of hyperspectral imagery for mapping grape varieties in the Barossa Valley, South Australia (http://hdl.handle.net/ 

2440/39292), Geoscience and remote sensing symposium (IGARSS'01) - IEEE 2001 International, vol.6 2875-2877p. 

doi:10.1109/IGARSS.2001.978191 
[5] Ferwerda, J.G. (2005), Charting the quality' of forage: measuring and mapping the variation of chemical components in foliage with 

hyperspectral remote sensing (http://www.itc.nl/library/Papers_2005/phd/ferwerda.pdf), Wageningen University , ITC Dissertation 126, 

166p. ISBN 90-8504-209-7 
[6] Tilling, A.K., et al., (2006) Remote sensing to detect nitrogen and water stress in wheat (http://www.regional.org.au/au/asa/2006/ 

plenary/technology/4584_tillingak.htm), The Australian Society of Agronomy 
[7] Fernandez Pierna, J. A., et al., 'Combination of Support Vector Machines (SVM) and Near Infrared (NIR) imaging spectroscopy for the 

detection of meat and bone meat (MBM) in compound feeds' Journal of Chemometrics 18 (2004) 341-349 
[8] Werff H. (2006), Knowledge based remote sensing of complex objects: recognition of spectral and spatial patterns resulting from natural 

hydrocarbon seepages (http://www.itc.nl/library/papers_2006/phd/vdwerff.pdf), Utrecht University, ITC Dissertation 131, 138p. ISBN 

90-6164-238-8 
[9] Noomen, M.F. (2007), Hyperspectral reflectance of vegetation affected by underground hydrocarbon gas seepage (http://www.itc.nl/ 

library/papers_2007/phd/noomen.pdf), Enschede, ITC 15 lp. ISBN 978-90-8504-671-4. 



Hyperspectral imaging 235 

External links 

• SpecTIR (http://www.spectir.com/) - Hyperspectral solutions and end to end global data collection & analysis 

• Specim, Spectral Imaging Ltd. (http://www.specim.fi/) Hyperspectral Imaging Solutions 

• Opticks (http://opticks.org/) - open source, remote sensing application and development framework. 

• ITT Visual Information Solutions - ENVI Hyperspectral Image Processing Software (http://www.ittvis.com/ 
ProductServices/ENVI.aspx) 

• A Hyperspectral Imaging Prototype (http://www.inrim.it/res/hyperspectral_imaging/) Fourier transform 
spectroscopy is combined with Fabry-Perot interferometry 

• Middleton Research (http://www.middletonresearch.com) Hyperspectral Imaging products, custom engineering 
solutions 

• Photon etc. (http://photonetc. com/index. php?lan=en&sec=300&subl=3000&sub2=1023) Hyperspectral 
Imaging Systems 

• UmBio - Evince. Hyperspectral image analysis in real-time. Visual information solutions, see industrial demo 
movies (http://beta.umbio.com/Public files/Products/Evince Image/Evincelmage.aspx) 

• A Matlab Hyperspectral Toolbox (http://matlabhyperspec.sourceforge.net/) 

• Telops Hyper-Cam (http://www.hyper-cam.com/) Commercial infrared hyperspectral camera 



2D-FT NMRI and Spectroscopy 



2D-FT Nuclear Magnetic resonance imaging (2D-FT NMRI), or Two-dimensional Fourier transform magnetic 
resonance imaging (NMRI), is primarily a non— invasive imaging technique most commonly used in biomedical 
research and medical radiology/nuclear medicine/MRI to visualize structures and functions of the living systems and 
single cells. For example it can provides fairly detailed images of a human body in any selected cross-sectional 
plane, such as longitudinal, transversal, sagital, etc. NMRI provides much greater contrast especially for the different 
soft tissues of the body than computed tomography (CT) as its most sensitive option observes the nuclear spin 
distribution and dynamics of highly mobile molecules that contain the naturally abundant, stable hydrogen isotope 
H as in plasma water molecules, blood, disolved metabolites and fats. This approach makes it most useful in 
cardiovascular, oncological (cancer), neurological (brain), musculoskeletal, and cartilage imaging. Unlike CT, it uses 
no ionizing radiation, and also unlike nuclear imaging it does not employ any radioactive isotopes. Some of the first 
MRI images reported were published in 1973 and the first study performed on a human took place on July 3, 
1977. Earlier papers were also published by Peter Mansfield in UK (Nobel Laureate in 2003), and R. Damadian 
in the USA, (together with an approved patent for magnetic imaging). Unpublished "high-resolution' (50 micron 
resolution) images of other living systems, such as hydrated wheat grains, were obtained and communicated in UK 
in 1977-1979, and were subsequently confirmed by articles published in Nature. 



2D-FT NMRI and Spectroscopy 



236 



NMRI Principle 

Certain nuclei such as H nuclei, or 
"fermions' have spin-1/2, because there 
are two spin states, referred to as "up" 
and "down" states. The nuclear 
magnetic resonance absorption 
phenomenon occurs when samples 
containing such nuclear spins are 
placed in a static magnetic field and a 
very short radiofrequency pulse is 
applied with a center, or carrier, 
frequency matching that of the 
transition between the up and down 
states of the spin-1/2 H nuclei that 
were polarized by the static magnetic 
field. Very low field schemes have 




Advanced clinical diagnostics and biomedical research NMR Imaging instrument. 



also been recently reported 



[5] 



Chemical Shifts 

NMR is a very useful family of techniques for chemical and biochemical research because of the chemical shift; this 

effect consists in a frequency shift of the nuclear magnetic resonance for specific chemical groups or atoms as a 

result of the partial shielding of the corresponding nuclei from the applied, static external magnetic field by the 

electron orbitals (or molecular orbitals) surrounding such nuclei present in the chemical groups. Thus, the higher the 

electron density surounding a specific nucleus the larger the chemical shift will be. The resulting magnetic field at 

the nucleus is thus lower than the applied external magnetic field and the resonance frequencies observed as a result 

of such shielding are lower than the value that would be observed in the absence of any electronic orbital shielding. 

Furthermore, in order to obtain a chemical shift value independent of the strength of the applied magnetic field and 

allow for the direct comparison of spectra obtained at different magnetic field values, the chemical shift is defined by 

the ratio of the strength of the local magnetic field value at the observed (electron orbital-shielded) nucleus by the 

external magnetic field strength, H, / H . The first NMR observations of the chemical shift, with the correct 

loc 

physical chemistry interpretation, were reported for F containing compounds in the early 1950's by Herbert S. 
Gutowsky and Charles P. Slichter from the University of Illinois at Urbana (USA). 



NMR Imaging Principles 

A number of methods have been devised for combining magnetic field gradients and radiofrequency pulsed 
excitation to obtain an image. Two major maethods involve either 2D -FT or 3D-FT reconstruction from 
projections, somewhat similar to Computed Tomography, with the exception of the image interpretation that in the 
former case must include dynamic and relaxation/contrast enhancement information as well. Other schemes involve 
building the NMR image either point-by-point or line-by-line. Some schemes use instead gradients in the rf field 

rather than in the static magnetic field. The majority of NMR images routinely obtained are either by the 

171 
Two-Dimensional Fourier Transform (2D-FT) technique (with slice selection), or by the Three-Dimensional 

Fourier Transform (3D— FT) techniques that are however much more time consuming at present. 2D-FT NMRI is 

sometime called in common parlance a "spin-warp". An NMR image corresponds to a spectrum consisting of a 

181 

number of "spatial frequencies' at different locations in the sample investigated, or in a patient. A two— dimensional 



2D-FT NMRI and Spectroscopy 237 

Fourier transformation of such a "real" image may be considered as a representation of such "real waves" by a matrix 
of spatial frequencies known as the k— space. We shall see next in some mathematical detail how the 2D-FT 
computation works to obtain 2D-FT NMR images. 

Two-dimensional Fourier transform imaging and spectroscopy 

A two-dimensional Fourier transform (2D-FT) is computed numerically or carried out in two stages, both involving 
"standard', one-dimensional Fourier transforms. However, the second stage Fourier transform is not the inverse 
Fourier transform (which would result in the original function that was transformed at the first stage), but a Fourier 
transform in a second variable— which is "shifted' in value— relative to that involved in the result of the first Fourier 
transform. Such 2D-FT analysis is a very powerful method for both NMRI and two-dimensional nuclear magnetic 
resonance spectroscopy (2D-FT NMRS) that allows the three-dimensional reconstruction of polymer and 
biopolymer structures at atomic resolution]]. for molecular weights (Mw) of dissolved biopolymers in aqueous 
solutions (for example) up to about 50,000 Mw. For larger biopolymers or polymers, more complex methods have 
been developed to obtain limited structural resolution needed for partial 3D-reconstructions of higher molecular 
structures, e.g. for up 900,000 Mw or even oriented microcrystals in aqueous suspensions or single crystals; such 
methods have also been reported for in vivo 2D-FT NMR spectroscopic studies of algae, bacteria, yeast and certain 
mammalian cells, including human ones. The 2D-FT method is also widely utilized in optical spectroscopy, such as 
2D-FT NIR hyperspectral imaging (2D-FT NIR-HS), or in MRI imaging for research and clinical, diagnostic 
applications in Medicine. In the latter case, 2D-FT NIR-HS has recently allowed the identification of single, 
malignant cancer cells surrounded by healthy human breast tissue at about 1 micron resolution, well-beyond the 
resolution obtainable 2D-FT NMRI for such systems in the limited time available for such diagnostic investigations 
(and also in magnetic fields up to the FDA approved magnetic field strength H of 4.7 T, as shown in the top image 
of the state-of-the-art NMRI instrument). A more precise mathematical definition of the "double' (2D) Fourier 
transform involved in both 2D NMRI and 2D-FT NMRS is specified next, and a precise example follows this 
generally accepted definition. 

2D-FT Definition 

A 2D-FT, or two-dimensional Fourier transform, is a standard Fourier transformation of a function of two variables, 
f(xi, £2)' can "i e d first in the first variable X\, followed by the Fourier transform in the second variable a^of the 
resulting function F(s\, X 2 ) ■ Note that in the case of both 2D-FT NMRI and 2D-FT NMRS the two independent 
variables in this definition are in the time domain, whereas the results of the two successive Fourier transforms have, 
of course, frequencies as the independent variable in the NMRS, and ultimately spatial coordinates for both 2D 
NMRI and 2D-FT NMRS following computer structural recontructions based on special algorithms that are different 
from FT or 2D-FT. Moreover, such structural algorithms are different for 2D NMRI and 2D-FT NMRS: in the 
former case they involve macroscopic, or anatomical structure detrmination, whereas in the latter case of 2D-FT 
NMRS the atomic structure reconstruction algorithms are based on the quantum theory of a microphysical (quantum) 
process such as nuclear Overhauser enhancement NOE, or specific magnetic dipole-dipole interactions between 
neighbor nuclei. 



2D-FT NMRI and Spectroscopy 238 

Example 1 

A 2D Fourier transformation and phase correction is applied to a set of 2D NMR (FID) signals : s(ti , ^yielding a 
real 2D-FT NMR "spectrum' (collection of ID FT-NMR spectra) represented by a matrix S whose elements are 

S(u h u 2 ) = Re / / cos{v 1 t 1 )exp { - iv ^ ) s{t 1 ,t 2 )dt 1 dt2 

where : l^iand : ^denote the discrete indirect double-quantum and single-quantum(detection) axes, respectively, 
in the 2D NMR experiments. Next, the \emph{covariance matrix} is calculated in the frequency domain according to 
the following equation 

C(i/ 2 , V2) = S S = 2_^[S(l>i,l> 2 )S(vi, ^2)], with : i/ 2; ^taking all possible single-quantum 

frequency values and with the summation carried out over all discrete, double quantum frequencies : V\ . 

Example 2 

ri2i 

Atomic Structure from 2D-FT STEM Images of electron distributions in a high-temperature cuprate 

superconductor "paracrystaf reveal both the domains (or "location') and the local symmetry of the 'pseudo-gap' in the 
electron-pair correlation band responsible for the high— temperature superconductivity effect (obtained at Cornell 
University). So far there have been three Nobel prizes awarded for 2D-FT NMR/MRI during 1992-2003, and an 
additional, earlier Nobel prize for 2D-FT of X-ray data ("CAT scans'); recently the advanced possibilities of 2D-FT 
techniques in Chemistry, Physiology and Medicine received very significant recognition. 

Brief explanation of NMRI diagnostic uses in Pathology 

As an example, a diseased tissue such as a malign tumor, can be detected by 2D-FT NMRI because the hydrogen 
nuclei of molecules in different tissues return to their equilibrium spin state at different relaxation rates, and also 
because of the manner in which a malign tumor spreads and grows rapidly along the blood vessels adjacent to the 
tumor, also inducing further vascularization to occur. By changing the pulse delays in the RF pulse sequence 
employed, and/or the RF pulse sequence itself, one may obtain a "relaxation— based contrast', or contrast 
enhancement between different types of body tissue, such as normal vs. diseased tissue cells for example. Excluded 
from such diagnostic observations by NMRI are all patients with ferromagnetic metal implants, (e.g., cochlear 
implants), and all cardiac pacemaker patients who cannot undergo any NMRI scan because of the very intense 
magnetic and RF fields employed in NMRI which would strongly interfere with the correct functioning of such 
pacemakers. It is, however, conceivable that future developments may also include along with the NMRI diagnostic 
treatments with special techniques involving applied magnetic fields and very high frequency RF. Already, surgery 
with special tools is being experimented on in the presence of NMR imaging of subjects. Thus, NMRI is used to 
image almost every part of the body, and is especially useful for diagnosis in neurological conditions, disorders of 
the muscles and joints, for evaluating tumors, such as in lung or skin cancers, abnormalities in the heart (especially 
in children with hereditary disorders), blood vessels, CAD, atherosclerosis and cardiac infarcts (courtesy of Dr. 
Robert R. Edelman) 

See also 



2D-FT NMRI and Spectroscopy 239 



Nuclear magnetic resonance (NMR) 

Medical imaging 

Protein nuclear magnetic resonance spectroscopy 

Kurt Wuthrich 

Chemical shift 

Computed tomography (CT) 

Fourier transform spectroscopy(FTS) 

Richard R. Ernst 



Magnetic resonance microscopy • Relaxation 

Solid-state NMR • Earth's field NMR (EFNMR) 

Herbert S. Gutowsky • Robinson oscillator 

John S. Waugh 

Charles P. Slichter 

FT-NIRS (NIR) 

Magnetic resonance elastography 



Reference list 



[I] Lauterbur, P.C., Nobel Laureate in 2003 (1973). "Image Formation by Induced Local Interactions: Examples of Employing Nuclear Magnetic 
Resonance". Nature 242: 190-1. doi:10.1038/242190a0. 

[2] [http://www.howstuffworks.com/mri.htm/printable Howstuffworks "How MRI Works" 

[3] Peter Mansfield. 2003. Nobel Laureate in Physiology and Medicine for (2D and 3D) MRI (http://www.parteqinnovations.com/pdf-doc/ 

fandr-Gazl006.pdf) 
[4] Antoine Abragam. 1968. Principles of Nuclear Magnetic Resonance., 895 pp., Cambridge University Press: Cambridge, UK. 
[5] Raftery D (August 2006). "MRI without the magnet" (http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez& 

artid=1568902). Proc Natl Acad Sci USA. 103 (34): 12657-8. doi:10.1073/pnas.0605625103. PMID 16912110. PMC 1568902. 
[6] Wu Y, Chesler DA, Glimcher MJ, et al (February 1999). "Multinuclear solid-state three-dimensional MRI of bone and synthetic calcium 

phosphates" (http://www.pnas. org/cgi/pmidlookup?view=long&pmid=9990066). Proc. Natl. Acad. Sci. U.S.A. 96 (4): 1574—8. 

doi:10.1073/pnas.96.4.1574. PMID 9990066. PMC 15521. . 
[7] http://www.math.cuhk.edu.hk/course/mat2071a/lecl_08.ppt 
[8] *Haacke, E Mark; Brown, Robert F; Thompson, Michael; Venkatesan, Ramesh (1999). Magnetic resonance imaging: physical principles and 

sequence design. New York: J. Wiley & Sons. ISBN 0-471-35128-8. 
[9] Richard R. Ernst. 1992. Nuclear Magnetic Resonance Fourier Transform (2D-FT) Spectroscopy. Nobel Lecture (http://nobelprize.org/ 

nobel_prizes/chemistry/laureates/1991/ernst-lecture.pdf), on December 9, 1992. 
[10] http://en.wikipedia.Org/wiki/Nuclear_magnetic_resonance#Nuclear_spin_and_magnets Kurt Wutrich in 1982-1986 : 2D-FT NMR of 

solutions 

[II] Charles P. Slichter. 1996. Principles of Magnetic Resonance. Springer: Berlin and New York, Third Edition., 651pp. ISBN 0-387-50157-6. 
[12] http://www.physorg.com/newsl29395045.html 

[13] http://nobelprize.org/nobel_prizes/chemistry/laureates/1991/ernst-lecture.pdf 

[14] Protein structure determination in solution by NMR spectroscopy (http://www.ncbi. nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve& 

db=pubmed&dopt=Abstract&list_uids=2266107&query_hl=33&itool=pubmed_docsum) Wuthrich K. J Biol Chem. 1990 December 

25;265(36):22059-62. 
[15] http://www.mr-tip.com/servl. php?type=img&img=Cardiac%20Infarct%20Short%20Axis%20Cine%204 



References 

• Antoine Abragam. 1968. Principles of Nuclear Magnetic Resonance., 895 pp., Cambridge University Press: 
Cambridge, UK. 

• Charles P. Slichter. 1996. Principles of Magnetic Resonance. Springer: Berlin and New York, Third Edition., 
651pp. ISBN 0-387-50157-6. 

• Kurt Wuthrich. 1986, NMR of Proteins and Nucleic Acids., J. Wiley and Sons: New York, Chichester, Brisbane, 
Toronto, Singapore. ( Nobel Laureate in 2002 for 2D-FT NMR Studies of Structure and Function of Biological 
Macromolecules (http://nobelprize.org/nobel_prizes/chemistry/laureates/2002/wutrich-lecture.pdf) 

• Protein structure determination in solution by NMR spectroscopy (http://www.ncbi.nlm.nih.gov/entrez/ 
query. fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=2266107&query_hl=33& 
itool=pubmed_docsum) Wuthrich K. J Biol Chem. 1990 December 25;265(36):22059-62 

• 2D-FT NMRI Instrument image: A JPG color image of a 2D-FT NMRI "monster' Instrument (http://upload. 
wikimedia. org/wikipedia/en/b/bf/HWB -NMRv900 . jpg) . 



2D-FT NMRI and Spectroscopy 240 

• Richard R. Ernst. 1992. Nuclear Magnetic Resonance Fourier Transform (2D-FT) Spectroscopy. Nobel Lecture 
(http://nobelprize.org/nobel_prizes/chemistry/laureates/1991/ernst-lecture.pdf), on December 9, 1992. 

• Peter Mansfield. 2003. Nobel Laureate in Physiology and Medicine for (2D and 3D) MRI (http://www. 
parteqinnovations.com/pdf-doc/fandr-Gazl006.pdf) 

• D. Benett. 2007. PhD Thesis. Worcester Polytechnic Institute. PDF of 2D-FT Imaging Applications to NMRI in 
Medical Research. (http://www.wpi.edu/Pubs/ETD/Available/etd-081707-080430/unrestricted/dbennett. 

pdf) Worcester Polytechnic Institute. (Includes many 2D-FT NMR images of human brains.) 

• Paul Lauterbur. 2003. Nobel Laureate in Physiology and Medicine for (2D and 3D) MRI. (http://nobelprize.org/ 
nobel_prizes/medicine/laureates/2003/) 

• Jean Jeener. 1971. Two-dimensional Fourier Transform NMR, presented at an Ampere International Summer 
School, Basko Polje, unpublished. A verbatim quote follows from Richard R. Ernst's Nobel Laureate Lecture 
delivered on December 2, 1992, "A new approach to measure two-dimensional (2D) spectra." has been proposed 
by Jean Jeener at an Ampere Summer School in Basko Polje, Yugoslavia, 1971 (Jean Jeneer,1971 }). He 
suggested a 2D Fourier transform experiment consisting of two $\pi/2$ pulses with a variable time $ t_l$ between 
the pulses and the time variable $t_2$ measuring the time elapsed after the second pulse as shown in Fig. 6 that 
expands the principles of Fig. 1. Measuring the response $s(t_l,t_2)$ of the two-pulse sequence and 
Fourier-transformation with respect to both time variables produces a two-dimensional spectrum $S(0_1,0_2)$ 
of the desired form. This two-pulse experiment by Jean Jeener is the forefather of a whole class of $2D$ 
experiments that can also easily be expanded to multidimensional spectroscopy. 

• Dudley, Robert, L (1993). "High-Field NMR Instrumentation". Ch. 10 in Physical Chemistry of Food Processes 
(New York: Van Nostrand-Reinhold) 2: 421-30. ISBN 0-442-00582-2. 

• Baianu, I.C.; Kumosinski, Thomas (August 1993). "NMR Principles and Applications to Structure and 
Hydration,". Ch.9 in Physical Chemistry of Food Processes (New York: Van Nostrand-Reinhold) 2: 338-420. 
ISBN 0-442-00582-2. 

• Haacke, E Mark; Brown, Robert F; Thompson, Michael; Venkatesan, Ramesh (1999). Magnetic resonance 
imaging: physical principles and sequence design. New York: J. Wiley & Sons. ISBN 0-471-35128-8. 

• Raftery D (August 2006). "MRI without the magnet" (http://www.pubmedcentral.nih.gov/articlerender. 
fcgi?tool=pmcentrez&artid=1568902). Proc Natl Acad Sci USA. 103 (34): 12657-8. 
doi:10.1073/pnas.0605625103. PMID 16912110. PMC 1568902. 

• Wu Y, Chesler DA, Glimcher MJ, et al (February 1999). "Multinuclear solid-state three-dimensional MRI of 
bone and synthetic calcium phosphates" (http://www.pnas. org/cgi/pmidlookup?view=long&pmid=9990066). 
Proc. Natl. Acad. Sci. U.S.A. 96 (4): 1574-8. doi:10.1073/pnas.96.4.1574. PMID 9990066. PMC 15521. 

External links 

• Cardiac Infarct or "heart attack" Imaged in Real Time by 2D-FT NMRI (http://www.mr-tip.com/exam_gifs/ 
cardiac_infarct_short_axis_cine_6.gif) 

• 3D Animation Movie about MRI Exam (http://www.patiencys.com/MRI/) 

• Interactive Flash Animation on MRI (http://www.e-mri.org) - Online Magnetic Resonance Imaging physics and 
technique course 

• International Society for Magnetic Resonance in Medicine (http://www.ismrm.org) 

• Danger of objects flying into the scanner (http://www.simplyphysics.com/flying_objects.html) 



2D-FT NMRI and Spectroscopy 241 

Related Wikipedia websites 

Medical imaging 

Computed tomography 

Magnetic resonance microscopy 

Fourier transform spectroscopy 

FT-NIRS 

Magnetic resonance elastography 

Nuclear magnetic resonance (NMR) 

Chemical shift 

Relaxation 

Robinson oscillator 

Earth's field NMR (EFNMR) 

Rabi cycle 

This article incorporates material by the original author from 2D -FT MR- Imaging and related Nobel awards (http:/ 
/planetphysics. org/ encyclopedia/ 2DFTImaging.html) on PlanetPhysics (http://planetphysics.org/), which is 
licensed under the GFDL. 



NMR microscopy 



Magnetic Resonance Microscopy (MRM, |jMRI) is Magnetic Resonance Imaging (MRI) at a microscopic level. A 
strict definition is MRI having voxel resolutions of better than 100 pm 3 

Nomenclature 

Many scientist in the field consider the name Magnetic Resonance Microscopy to be a misnomer, since the images 
produced are much worse than those produced by even a marginal optical or electron microscope. As such, the name 
High Resolution Magnetic Resonance Imaging is often preferred in scientific literature on the subject. In fact, the 
term is most widely used by the High Resolution Magnetic Resonance Imaging group from Duke University, headed 
by Allan Johnson. 

Differences between MRI and MRM 

• Resolution: Typical medical MRI resolution is about 1 mm 3 ; the desired resolution of MRM is 100 |jm 3 or 
smaller. 

• Specimen size: Medical MRI machines are designed so that a patient may fit inside. MRM chambers are usually 
small, typically less than 1 cm 3 . 

Current status of MRM 

Although MRI is very common for medical applications, MRM is still developed in laboratories. The major barriers 
for practical MRM include: 

• Magnetic field gradient: High gradient focus the magnetic resonance in a smaller volume (smaller point spread 
function), results in a better spatial resolution. The gradients for MRM are typically 50 to 100 times those of 
clinical systems. However, the construction of radio frequency (RF) coil used in MRM does not allow ultrahigh 
gradient. 

• Sensitivity: Because the voxels for MRM can be 1/100,000 of those in MRI, the signal will be proportionately 
weaker 



NMR microscopy 242 

Alternative MRM 

Magnetic Resonance Force Microscopy (MRFM) is claimed to have nm 3 -scale resolutions. It improves the 
sensitivity issue by introducing microfabricated cantilever to measure tiny signals. The magnetic gradient is 
generated by a micrometre-scale magnetic tip, yielding a typical gradient 10 million times larger than those of 
clinical systems. This technique is still in the beginning stage. Because the specimen need to be in high vacuum at 
cryogenic temperatures, MRFM can be only used for solid state matters. 

References 

[1] P. Glover and P. Mansfield, Limits to magnetic resonance microscopy, Rep. Prog. Phys. 65 1489—1511, 2002 
[2] R. Maronpot Applications of Magnetic Resonance Microscopy, Toxicologic Pathology, 32(Suppl. 2):42^8, 2004 

External links 

• Introduction to Magnetic Resonance Microscopy (http://cbaweb2.med.unc.edu/henson_mrm/pages/mrmfaq. 
html#MRMAnchor) Auditory Research Laboratory at the Univ. of North Carolina. 



Chemical imaging 



Chemical imaging (as quantitative - chemical mapping) is the analytical capability to create a visual image of 
components distribution from simultaneous measurement of spectra and spatial, time informations. 

The main idea - for chemical imaging, the analyst may choose to take as many data spectrum measured at a 
particular chemical component in spatial location at time; this is useful for chemical identification and quantification. 
Alternatively, selecting an image plane at a particular data spectrum (PCA - multivariate data of wavelength, spatial 
location at time) can mapp the spatial distribution of sample components, provided that their spectral signatures are 
different at the selected data spectrum. 

Software for chemical imaging is most specific and distinguished from chemical methods as the chemometrics. 

T31 
Imaging technique is most often applied to either solid or gel samples, and has applications in chemistry, biology 

, medicine , pharmacy (see also for example: Chemical Imaging Without Dyeing ), food 

science, biotechnology , agriculture and industry (see for example:NIR Chemical Imaging in Pharmaceutical 

Industry and Pharmaceutical Process Analytical Technology: ). NIR, IR and Raman chemical imaging is also 

referred to as hyperspectral, spectroscopic, spectral or multispectral imaging (also see microspectroscopy). However, 

other ultra-sensitive and selective imaging techniques are also in use that involve either UV-visible or fluorescence 

microspectroscopy. Many imaging techniques can be used to analyze samples of all sizes, from the single 

molecule to the cellular level in biology and medicine , and to images of planetary systems in 

astronomy, but different instrumentation is employed for making observations on such widely different systems. 

Imaging instrumentation is composed of three components: a radiation source to illuminate the sample, a spectrally 
selective element, and usually a detector array (the camera) to collect the images. When many stacked spectral 
channels (wavelengths) are collected for different locations of the microspectrometer focus on a line or planar array 
in the focal plane, the data is called hyperspectral; fewer wavelength data sets are called multispectral. The data 
format is called a hypercube. The data set may be visualized as a three-dimensional block of data spanning two 
spatial dimensions (x and y), with a series of wavelengths (lambda) making up the third (spectral) axis. The 
hypercube can be visually and mathematically treated as a series of spectrally resolved images (each image plane 
corresponding to the image at one wavelength) or a series of spatially resolved spectra. 

Many materials, both manufactured and naturally occurring, derive their functionality from the spatial distribution of 
sample components. For example, extended release pharmaceutical formulations can be achieved by using a coating 



Chemical imaging 243 

that acts as a barrier layer. The release of active ingredient is controlled by the presence of this barrier, and 
imperfections in the coating, such as discontinuities, may result in altered performance. In the semi-conductor 
industry, irregularities or contaminants in silicon wafers or printed micro-circuits can lead to failure of these 
components. The functionality of biological systems is also dependent upon chemical gradients — a single cell, 
tissue, and even whole organs function because of the very specific arrangement of components. It has been shown 
that even small changes in chemical composition and distribution may be an early indicator of disease. 

Any material that depends on chemical gradients for functionality may be amenable to study by an analytical 
technique that couples spatial and chemical characterization. To efficiently and effectively design and manufacture 
such materials, the 'what' and the 'where' must both be measured. The demand for this type of analysis is increasing 
as manufactured materials become more complex. Chemical imaging techniques is critical to understanding modern 
manufactured products and in some casses is a non-destructive technique so that samples are preserved for further 
testing. 

History 

Commercially available laboratory-based chemical imaging systems emerged in the early 1990s (ref. 1-5). In 
addition to economic factors, such as the need for sophisticated electronics and extremely high-end computers, a 
significant barrier to commercialization of infrared imaging was that the focal plane array (FPA) needed to read IR 
images were not readily available as commercial items. As high-speed electronics and sophisticated computers 
became more commonplace, and infrared cameras became readily commercially available, laboratory chemical 
imaging systems were introduced. 

Initially used for novel research in specialized laboratories, chemical imaging became a more commonplace 
analytical technique used for general R&D, quality assurance (QA) and quality control (QC) in less than a decade. 
The rapid acceptance of the technology in a variety of industries (pharmaceutical, polymers, semiconductors, 
security, forensics and agriculture) rests in the wealth of information characterizing both chemical composition and 
morphology. The parallel nature of chemical imaging data makes it possible to analyze multiple samples 
simultaneously for applications that require high throughput analysis in addition to characterizing a single sample. 

Principles 

Chemical imaging shares the fundamentals of vibrational spectroscopic techniques, but provides additional 
information by way of the simultaneous acquisition of spatially resolved spectra. It combines the advantages of 
digital imaging with the attributes of spectroscopic measurements. Briefly, vibrational spectroscopy measures the 
interaction of light with matter. Photons that interact with a sample are either absorbed or scattered; photons of 
specific energy are absorbed, and the pattern of absorption provides information, or a fingerprint, on the molecules 
that are present in the sample. 

On the other hand, in terms of the observation setup, chemical imaging can be carried out in one of the following 
modes: (optical) absorption, emission (fluorescence), (optical) transmission or scattering (Raman). A consensus 
currently exists that the fluorescence (emission) and Raman scattering modes are the most sensitive and powerful, 
but also the most expensive. 

In a transmission measurement, the radiation goes through a sample and is measured by a detector placed on the far 
side of the sample. The energy transferred from the incoming radiation to the molecule(s) can be calculated as the 
difference between the quantity of photons that were emitted by the source and the quantity that is measured by the 
detector. In a diffuse reflectance measurement, the same energy difference measurement is made, but the source and 
detector are located on the same side of the sample, and the photons that are measured have re-emerged from the 
illuminated side of the sample rather than passed through it. The energy may be measured at one or multiple 
wavelengths; when a series of measurements are made, the response curve is called a spectrum. 



Chemical imaging 244 

A key element in acquiring spectra is that the radiation must somehow be energy selected — either before or after 
interacting with the sample. Wavelength selection can be accomplished with a fixed filter, tunable filter, 
spectrograph, an interferometer, or other devices. For a fixed filter approach, it is not efficient to collect a significant 
number of wavelengths, and multispectral data are usually collected. Interferometer-based chemical imaging requires 
that entire spectral ranges be collected, and therefore results in hyperspectral data. Tunable filters have the flexibility 
to provide either multi- or hyperspectral data, depending on analytical requirements. 

Spectra may be measured one point at a time using a single element detector (single-point mapping), as a line-image 
using a linear array detector (typically 16 to 28 pixels) (linear array mapping), or as a two-dimensional image using a 
Focal Plane Array (FPA)(typically 256 to 16,384 pixels) (FPA imaging). For single-point the sample is moved in the 
x and y directions point-by-point using a computer-controlled stage. With linear array mapping, the sample is moved 
line-by-line with a computer-controlled stage. FPA imaging data are collected with a two-dimensional FPA detector, 
hence capturing the full desired field-of-view at one time for each individual wavelength, without having to move 
the sample. FPA imaging, with its ability to collected tens of thousands of spectra simultaneously is orders of 
magnitude faster than linear arrays which are can typically collect 16 to 28 spectra simultaneously, which are in turn 
much faster than single-point mapping. 

Terminology 

Some words common in spectroscopy, optical microscopy and photography have been adapted or their scope 
modified for their use in chemical imaging. They include: resolution, field of view and magnification. There are two 
types of resolution in chemical imaging. The spectral resolution refers to the ability to resolve small energy 
differences; it applies to the spectral axis. The spatial resolution is the minimum distance between two objects that is 
required for them to be detected as distinct objects. The spatial resolution is influenced by the field of view, a 
physical measure of the size of the area probed by the analysis. In imaging, the field of view is a product of the 
magnification and the number of pixels in the detector array. The magnification is a ratio of the physical area of the 
detector array divided by the area of the sample field of view. Higher magnifications for the same detector image a 
smaller area of the sample. 

Types of vibrational chemical imaging instruments 

Chemical imaging has been implemented for mid-infrared, near-infrared spectroscopy and Raman spectroscopy. As 
with their bulk spectroscopy counterparts, each imaging technique has particular strengths and weaknesses, and are 
best suited to fulfill different needs. 

Mid-infrared chemical imaging 

Mid-infrared (MIR) spectroscopy probes fundamental molecular vibrations, which arise in the spectral range 
2,500-25,000 nm. Commercial imaging implementations in the MIR region typically employ Fourier Transform 
Infrared (FT-IR) interferometers and the range is more commonly presented in wavenumber, 4,000 — 400 cm" . The 
MIR absorption bands tend to be relatively narrow and well-resolved; direct spectral interpretation is often possible 
by an experienced spectroscopist. MIR spectroscopy can distinguish subtle changes in chemistry and structure, and is 
often used for the identification of unknown materials. The absorptions in this spectral range are relatively strong; 
for this reason, sample presentation is important to limit the amount of material interacting with the incoming 
radiation in the MIR region. Most data collected in this range is collected in transmission mode through thin sections 
(-10 micrometres) of material. Water is a very strong absorber of MIR radiation and wet samples often require 
advanced sampling procedures (such as attenuated total reflectance). Commercial instruments include point and line 
mapping, and imaging. All employ an FT-IR interferometer as wavelength selective element and light source. 



Chemical imaging 



245 




For types of MIR microscope, see 
Microscopy#Infrared microscopy. 

Atmospheric windows in the infrared 

spectrum are also employed to perform 

chemical imaging remotely. In these spectral 

regions the atmospheric gases (mainly water 

and CO ) present low absorption and allow 

infrared viewing over kilometer distances. 

Target molecules can then be viewed using 

the selective absorption/emission processes 

described above. An example of the chemical imaging of a simultaneous release of SF and NH is shown in the 

image. 



Remote chemical imaging of a simultaneous release of SF and NH at 1.5km using 



the FIRST imaging spectrometer 



I-- 



Near-infrared chemical imaging 

The analytical near infrared (NIR) region spans the range from approximately 700-2,500 nm. The absorption bands 
seen in this spectral range arise from overtones and combination bands of O-H, N-H, C-H and S-H stretching and 
bending vibrations. Absorption is one to two orders of magnitude smaller in the NIR compared to the MIR; this 
phenomenon eliminates the need for extensive sample preparation. Thick and thin samples can be analyzed without 
any sample preparation, it is possible to acquire NIR chemical images through some packaging materials, and the 
technique can be used to examine hydrated samples, within limits. Intact samples can be imaged in transmittance or 
diffuse reflectance. 

The lineshapes for overtone and combination bands tend to be much broader and more overlapped than for the 
fundamental bands seen in the MIR. Often, multivariate methods are used to separate spectral signatures of sample 
components. NIR chemical imaging is particularly useful for performing rapid, reproducible and non-destructive 

[231 [24] 

analyses of known materials . NIR imaging instruments are typically based on one of two platforms: imaging 

using a tunable filter and broad band illumination, and line mapping employing an FT-IR interferometer as the 
wavelength filter and light source. 

Raman chemical imaging 

The Raman shift chemical imaging spectral range spans from approximately 50 to 4,000 cm" ; the actual spectral 
range over which a particular Raman measurement is made is a function of the laser excitation frequency. The basic 
principle behind Raman spectroscopy differs from the MIR and NIR in that the x-axis of the Raman spectrum is 
measured as a function of energy shift (in cm" ) relative to the frequency of the laser used as the source of radiation. 
Briefly, the Raman spectrum arises from inelastic scattering of incident photons, which requires a change in 
polarizability with vibration, as opposed to infrared absorption, which requires a change in dipole moment with 
vibration. The end result is spectral information that is similar and in many cases complementary to the MIR. The 

7 

Raman effect is weak - only about one in 10 photons incident to the sample undergoes Raman scattering. Both 
organic and inorganic materials possess a Raman spectrum; they generally produce sharp bands that are chemically 
specific. Fluorescence is a competing phenomenon and, depending on the sample, can overwhelm the Raman signal, 
for both bulk spectroscopy and imaging implementations. 

Raman chemical imaging requires little or no sample preparation. However, physical sample sectioning may be used 
to expose the surface of interest, with care taken to obtain a surface that is as flat as possible. The conditions required 
for a particular measurement dictate the level of invasiveness of the technique, and samples that are sensitive to high 
power laser radiation may be damaged during analysis. It is relatively insensitive to the presence of water in the 
sample and is therefore useful for imaging samples that contain water such as biological material. 



Chemical imaging 246 

Fluorescence imaging (visible and NIR) 

This emission microspectroscopy mode is the most sensitive in both visible and FT-NIR microspectroscopy, and has 
therefore numerous biomedical, biotechnological and agricultural applications. There are several powerful, highly 
specific and sensitive fluorescence techniques that are currently in use, or still being developed; among the former 
are FLIM, FRAP, FRET and FLIM-FRET; among the latter are NIR fluorescence and probe-sensitivity enhanced 
NIR fluorescence microspectroscopy and nanospectroscopy techniques (see "Further reading" section). 

Sampling and samples 

The value of imaging lies in the ability to resolve spatial heterogeneities in solid-state or gel/gel-like samples. 
Imaging a liquid or even a suspension has limited use as constant sample motion serves to average spatial 
information, unless ultra-fast recording techniques are employed as in fluorescence correlation microspectroscopy or 
FLIM obsevations where a single molecule may be monitored at extremely high (photon) detection speed. 
High-throughput experiments (such as imaging multi-well plates) of liquid samples can however provide valuable 
information. In this case, the parallel acquisition of thousands of spectra can be used to compare differences between 
samples, rather than the more common implementation of exploring spatial heterogeneity within a single sample. 

Similarly, there is no benefit in imaging a truly homogeneous sample, as a single point spectrometer will generate 
the same spectral information. Of course the definition of homogeneity is dependent on the spatial resolution of the 
imaging system employed. For MIR imaging, where wavelengths span from 3-10 micrometres, objects on the order 
of 5 micrometres may theoretically be resolved. The sampled areas are limited by current experimental 
implementations because illumination is provided by the interferometer. Raman imaging may be able to resolve 
particles less than 1 micrometre in size, but the sample area that can be illuminated is severely limited. With Raman 
imaging, it is considered impractical to image large areas and, consequently, large samples. FT-NIR 
chemical/hyperspectral imaging usually resolves only larger objects (>10 micrometres), and is better suited for large 

samples because illumination sources are readily available. However, FT-NIR microspectroscopy was recently 

T251 
reported to be capable of about 1.2 micron (micrometer) resolution in biological samples Furthermore, 

two-photon excitation FCS experiments were reported to have attained 15 nanometer resolution on biomembrane 

thin films with a special coincidence photon-counting setup. 

Detection limit 

The concept of the detection limit for chemical imaging is quite different than for bulk spectroscopy, as it depends 
on the sample itself. Because a bulk spectrum represents an average of the materials present, the spectral signatures 
of trace components are simply overwhelmed by dilution. In imaging however, each pixel has a corresponding 
spectrum. If the physical size of the trace contaminant is on the order of the pixel size imaged on the sample, its 
spectral signature will likely be detectable. If however, the trace component is dispersed homogeneously (relative to 
pixel image size) throughout a sample, it will not be detectable. Therefore, detection limits of chemical imaging 
techniques are strongly influenced by particle size, the chemical and spatial heterogeneity of the sample, and the 
spatial resolution of the image. 

Data analysis 

Data analysis methods for chemical imaging data sets typically employ mathematical algorithms common to single 
point spectroscopy or to image analysis. The reasoning is that the spectrum acquired by each detector is equivalent to 
a single point spectrum; therefore pre-processing, chemometrics and pattern recognition techniques are utilized with 
the similar goal to separate chemical and physical effects and perform a qualitative or quantitative characterization of 
individual sample components. In the spatial dimension, each chemical image is equivalent to a digital image and 
standard image analysis and robust statistical analysis can be used for feature extraction. 



Chemical imaging 247 

See also 

• Multispectral image 

• Microspectroscopy 

• Imaging spectroscopy 

References 

[I] http://www.imaging.net/chemical-imaging/Chemical imaging 

[2] http://www.malvern.com/LabEng/products/sdi/bibliography/sdi_bibliography.htm E. N. Lewis, E. Lee and L. H. Kidder, Combining 

Imaging and Spectroscopy: Solving Problems with Near-Infrared Chemical Imaging. Microscopy Today, Volume 12, No. 6, 11/2004. 
[3] C.L. Evans and X.S. Xie.2008. Coherent Anti-Stokes Raman Scattering Microscopy: Chemical Imaging for Biology and Medicine., 

doi:10.1146/annurev.anchem.l.031207. 112754 Annual Review of Analytical Chemistry, 1: 883-909. 
[4] Diaspro, A., and Robello, M. (1999). Multi-photon Excitation Microscopy to Study Biosystems. European Microscopy and Analysis., 5:5-7. 
[5] D.S. Mantus and G. H. Morrison. 1991. Chemical imaging in biology and medicine using ion microscopy., Microchimica Acta, 104, (1-6) 

January 1991, doi: 10.1007/BF01245536 
[6] Bagatolli, L.A., and Gratton, E. (2000). Two-photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of 

binary phospholipid mixtures. Biophys J., 78:290-305. 
[7] Schwille, P., Haupts, U., Maiti, S., and Webb. W.(1999). Molecular dynamics in living cells observed by fluorescence correlation 

spectroscopy with one- and two-photon excitation. Biophysical Journal, 77(10):2251-2265. 
[8] l.Lee, S. C. et al., (2001). One Micrometer Resolution NMR Microscopy. J. Magn. Res., 150: 207-213. 
[9] Near Infrared Microspectroscopy, Fluorescence Microspectroscopy, Infrared Chemical Imaging and High Resolution Nuclear Magnetic 

Resonance Analysis of Soybean Seeds, Somatic Embryos and Single Cells., Baianu, I.C. et al. 2004., In Oil Extraction and Analysis. , D. 

Luthria, Editor pp.241-273, AOCS Press., Champaign, IL. 
[10] Single Cancer Cell Detection by Near Infrared Microspectroscopy, Infrared Chemical Imaging and Fluorescence Microspectroscopy. 2004.1. 

C. Baianu, D. Costescu, N. E. Hofmann and S. S. Korban, q-bio/0407006 (July 2004) (http://arxiv.org/abs/q-bio/0407006) 

[II] J. Dubois, G. Sando, E. N. Lewis, Near-Infrared Chemical Imaging, A Valuable Tool for the Pharmaceutical Industry, G.I.T. Laboratory 
Journal Europe, No. 1-2, 2007. 

[12] http://witec.de/en/download/Raman/ImagingMicroscopy04.pdf 

[13] Raghavachari, R., Editor. 2001. Near-Infrared Applications in Biotechnology, Marcel-Dekker, New York, NY. 

[14] Applications of Novel Techniques to Health Foods, Medical and Agricultural Biotechnology. (June 2004) I. C. Baianu, P. R. Lozano, V. I. 

Prisecaru and H. C. Lin q-bio/0406047 (http://arxiv.org/abs/q-bio/0406047) 
[15] http://www.spectroscopyeurope.com/NIR_14_3.pdf 
[16] http://www.fda.gov/cder/OPS/PAT.htm 
[17] Eigen, M., and Rigler, R. (1994). Sorting single molecules: Applications to diagnostics and evolutionary biotechnology, Proc. Natl. Acad. 

Sci. USA 91:5740. 
[18] Rigler R. and Widengren J. (1990). Ultrasensitive detection of single molecules by fluorescence correlation spectroscopy, BioScience (Ed. 

Klinge & Owman) p. 180. 
[19] Single Cancer Cell Detection by Near Infrared Microspectroscopy, Infrared Chemical Imaging and Fluorescence Microspectroscopy. 2004.1. 

C. Baianu, D. Costescu, N. E. Hofmann, S. S. Korban and et al., q-bio/0407006 (July 2004) (http://arxiv.org/abs/q-bio/0407006) 
[20] Oehlenschlager F., Schwille P. and Eigen M. (1996). Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with 

fluorescence correlation spectroscopy, Proc. Natl. Acad. Sci. USA 93:1281. 
[21] Near Infrared Microspectroscopy, Fluorescence Microspectroscopy, Infrared Chemical Imaging and High Resolution Nuclear Magnetic 

Resonance Analysis of Soybean Seeds, Somatic Embryos and Single Cells., Baianu, I.C. et al. 2004., In Oil Extraction and Analysis.,!). 

Luthria, Editor pp.241-273, AOCS Press., Champaign, IL. 
[22] M. Chamberland, V. Farley, A. Vallieres, L. Belhumeur, A. Villemaire, J. Giroux et J. Legault, High-Performance Field-Portable Imaging 

Radiometric Spectrometer Technology For Hyperspectral imaging Applications, Proc. SPIE 5994, 59940N, September 2005. 
[23] Novel Techniques for Microspectroscopy and Chemical Imaging Analysis of Soybean Seeds and Embryos. (2002). Baianu, I.C., Costescu, 

D.M., and You, T. Soy2002 Conference, Urbana, Illinois. 
[24] Near Infrared Microspectroscopy, Chemical Imaging and NMR Analysis of Oil in Developing and Mutagenized Soybean Embryos in 

Culture. (2003). Baianu, I.C., Costescu, D.M., Hofmann, N., and Korban, S.S. AOCS Meeting, Analytical Division. 
[25] Near Infrared Microspectroscopy, Fluorescence Microspectroscopy, Infrared Chemical Imaging and High Resolution Nuclear Magnetic 

Resonance Analysis of Soybean Seeds, Somatic Embryos and Single Cells., Baianu, I.C. et al. 2004., In Oil Extraction and Analysis. , D. 

Luthria, Editor pp.241-273, AOCS Press., Champaign, IL. 



Chemical imaging 248 

Further reading 

1. E. N. Lewis, P. J. Treado, I. W. Levin, Near-Infrared and Raman Spectroscopic Imaging, American Laboratory, 
06/1994:16(1994) 

2. E. N. Lewis, P. J. Treado, R. C. Reeder, G. M. Story, A. E. Dowrey, C. Marcott, I. W. Levin, FTIR spectroscopic 
imaging using an infrared focal-plane array detector, Analytical Chemistry, 67:3377 (1995) 

3. P. Colarusso, L. H. Kidder, I. W. Levin, J. C. Fraser, E. N. Lewis Infrared Spectroscopic Imaging: from Planetary 
to Cellular Systems, Applied Spectroscopy, 52 (3):106A (1998) 

4. P. J. Treado I. W. Levin, E. N. Lewis, Near-Infrared Spectroscopic Imaging Microscopy of Biological Materials 
Using an Infrared Focal -Plane Array and an Acousto-Optic Tunable Filter (AOTF), Applied Spectroscopy, 48:5 
(1994) 

5. Hammond, S.V., Clarke, F. C, Near-infrared microspectroscopy. In: Handbook of Vibrational Spectroscopy, 
Vol. 2, J.M. Chalmers and P.R. Griffiths Eds. John Wiley and Sons, West Sussex, UK, 2002, p.1405-1418 

6. L.H. Kidder, A.S. Haka, E.N. Lewis, Instrumentation for FT-IR Imaging. In: Handbook of Vibrational 
Spectroscopy, Vol. 2, J.M. Chalmers and P.R. Griffiths Eds. John Wiley and Sons, West Sussex, UK, 2002, 
pp. 1386-1404 

7. J. Zhang; A. O'Connor; J. F. Turner II, Cosine Histogram Analysis for Spectral Image Data 
Classification,Applied Spectroscopy, Volume 58, Number 11, November 2004, pp. 1318-1324(7) 

8. J. F. Turner II; J. Zhang; A. O'Connor, A Spectral Identity Mapper for Chemical Image Analysis, Applied 
Spectroscopy, Volume 58, Number 11, November 2004, pp. 1308-1317(10) 

9. H. R. MORRIS, J. F. TURNER II, B. MUNRO, R. A. RYNTZ, P. J. TREADO, Chemical imaging of 
thermoplastic olefin (TPO) surface architecture, Langmuir, 1999, vol. 15, no8, pp. 2961-2972 

10. J. F. Turner II, Chemical imaging and spectroscopy using tunable filters: Instrumentation, methodology, and 
multivariate analysis, Thesis (PhD). UNIVERSITY OF PITTSBURGH, Source DAI-B 59/09, p. 4782, Mar 1999, 
286 pages. 

11. P. Schwille.(2001). in Fluorescence Correlation Spectroscopy. Theory and applications. R. Rigler & E.S. Elson, 
eds., p. 360. Springer Verlag: Berlin. 

12. Schwille P., Oehlenschlager F. and Walter N. (1996). Analysis of RNA-DNA hybridization kinetics by 
fluorescence correlation spectroscopy, Biochemistry 35:10182. 

13. FLIM I Fluorescence Lifetime Imaging Microscopy: Fluorescence, fluorophore chemical imaging, confocal 
emission microspectroscopy, FRET, cross-correlation fluorescence microspectroscopy (http://www. 
nikoninstruments . com/infocenter.php ?n=FLIM) . 

14. FLIM Applications: (http://www. nikoninstruments. com/infocenter.php ?n=FLIM) "FLIM is able to 
discriminate between fluorescence emanating from different fluorophores and autoflorescing molecules in a 
specimen, even if their emission spectra are similar. It is, therefore, ideal for identifying fluorophores in 
multi-label studies. FLIM can also be used to measure intracellular ion concentrations without extensive 
calibration procedures (for example, Calcium Green) and to obtain information about the local environment of a 
fluorophore based on changes in its lifetime." FLIM is also often used in microspectroscopic/chemical imaging, 
or microscopic, studies to monitor spatial and temporal protein-protein interactions, properties of membranes and 
interactions with nucleic acids in living cells. 

15. Gadella TW Jr., FRET and FLIM techniques, 33. Imprint: Elsevier, ISBN 978-0-08-054958-3. (2008) 560 pages 

16. Langel FD, et al., Multiple protein domains mediate interaction between BcllO and Maltl, /. Biol. Chem., 
(2008)283(47):32419-31 

17. Clayton AH. , The polarized AB plot for the frequency-domain analysis and representation of fluorophore 
rotation and resonance energy homotransfer. J Microscopy. (2008) 232(2):306-12 

18. Clayton AH, et al., Predominance of activated EGFR higher-order oligomers on the cell surface. Growth 
Factors (2008) 20:1 



Chemical imaging 



249 



19. Plowman et al., Electrostatic Interactions Positively Regulate K-Ras Nanocluster Formation and Function. 
Molecular and Cellular Biology (2008) 4377-4385 

20. Belanis L, et al., Galectin-1 Is a Novel Structural Component and a Major Regulator of H-Ras Nanoclusters. 
Molecular Biology of the Cell (2008) 19:1404-1414 

21. Van Manen HJ, Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime 
imaging microscopy. Biophys J. (2008) 94(8):L67-9 

22. Van der Krogt GNM, et al., A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: 
Application to the Epac cAMP Sensor as an Example, PLoS ONE, (2008) 3(4):el916 

23. Dai X, et al., Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in 
living cells. Nanomedicine. (2008) 4(l):49-56. 

External links 

• NIR Chemical Imaging in Pharmaceutical Industry (http://www.spectroscopyeurope.com/NIR_14_3.pdf) 

• Pharmaceutical Process Analytical Technology: (http://www.fda.gov/cder/OPS/PAT.htm) 

• NIR Chemical Imaging for Counterfeit Pharmaceutical Product Analysis (http://www.spectroscopymag.com/ 
spectroscopy /Near-IR+Spectroscopy/NIR-Chemical-Imaging-for-Counterfeit-Pharmaceutica/ArticleStandard/ 
Article/detail/406629) 

• Chemical Imaging: Potential New Crime Busting Tool (http://www.sciencedaily.com/releases/2007/08/ 
070802103435.htm) 

• Applications of Chemical Imaging in Research (http://www3.imperial.ac.uk/ 
vibrationalspectroscopyandchemicalimaging/research) 



Fluorescence microscopy 



A fluorescence microscope (colloquially synonymous with 
epifluorescence microscope) is an optical microscope used to study 
properties of organic or inorganic substances using the phenomena of 
fluorescence and phosphorescence instead of, or in addition to, 



reflection and absorption 



[1] [2] 




An upright fluorescence microscope (Olympus 

BX61) with the fluorescent filter cube turret 
above the objective lenses, coupled with a digital 



Fluorescence microscopy 



250 



Technique 

In most cases, a component of interest in the specimen can be labeled 
specifically with a fluorescent molecule called a fluorophore (such as 
green fluorescent protein (GFP), fluorescein or DyLight 488). The 
specimen is illuminated with light of a specific wavelength (or 
wavelengths) which is absorbed by the fluorophores, causing them to 
emit light of longer wavelengths (i.e. of a different color than the 
absorbed light). The illumination light is separated from the much 
weaker emitted fluorescence through the use of a spectral emission 
filter. Typical components of a fluorescence microscope are the light 
source (xenon arc lamp or mercury-vapor lamp), the excitation filter, 
the dichroic mirror (or dichromatic beamsplitter), and the emission 
filter (see figure below). The filters and the dichroic are chosen to 
match the spectral excitation and emission characteristics of the 
fluorophore used to label the specimen. In this manner, the 
distribution of a single fluorophore (color) is imaged at a time. 
Multi-color images of several types of fluorophores must be composed 



by combining several single-color images 



[l] 




An inverted fluorescence microscope (Nikon 

TE2000) with the fluorescent filter cube turret 

below the stage. Note the orange plate that allows 

the user to look at a sample while protecting their 

eyes from the UV light. 



Most fluorescence microscopes in use are epifluorescence microscopes 

(i.e. excitation and observation of the fluorescence are from above {epi—) the specimen). These microscopes have 
become an important part in the field of biology, opening the doors for more advanced microscope designs, such as 
the confocal microscope and the total internal reflection fluorescence microscope (TIRF). 

Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching. Special care 
must be taken to prevent photobleaching through the use of more robust fluorophores, by minimizing illumination, 
or by introducing a scavenger system to reduce the rate of photobleaching. 



Epifluorescence microscopy 



1 1 


dichroic mirror / 









objective 



T 



emission filter 



light source 



excitation filter 



H 



specimen 
Schematic of a fluorescence microscope. 



Epifluorescence microscopy is a method of fluorescence microscopy 
that is widely used in life sciences. The excitatory light is passed from 
above (or, for inverted microscopes, from below), through the 
objective lens and then onto the specimen instead of passing it first 
through the specimen. The fluorescence in the specimen gives rise to 
emitted light which is focused to the detector by the same objective 
that is used for the excitation. Since most of the excitatory light is 
transmitted through the specimen, only reflected excitatory light 
reaches the objective together with the emitted light and this method 
therefore gives an improved signal to noise ratio. An additional filter 
between the objective and the detector can filter out the remaining 
excitation light from fluorescent light. A common use in biology is to 
apply fluorescent or fluorochrome stains to the specimen in order to 
image distributions of proteins or other molecules of interest. 



Fluorescence microscopy 25 1 

Improvements and sub-diffraction techniques 

The nature of light limits the size of the spot to which light can be focused. According to the diffraction limit a 
focused light distribution cannot be made smaller than approximately half of the wavelength of the used light. 
Uncovered in the 19th century by Ernst Abbe this has been a barrier of the achievable resolution of fluorescence 
light microscopes for a long time. While resolution is denoted by the ability to discern different objects of the same 
kind, localizing or tracking of single particles have been performed with a precision much below the diffraction 
limit. 

Several improvements in microscopy techniques have been invented in the 20th century and have resulted in 
increased resolution and contrast to some extent. However they did not overcome the diffraction limit. In 1978 first 
theoretical ideas have been developed to break this barrier by using a 4Pi microscope as a confocal laser scanning 
fluorescence microscope where the light is focused ideally from all sides to a common focus which is used to scan 
the object by 'point-by-point' excitation combined with 'point-by-point' detection . However, the first experimental 
demonstration of the 4pi microscope took place in 1994 . The 4Pi microscopy is maximizing the amount of 
available focusing directions by using two opposing objective lenses or Multi-photon microscopy using redshifted 
light and multi-photon excitation. 

The first technique to really achieve a sub-diffraction resolution was STED microscopy, proposed in 1994. This 
method and all techniques following the RESOLFT concept rely on a strong non-linear interaction between light and 
fluorescing molecules. The molecules are driven strongly between distinguishable molecular states at each specific 
location, so that finally light can be emitted at only a small fraction of space, hence an increased resolution. 

As well in the 1990s another super resolution microscopy method based on wide field microscopy has been 
developed. Substantially improved size resolution of cellular nanostructures stained with a fluorescent marker was 
achieved by development of SPDM localization microscopy and the structured laser illumination (spatially 
modulated illumination, SMI) . Combining the principle of SPDM with SMI resulted in the development of the 
Vertico SMI microscope . Single molecule detection of normal blinking fluorescent dyes like GFP can be 

achieved by using a further development of SPDM the so-called SPDMphymod technology which makes it possible 
to detect and count two different fluorescent molecule types at the molecular level (this technology is referred to as 

ro] 

2CLM, 2 Color Localization Microscopy) 

Alternatively, the advent of photoactivated localization microscopy could achieve similar results by relying on 
blinking or switching of single molecules, where the fraction of fluorescing molecules is very small at each time. 
This stochastic response of molecules on the applied light corresponds also to a highly nonlinear interaction, leading 
to subdiffraction resolution. 



Fluorescence microscopy 



252 



Gallery 











EJ 




Epifluorescent imaging of the 

three components in a dividing 

human cancer cell. DNA is 

stained blue, a protein called 

INCENP is green, and the 

microtubules are red. Each 

fluorophore is imaged separately 

using a different combination of 

excitation and emission filters, 

and the images are captured 

sequentially using a digital CCD 

camera, then overlaid to give a 

complete image. 




Endothelial cells under the 

microscope. Nuclei are stained 

blue with DAPI, microtubules are 

marked green by an antibody 

bound to FITC and actin filaments 

are labeled red with phalloidin 

bound to TRITC. Bovine 

pulmonary artery endothelial 

(BPAE) cells 




Human lymphocyte nucleus stained with 
DAPI with chromosome 13 (green) and 21 

(red) centromere probes hybridized 
(Fluorescent in situ hybridization (FISH)) 




Yeast cell membrane visualized 

by some membrane proteins 

fused with RFP and GFP 

fluorescent markers. Imposition 

of light from both of markers 

results in yellow color. 





D 

200rtm 

• 
t 

* 

** 
+ 




15nm ♦ T\ 

* *l4nrr 
— * 
30im 


Mic 

mo 

huma 

dista 

the 

St 

Vertic 


super Resolution 
■■oscopy: Single ' 
ecule detection 
n cancer cell. Ty 
nee measuremen 
15 nm range (5 
andard deviatior 
measured with a 
o-SMI/SPDMph 
microscope 


fFP 

n a 
pical 
ts in 

am 
) 

ymod 




Super Resolution Microscopy: Co-localzation 

microscopy (2CLM) with GFP and RFP 

fusion proteins (nucleus of a bone cancer cell) 

120.000 localized molecules in a wide-field 
2 
area (470 um~) measured with a 

Vertico-SMI/SPDMphymod micrsocpe 



Fluorescence microscopy 



253 





# "> ■ 

/ 




<**! 


nni 



Fluorescence microscopy of 
DNA Expression in the 
Human Wild-Type and 
P239S Mutant Palladin. 




Fluorescence microscopy images of sun flares 

pathology in a blood cell showing the affected 

areas in red. 



See also 

• Microscope 

• Mercury-vapor lamp, Xenon arc lamp 

• Stokes shift 



References 

[1] Spring KR, Davidson MW. "Introduction to Fluorescence Microscopy" (http://www.microscopyu.com/articles/fluorescence/ 

fluorescenceintro.html). Nikon MicroscopyU . . Retrieved 2008-09-28. 
[2] "The Fluorescence Microscope" (http://nobelprize.org/educational_games/physics/microscopes/fluorescence/). Microscopes — Help 

Scientists Explore Hidden Worlds. The Nobel Foundation. . Retrieved 2008-09-28. 
[3] Considerations on a laser-scanning-microscope with high resolution and depth of field: C. Cremer and T. Cremer in M1CROSCOPICA 

ACTA VOL. 81 NUMBER 1 September.pp. 31—44 (1978) 
[4] S.W. Hell, E.H.K. Stelzer, S. Lindek, C. Cremer (1994). "Confocal microscopy with an increased detection aperture: type-B 4Pi confocal 

microscopy" (http://www.opticsinfobase.org/viewmedia.cfm?uri=ol-19-3-222&seq=0). Optics Letters 19: 222—224. 

doi:10.1364/OL. 19.000222. . 
[5] M. Hausmann, B. Schneider, J. Bradl, C. Cremer (1997): High-precision distance microscopy of 3D-nanostructures by a spatially modulated 

excitation fluorescence microscope. In: Optical Biopsies and Microscopic Techniques II (Edts Bigio IJ, Schneckenburger H, Slavik J, 

Svanberg K, Viallet PM), Proc. SPIE 3197: 217-222 
[6] High precision structural analysis of subnuclear complexes in fixed and live cells via Spatially Modulated Illumination (SMI) microscopy: J. 

Reymann, D. Baddeley, P. Lemmer, W. Stadter, T. Jegou, K. Rippe, C. Cremer, U. Birk in CHROMOSOME RESEARCH, Vol. 16, pp. 367 

-382 (2008) 
[7] Nano-structure analysis using Spatially Modulated Illumination microscopy: D. Baddeley, C. Batram, Y. Weiland, C. Cremer, U.J. Birk in 

NATURE PROTOCOLS, Vol 2, pp. 2640 - 2646 (2007) 
[8] Manuel Gunkel, Fabian Erdel, Karsten Rippe, Paul Lemmer, Rainer Kaufmann, Christoph Hormann, Roman Amberger and Christoph 

Cremer: Dual color localization microscopy of cellular nanostructures. In: Biotechnology Journal, 2009, 4, 927-938. ISSN 1860-6768 



External links 

• Fluorophores.org (http://www.fluorophores.org) - Database of fluorescent dyes. 



Fluorescence correlation spectroscopy 254 

Fluorescence correlation spectroscopy 

Fluorescence correlation spectroscopy (FCS) is a correlation analysis of fluctuation of the fluorescence intensity. 
The analysis provides parameters of the physics under the fluctuations. One of the interesting applications of this is 
an analysis of the concentration fluctuations of fluorescent particles (molecules) in solution. In this application, the 
fluorescence emitted from a very tiny space in solution containing a small number of fluorescent particles 
(molecules) is observed. The fluorescence intensity is fluctuating due to Brownian motion of the particles. In other 
words, the number of the particles in the sub-space defined by the optical system is randomly changing around the 
average number. The analysis gives the average number of fluorescent particles and average diffusion time, when the 
particle is passing through the space. Eventually, both the concentration and size of the particle (molecule) are 
determined. Since the method is observing a small number of molecule in a very tiny spot, it is a very sensitive 
analytical tool. Both parameters are important in biochemical research, biophysics, and chemistry. In contrast to 
other methods, such as HPLC analysis, FCS has no physical separation process and has a good spatial resolution 
determined by the optics. These are of great advantage. Moreover, the method enables us to observe 
fluorescence-tagged molecules in the biochemical pathway in intact living cells. This opens a new area, "in situ or in 
vivo biochemistry": tracing the biochemical pathway in intact cells and organs. 

Commonly, FCS is employed in the context of optical microscopy, in particular confocal or two-photon microscopy. 
In these techniques light is focused on a sample and the measured fluorescence intensity fluctuations (due to 
diffusion, physical or chemical reactions, aggregation, etc.) are analyzed using the temporal autocorrelation. Because 
the measured property is essentially related to the magnitude and/or the amount of fluctuations, there is an optimum 
measurement regime at the level when individual species enter or exit the observation volume (or turn on and off in 
the volume). When too many entities are measured at the same time the overall fluctuations are small in comparison 
to the total signal and may not be resolvable — in the other direction, if the individual fluctuation-events are too 
sparse in time, one measurement may take prohibitively too long. FCS is in a way the fluorescent counterpart to 
dynamic light scattering, which uses coherent light scattering, instead of (incoherent) fluorescence. 

When an appropriate model is known, FCS can be used to obtain quantitative information such as 

• diffusion coefficients 

• hydrodynamic radii 

• average concentrations 

• kinetic chemical reaction rates 

• singlet-triplet dynamics 

Because fluorescent markers come in a variety of colors and can be specifically bound to a particular molecule (e.g. 
proteins, polymers, metal-complexes, etc.), it is possible to study the behavior of individual molecules (in rapid 
succession in composite solutions). With the development of sensitive detectors such as avalanche photodiodes the 
detection of the fluorescence signal coming from individual molecules in highly dilute samples has become practical. 
With this emerged the possibility to conduct FCS experiments in a wide variety of specimens, ranging from 
materials science to biology. The advent of engineered cells with genetically tagged proteins (like green fluorescent 
protein) has made FCS a common tool for studying molecular dynamics in living cells. 



Fluorescence correlation spectroscopy 255 

History 

Signal-correlation techniques were first experimentally applied to fluorescence in 1972 by Magde, Elson, and 
Webb , who are therefore commonly credited as the "inventors" of FCS. The technique was further developed in a 
group of papers by these and other authors soon after, establishing the theoretical foundations and types of 
applications. See Thompson (1991) for a review of that period. 

Beginning in 1993 , a number of improvements in the measurement techniques — notably using confocal 

microscopy, and then two-photon microscopy — to better define the measurement volume and reject 

T71 rsi 
background — greatly improved the signal-to-noise ratio and allowed single molecule sensitivity. Since then, 

there has been a renewed interest in FCS, and as of August 2007 there have been over 3,000 papers using FCS found 

in Web of Science. See Krichevsky and Bonnet for a recent review. In addition, there has been a flurry of activity 

extending FCS in various ways, for instance to laser scanning and spinning-disk confocal microscopy (from a 

stationary, single point measurement), in using cross-correlation (FCCS) between two fluorescent channels instead 

of autocorrelation, and in using Forster Resonance Energy Transfer (FRET) instead of fluorescence. 

Typical FCS setup 

The typical FCS setup consists of a laser line (wavelengths ranging typically from 405—633 nm (cw), and from 
690—1100 nm (pulsed)), which is reflected into a microscope objective by a dichroic mirror. The laser beam is 
focused in the sample, which contains fluorescent particles (molecules) in such high dilution, that only a few are 
within the focal spot (usually 1—100 molecules in one fL). When the particles cross the focal volume, they fluoresce. 
This light is collected by the same objective and, because it is red-shifted with respect to the excitation light it passes 
the dichroic mirror reaching a detector, typically a photomultiplier tube or avalanche photodiode detector. The 
resulting electronic signal can be stored either directly as an intensity versus time trace to be analyzed at a later point, 
or computed to generate the autocorrelation directly (which requires special acquisition cards). The FCS curve by 
itself only represents a time-spectrum. Conclusions on physical phenomena have to be extracted from there with 
appropriate models. The parameters of interest are found after fitting the autocorrelation curve to modeled functional 
forms. 

The measurement volume 

The measurement volume is a convolution of illumination (excitation) and detection geometries, which result from 
the optical elements involved. The resulting volume is described mathematically by the point spread function (or 
PSF), it is essentially the image of a point source. The PSF is often described as an ellipsoid (with unsharp 
boundaries) of few hundred nanometers in focus diameter, and almost one micrometre along the optical axis. The 
shape varies significantly (and has a large impact on the resulting FCS curves) depending on the quality of the 
optical elements (it is crucial to avoid astigmatism and to check the real shape of the PSF on the instrument). In the 
case of confocal microscopy, and for small pinholes (around one Airy unit), the PSF is well approximated by 
Gaussians: 

PSF(r, z) = I oe - 2r2 /< e - 2z2 /^ 
where / is the peak intensity, r and z are radial and axial position, and ^-^and UJ z are the radial and axial radii, 
and UJ Z > UJ x y . This Gaussian form is assumed in deriving the functional form of the autocorrelation. 
Typically <^> X yis 200—300 nm, and U) z is 2—6 times larger. One common way of calibrating the measurement 
volume parameters is to perform FCS on a species with known diffusion coefficient and concentration (see below). 
Diffusion coefficients for common fluorophores in water are given in a later section. 
The Gaussian approximation works to varying degrees depending on the optical details, and corrections can 

ri2i 

sometimes be applied to offset the errors in approximation. 



Fluorescence correlation spectroscopy 256 

Autocorrelation function 

The (temporal) autocorrelation function is the correlation of a time series with itself shifted by time t, as a function 
of r: 

_ (SI(t)6I(t + T)) (I(t)I(t + T)) _ 

[ } <w m) 2 

where 5I(t) = lit) — (I(t)) is the deviation from the mean intensity. The normalization (denominator) here is 
the most commonly used for FCS, because then the correlation at 7- = 0, G(0), is related to the average number of 
particles in the measurement volume. 

Interpreting the autocorrelation function 

To extract quantities of interest, the autocorrelation data can be fitted, typically using a nonlinear least squares 
algorithm. The fit's functional form depends on the type of dynamics (and the optical geometry in question). 

Normal diffusion 

The fluorescent particles used in FCS are small and thus experience thermal motions in solution. The simplest FCS 
experiment is thus normal 3D diffusion, for which the autocorrelation is: 

«M = c (»)(TT(^IiW^P + G(oo) 

where a = CV z /uj x yis the ratio of axial to radial e ~ 2 radii of the measurement volume, and Tf>is the 

characteristic residence time. This form was derived assuming a Gaussian measurement volume. Typically, the fit 
would have three free parameters— G(0), G(oo), and Tjy— from which the diffusion coefficient and fluorophore 

concentration can be obtained. 

With the normalization used in the previous section, G(0) gives the mean number of diffusers in the volume <N>, or 

equivalently — with knowledge of the observation volume size — the mean concentration: 

G{0) = W) = ^wy 

where the effective volume is found from integrating the Gaussian form of the measurement volume and is given by: 

v eS = ^W xy . z . 

T£> gives the diffusion coefficient: 
,2 



D = lo 2 JAt d . 



Anomalous diffusion 

If the diffusing particles are hindered by obstacles or pushed by a force (molecular motors, flow, etc.) the dynamics 
is often not sufficiently well-described by the normal diffusion model, where the mean squared displacement (MSD) 
grows linearly with time. Instead the diffusion may be better described as anomalous diffusion, where the temporal 
dependenc of the MSD is non-linear as in the power-law: 

MSD = 6D a t a 

where D a is an anomalous diffusion coefficient. "Anomalous diffusion" commonly refers only to this very generic 
model, and not the many other possibilities that might be described as anomalous. Also, a power law is, in a strict 
sense, the expected form only for a narrow range of rigorously defined systems, for instance when the distribution of 
obstacles is fractal. Nonetheless a power law can be a useful approximation for a wider range of systems. 
The FCS autocorrelation function for anomalous diffusion is: 



Fluorescence correlation spectroscopy 257 

G(t) = G(0)- , . — r- in —. — — — - + G(oo), 

(1 + (r/r D ) a )(l + a- 2 (r/r D )«)V2 

where the anomalous exponent a is the same as above, and becomes a free parameter in the fitting. 

Using FCS, the anomalous exponent has been shown to be an indication of the degree of molecular crowding (it is 

ri3i 

less than one and smaller for greater degrees of crowding) 

Polydisperse diffusion 

If there are diffusing particles with different sizes (diffusion coefficients), it is common to fit to a function that is the 
sum of single component forms: 

g(t) = g(o) J2 K^r-/ — wi + -v i — iw* + G(oo) 

i (1 + [T/T Dt i)){l + a 2 (r/r Ai )) 1/2 
where the sum is over the number different sizes of particle, indexed by i, and Ctj gives the weighting, which is 
related to the quantum yield and concentration of each type. This introduces new parameters, which makes the fitting 
more difficult as a higher dimensional space must be searched. Nonlinear least square fitting typically becomes 
unstable with even a small number of T~D,i s. A more robust fitting scheme, especially useful for polydisperse 

[141 

samples, is the Maximum Entropy Method 

Diffusion with flow 

With diffusion together with a uniform flow with velocity v in the lateral direction, the autocorrelation is : 
where t v = u> xy /vis the average residence time if there is only a flow (no diffusion). 

Chemical relaxation 

A wide range of possible FCS experiments involve chemical reactions that continually fluctuate from equilibrium 
because of thermal motions (and then "relax"). In contrast to diffusion, which is also a relaxation process, the 
fluctuations cause changes between states of different energies. One very simple system showing chemical relaxation 
would be a stationary binding site in the measurement volume, where particles only produce signal when bound (e.g. 
by FRET, or if the diffusion time is much faster than the sampling interval). In this case the autocorrelation is: 

G(t) = G(0) exp(-T/V B ) + C7(oo) 
where 

TB = (feon + feoff)" 1 

is the relaxation time and depends on the reaction kinetics (on and off rates), and: 

is related to the equilibrium constant K. 

Most systems with chemical relaxation also show measureable diffusion as well, and the autocorrelation function 
will depend on the details of the system. If the diffusion and chemical reaction are decoupled, the combined 
autocorrelation is the product of the chemical and diffusive autocorrelations. 

Triplet state correction 

The autocorrelations above assume that the fluctuations are not due to changes in the fluorescent properties of the 
particles. However, for the majority of (bio)organic fluorophores— e.g. green fluorescent protein, rhodamine, Cy3 and 
Alexa Fluor dyes— some fraction of illuminated particles are excited to a triplet state (or other non-radiative decaying 
states) and then do not emit photons for a characteristic relaxation time Tp. Typically Tpis on the order of 
microseconds, which is usually smaller than the dynamics of interest (e.g. Tjj) but large enough to be measured. A 



Fluorescence correlation spectroscopy 



258 



multiplicative term is added to the autocorrelation account for the triplet state. For normal diffusion: 

where ^is the fraction of particles that have entered the triplet state and Tpis the corresponding triplet state 
relaxation time. If the dynamics of interest are much slower than the triplet state relaxation, the short time 
component of the autocorrelation can simply be truncated and the triplet term is unnecessary. 

Common fluorescent probes 

The fluorescent species used in FCS is typically a biomolecule of interest that has been tagged with a fluorophore 
(using immunohistochemistry for instance), or is a naked fluorophore that is used to probe some environment of 
interest (e.g. the cytoskeleton of a cell). The following table gives diffusion coefficients of some common 
fluorophores in water at room temperature, and their excitation wavelengths. 



Fluorescent dye 


D(xlO" 10 m 2 s" 1 ) 


Excitation wavelength (nm) 


Reference 


Rhodamine 6G 


2.8, 3.0, 4.14 ± 0.05 @ 25.00 °C 


514 


[16] [17] [18] 


Rhodamine 110 


2.7 


488 


[19] 


Tetramethyl rhodamine 


2.6 


543 




Cy3 


2.8 


543 




Cy5 


2.5,3.7 + 0.15 @ 25.00 °C 


633 


[20] [21] 


carboxyfluorescein 


3.2 


488 




Alexa-488 


1.96,4.35 @ 22.5±0.5 °C 


488 


[22] [23] 


Atto655-maleimide 


4.07±0.1 @ 25.00 °C 


663 


[24] 


Atto655-carboxylicacid 


4.26 ± 0.08 @ 25.00 °C 


663 


[25] 


2', 7'-difluorofluorescein (Oregon Green488) 


4.11+0.06 @ 25.00 °C 


498 


[26] 



Variations of FCS 

FCS almost always refers to the single point, single channel, temporal autocorrelation measurement, although the 
term "fluorescence correlation spectroscopy" out of its historical scientific context implies no such restriction. FCS 
has been extended in a number of variations by different researchers, with each extension generating another name 
(usually an acronym). 



Fluorescence cross-correlation spectroscopy (FCCS) 

FCS is sometimes used to study molecular interactions using differences in diffusion times (e.g. the product of an 
association reaction will be larger and thus have larger diffusion times than the reactants individually); however, 
FCS is relatively insensitive to molecular mass as can be seen from the following equation relating molecular mass 
to the diffusion time of globular particles (e.g. proteins): 

T D = ^(M)V3 

2kT v J 
where ??is the viscosity of the sample and j\/f is the molecular mass of the fluorescent species. In practice, the 

diffusion times need to be sufficiently different— a factor of at least 1.6— which means the molecular masses must 

[27] 

differ by a factor of 4. Dual color fluorescence cross-correlation spectroscopy (FCCS) measures interactions by 
cross-correlating two or more fluorescent channels (one channel for each reactant), which distinguishes interactions 
more sensitively than FCS, particularly when the mass change in the reaction is small. 



Fluorescence correlation spectroscopy 259 

Brightness analysis methods (N&B, [28] PCH, [29] FIDA, [30] Cumulant Analysis 1311 ) 

Fluorescence cross correlation spectroscopy overcomes the weak dependence of diffusion rate on molecular mass by 
looking at multicolor coincidence. What about homo-interactions? The solution lies in brightness analysis. These 
methods use the heterogeneity in the intensity distribution of fluorescence to measure the molecular brightness of 
different species in a sample. Since dimers will contain twice the number of fluorescent labels as monomers, their 
molecular brightness will be approximately double that of monomers. As a result, the relative brightness is sensitive 
a measure of oligomerization. The average molecular brightness ( {V\ ) is related to the variance ( <j 2 ) and the 
average intensity ( (J\ ) as follows: 

Here f+ and £{ are the fractional intensity and molecular brigthness, respectively, of species <j . 

Two- and three- photon FCS excitation 

Several advantages in both spatial resolution and minimizing photodamage/photobleaching in organic and/or 
biological samples are obtained by two-photon or three-photon excitation FCS 

FRET-FCS 

Another FCS based approach to studying molecular interactions uses fluorescence resonance energy transfer (FRET) 
instead of fluorescence, and is called FRET-FCS. With FRET, there are two types of probes, as with FCCS; 
however, there is only one channel and light is only detected when the two probes are very close — close enough to 
ensure an interaction. The FRET signal is weaker than with fluorescence, but has the advantage that there is only 
signal during a reaction (aside from autofluorescence). 

Image correlation spectroscopy (ICS) 

When the motion is slow (in biology, for example, diffusion in a membrane), getting adequate statistics from a 

single-point FCS experiment may take a prohibitively long time. More data can be obtained by performing the 

experiment in multiple spatial points in parallel, using a laser scanning confocal microscope. This approach has been 

[39] 
called Image Correlation Spectroscopy (ICS) . The measurements can then be averaged together. 

Another variation of ICS performs a spatial autocorrelation on images, which gives information about the 
concentration of particles . The correlation is then averaged in time. 

A natural extension of the temporal and spatial correlation versions is spatio-temporal ICS (STICS) .In STICS 
there is no explicit averaging in space or time (only the averaging inherent in correlation). In systems with 
non-isotropic motion (e.g. directed flow, asymmetric diffusion), STICS can extract the directional information. A 
variation that is closely related to STICS (by the Fourier transform) is fc-space Image Correlation Spectroscopy 

(kICS). [42] 

There are cross-correlation versions of ICS as well. 

Scanning FCS variations 

Some variations of FCS are only applicable to serial scanning laser microscopes. Image Correlation Spectroscopy 
and its variations all were implemented on a scanning confocal or scanning two photon microscope, but transfer to 

other microscopes, like a spinning disk confocal microscope. Raster ICS (RICS) , and position sensitive FCS 

T441 
(PSFCS) incorporate the time delay between parts of the image scan into the analysis. Also, low dimensional 

scans (e.g. a circular ring) — only possible on a scanning system — can access time scales between single point 

and full image measurements. Scanning path has also been made to adaptively follow particles. 



Fluorescence correlation spectroscopy 260 

Spinning disk FCS, and spatial mapping 

Any of the image correlation spectroscopy methods can also be performed on a spinning disk confocal microscope, 
which in practice can obtain faster imaging speeds compared to a laser scanning confocal microscope. This approach 

has recently been applied to diffusion in a spatially varying complex environment, producing a pixel resolution map 

T471 
of a diffusion coefficient. . The spatial mapping of diffusion with FCS has subsequently been extended to the 

TIRF system. Spatial mapping of dynamics using correlation techniques had been applied before, but only at 

sparse points or at coarse resolution 

Total internal reflection FCS 

Total internal reflection fluorescence (TIRF) is a microscopy approach that is only sensitive to a thin layer near the 
surface of a coverslip, which greatly minimizes background fluorscence. FCS has been extended to that type of 
microscope, and is called TIR-FCS . Because the fluorescence intensity in TIRF falls off exponentially with 
distance from the coverslip (instead of as a Gaussian with a confocal), the autocorrelation function is different. 

Other fluorescent dynamical approaches 

There are two main non-correlation alternatives to FCS that are widely used to study the dynamics of fluorescent 
species. 

Fluorescence recovery after photobleaching (FRAP) 

In FRAP, a region is briefly exposed to intense light, irrecoverably photobleaching fluorophores, and the 
fluorescence recovery due to diffusion of nearby (non-bleached) fluorophores is imaged. A primary advantage of 
FRAP over FCS is the ease of interpreting qualitative experiments common in cell biology. Differences between cell 
lines, or regions of a cell, or before and after application of drug, can often be characterized by simple inspection of 
movies. FCS experiments require a level of processing and are more sensitive to potentially confounding influences 
like: rotational diffusion, vibrations, photobleaching, dependence on illumination and fluorescence color, inadequate 
statistics, etc. It is much easier to change the measurement volume in FRAP, which allows greater control. In 
practice, the volumes are typically larger than in FCS. While FRAP experiments are typically more qualitative, some 
researchers are studying FRAP quantitatively and including binding dynamics. A disadvantage of FRAP in cell 
biology is the free radical perturbation of the cell caused by the photobleaching. It is also less versatile, as it cannot 
measure concentration or rotational diffusion, or co-localization. FRAP requires a significantly higher concentration 
of fluorophores than FCS. 

Particle tracking 

In particle tracking, the trajectories of a set of particles are measured, typically by applying particle tracking 
algorithms to movies. [52] Particle tracking has the advantage that all the dynamical information is maintained in the 
measurement, unlike FCS where correlation averages the dynamics to a single smooth curve. The advantage is 
apparent in systems showing complex diffusion, where directly computing the mean squared displacement allows 
straightforward comparison to normal or power law diffusion. To apply particle tracking, the particles have to be 
distinguishable and thus at lower concentration than required of FCS. Also, particle tracking is more sensitive to 
noise, which can sometimes affect the results unpredictably. 



Fluorescence correlation spectroscopy 261 

See also 

• Confocal microscopy 

• Fluorescence cross-correlation spectroscopy ,FCCS 

• FRET 

• Dynamic light scattering 

• Diffusion coefficient 

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Fluorescence correlation spectroscopy 263 

Further reading 

• Rigler R. and Widengren J. (1990). Ultrasensitive detection of single molecules by fluorescence correlation 
spectroscopy, BioScience (Ed. Klinge & Owman) p. 180 

• Oehlenschlager F., Schwille P. and Eigen M. (1996). Detection of HIV-1 RNA by nucleic acid sequence-based 
amplification combined with fluorescence correlation spectroscopy, Proc. Natl. Acad. Sci. USA 93:1281. 

External links 

• Single-molecule spectroscopic methods (http://dx.doi.org/10. 1016/j.sbi.2004.09.004) 

• FCS Classroom (http://www.fcsxpert.com/classroom) 

• Stowers Institute FCS Tutorial (http://research.stowers-institute.org/microscopy/external/Technology/FCS/ 
index.htm) 

• Cell Migration Consortium FCS Tutorial (http://www.cellmigration.org/resource/imaging/ 
imaging_approaches_correlation_microscopy.shtml) 

Fluorescence cross-correlation spectroscopy 

Fluorescence cross-correlation spectroscopy (FCCS) was introduced by Eigen and Rigler in 1994 and 
experimentally realized by Schwille in 1997. It extends the fluorescence correlation spectroscopy (FCS) procedure 
by introducing high sensitivity for distinguishing fluorescent particles which have a similar diffusion coefficient. 
FCCS uses two species which are independently labelled with two spectrally separated fluorescent probes. These 
fluorescent probes are excited and detected by two different laser light sources and detectors commonly known as 
green and red respectively. Both laser light beams are focused into the sample and tuned so that they overlap to form 
a superimposed confocal observation volume. 

The normalized cross-correlation function is defined for two fluorescent species Q and ft which are independent 
green, G and red, R channels as follows: 

where differential fluorescent signals SIq at a specific time, f and 5Ir at a delay time, rlater is correlated with 
each other. 

Modeling 

Cross-correlation curves are modeled according to a slightly more complicated mathematical function than applied 
in FCS. First of all, the effective superimposed observation volume in which the G and R channels form a single 
observation volume, V e ff eg m me solution: 

V eff , RG = ^ 2 «, G + < R )« G + <J 1/2 /2 3/2 

2 2 

where uj G and uj x R are radial parameters and ^ z ,Gand ^,i?are the axial parameters for the G and R 

channels respectively. 

The diffusion time, t~d,GR for a doubly (G and R) fluorescent species is therefore described as follows: 



r D,GR 



■ ,2 i , ,2 



8D GR 
where Dgr is the diffusion coefficient of the doubly fluorescent particle. 

The cross-correlation curve generated from diffusing doubly labelled fluorescent particles can be modelled in 
separate channels as follows: 

(< C G > Diff k (r)+ < C GR > Differ)) 



G g {t) = 1 



VeffGR(< C G > + < C GR >) S 



Fluorescence cross-correlation spectroscopy 264 

n( , 1 , (< Cr > Diffk(r)+ < C GR > Differ)) 

v ' VeffM< c R > + < c GR >y 

In the ideal case, the cross-correlation function is proportional to the concentration of the doubly labeled fluorescent 
complex: 

with Diffk(r) 



Contrary to FCS, the intercept of the cross-correlation curve does not yield information about the doubly labelled 
fluorescent particles in solution. 

See also 

• Fluorescence correlation spectroscopy 

• Dynamic light scattering 

• Fluorescence spectroscopy 

• Diffusion coefficient 

External links 

• FCS Classroom [1] 



References 

[1] http://www.fcsxpert.com/classroom 

Circular dichroism 



First pioneered by Jean-Baptiste Biot, Augustin Fresnel, and Aime Cotton , circular dichroism (CD) refers to the 

differential absorption of left and right circularly polarized light. . This phenomenon is exhibited in the 

absorption bands of optically active chiral molecules. CD spectroscopy has a wide range of applications in many 

mi 
different fields. Most notably, UV CD is used to investigate the secondary structure of proteins . UV/Vis CD is 

used to investigate charge-transfer transitions . Near-infrared CD is used to investigate geometric and electronic 

structure by probing metal d— >d transitions . Vibrational circular dichroism, which uses light from the infrared 

energy region, is used for structural studies of small organic molecules, and most recently proteins and DNA . 

Physical Principles 
Circular polarization of light 

Electromagnetic radiation consists of an electric and magnetic field that oscillate perpendicular to one another and to 

ro] 

the propagating direction . While linearly polarized light occurs when the electric field vector oscillates only in 
one plane and changes in magnitude, circularly polarized light occurs when the electric field vector rotates about its 
propagation direction and retains constant magnitude. Hence, it forms a helix in space while propagating. For left 
circularly polarized light (LCP) with propagation towards the observer, the electric vector rotates counterclockwise 
. For right circularly polarized light (RCP), the electric vector rotates clockwise. 



Circular dichroism 



265 



Linearly polarized 



Circularly polari; 




Interaction of circularly polarized light with matter 

When circularly polarized light passes through an absorbing optically active medium, the speeds between right and 

left polarizations differ (c * c ) as well as their wavelength (X * X ) and the extent to which they are absorbed 

rioi 
(e *e ). Circular dichroism is the difference Ae = e - e . The electric field of a light beam causes a linear 

L R L R 

displacement of charge when interacting with a molecule (electric dipole), whereas the magnetic field of it causes a 
circulation of charge (magnetic dipole). These two motions combined cause an excitation of an electron in a helical 
motion, which includes translation and rotation and their associated operators. The experimentally determined 
relationship between the rotational strength (R) of a sample and the Ae is given by 

3hcl0 3 ln(10) f Ae 



R 



exp 



-dv 



32n 3 N A J v 
The rotational strength has also been determined theoretically, 

We see from these two equations that in order to have non-zero /\£ , the electric and magnetic dipole moment 
operators ( M/ i a- i ) an( ^ M( d' I 1^ must transform as the same irreducible representation. C^and D n 
are the only point groups where this can occur, making only chiral molecules CD active. 

Simply put, since circularly polarized light itself is "chiral", it interacts differently with chiral molecules. That is, the 
two types of circularly polarized light are absorbed to different extents. In a CD experiment, equal amounts of left 
and right circularly polarized light of a selected wavelength are alternately radiated into a (chiral) sample. One of the 
two polarizations is absorbed more than the other one, and this wavelength-dependent difference of absorption is 
measured, yielding the CD spectrum of the sample. Due to the interaction with the molecule, the electric field vector 
of the light traces out an elliptical path after passing through the sample. 

Delta absorbance 

By definition, 

AA = A L - A R 

where AA (Delta Absorbance) is the difference between absorbance of left circularly polarized (LCP) and right 
circularly polarized (RCP) light (this is what is usually measured). AA is a function of wavelength, so for a 
measurement to be meaningful the wavelength at which it was performed must be known. 



Circular dichroism 



266 



Molar circular dichroism 

It can also be expressed, by applying Beer's law, as: 

A A = (e L - e R )Cl 



where 



8 and e are the molar extinction coefficients for LCP and RCP light, 
C is the molar concentration 
I is the path length in centimeters (cm). 
Then 

Ae = e L - e R 
is the molar circular dichroism. This intrinsic property is what is usually meant by the circular dichroism of the 
substance. Since /\^ is a function of wavelength, a molar circular dichroism value ( /\^ ) must specify the 
wavelength at which it is valid. 

Extrinsic effects on circular dichroism 

In many practical applications of circular dichroism (CD), as discussed below, the measured CD is not simply an 
intrinsic property of the molecule, but rather depends on the molecular conformation. In such a case the CD may also 
be a function of temperature, concentration, and the chemical environment, including solvents. In this case the 
reported CD value must also specify these other relevant factors in order to be meaningful. 

Molar ellipticity 

Although AA is usually measured, for historical reasons most measurements are reported in degrees of ellipticity. 
Molar circular dichroism and molar ellipticity, [6], are readily interconverted by the equation: 




Er-E l 



Elliptical polarized light (purple) is composed of 

unequal contributions of right (blue) and left (red) 

circular polarized light. 



[0] = 3298.2 Ae. 
This relationship is derived by defining the ellipticity of the polarization as: 

Er — E L 



tan 9 = 



E R + E L 



where 



Circular dichroism 267 

E and E are the magnitudes of the electric field vectors of the right-circularly and left-circularly polarized 

R h, 

light, respectively. 

When E equals E (when there is no difference in the absorbance of right- and left-circular polarized light), 6 is 0° 
and the light is linearly polarized. When either E or E is equal to zero (when there is complete absorbance of the 

R L 

circular polarized light in one direction), 6 is 45° and the light is circularly polarized. 

Generally, the circular dichroism effect is small, so tan6 is small and can be approximated as 6 in radians. Since the 
intensity or irradiance, I, of light is proportional to the square of the electric-field vector, the ellipticity becomes: 

f/ l/2 _ jl/2, 

9 (radians) = ^j= \zj- 

Then by substituting for I using Beer's law in natural logarithm form: 

I = I e- Ainl ° 
The ellipticity can now be written as: 

/g^lnlO _ e =4^1nl0N e AA i ^_ l 

0(radians) = —7377— _^. — = -TTbTio 7 

(e 2 lnl0 + e a lnl °) e AA ^r + l 

Since A A « 1, this expression can be approximated by expanding the exponentials in a Taylor series to first-order 

and then discarding terms of AA in comparison with unity and converting from radians to degrees: 

lnl0\ /18Cf 



# (degrees) = A A 



4 J \ n 
The linear dependence of solute concentration and pathlength is removed by defining molar ellipticity as, 

L J CI 

Then combining the last two expression with Beer's law, molar ellipticity becomes: 



= mAe (^\m\ = 3298.2 A, 



\ 4 J \ n / 

Mean residue ellipticity 

Methods for estimating secondary structure in polymers, proteins and polypeptides in particular, often require that 
the measured molar ellipticity spectrum be converted to a normalized value, specifically a value independent of the 
polymer length. Mean residue ellipticity is used for this purpose; it is simply the measured molar ellipticity of the 
molecule divided by the number of monomer units (residues) in the molecule. 

Application to biological molecules 

In general, this phenomenon will be exhibited in absorption bands of any optically active molecule. As a 
consequence, circular dichroism is exhibited by biological molecules, because of their dextrorotary and levorotary 
components. Even more important is that a secondary structure will also impart a distinct CD to its respective 
molecules. Therefore, the alpha helix of proteins and the double helix of nucleic acids have CD spectral signatures 
representative of their structures. The capacity of CD to give a representative structural signature makes it a powerful 
tool in modern biochemistry with applications that can be found in virtually every field of study. 

CD is closely related to the optical rotatory dispersion (ORD) technique, and is generally considered to be more 
advanced. CD is measured in or near the absorption bands of the molecule of interest, while ORD can be measured 
far from these bands. CD's advantage is apparent in the data analysis. Structural elements are more clearly 
distinguished since their recorded bands do not overlap extensively at particular wavelengths as they do in ORD. In 
principle these two spectral measurements can be interconverted through an integral transform (Kramers— Kronig 



Circular dichroism 268 

relation), if all the absorptions are included in the measurements. 

The far-UV (ultraviolet) CD spectrum of proteins can reveal important characteristics of their secondary structure. 
CD spectra can be readily used to estimate the fraction of a molecule that is in the alpha-helix conformation, the 
beta-sheet conformation, the beta-turn conformation, or some other (e.g. random coil) conformation. These 

fractional assignments place important constraints on the possible secondary conformations that the protein can be 
in. CD cannot, in general, say where the alpha helices that are detected are located within the molecule or even 
completely predict how many there are. Despite this, CD is a valuable tool, especially for showing changes in 
conformation. It can, for instance, be used to study how the secondary structure of a molecule changes as a function 
of temperature or of the concentration of denaturing agents, e.g. Guanidinium hydrochloride or urea. In this way it 
can reveal important thermodynamic information about the molecule (such as the enthalpy and Gibbs free energy of 
denaturation) that cannot otherwise be easily obtained. Anyone attempting to study a protein will find CD a valuable 
tool for verifying that the protein is in its native conformation before undertaking extensive and/or expensive 
experiments with it. Also, there are a number of other uses for CD spectroscopy in protein chemistry not related to 
alpha-helix fraction estimation. 

The near-UV CD spectrum (>250 nm) of proteins provides information on the tertiary structure. The signals obtained 
in the 250-300 nm region are due to the absorption, dipole orientation and the nature of the surrounding environment 
of the phenylalanine, tyrosine, cysteine (or S-S disulfide bridges) and tryptophan amino acids. Unlike in far-UV CD, 
the near-UV CD spectrum cannot be assigned to any particular 3D structure. Rather, near-UV CD spectra provide 
structural information on the nature of the prosthetic groups in proteins, e.g., the heme groups in hemoglobin and 
cytochrome c. 

Visible CD spectroscopy is a very powerful technique to study metal— protein interactions and can resolve individual 
d-d electronic transitions as separate bands. CD spectra in the visible light region are only produced when a metal 
ion is in a chiral environment, thus, free metal ions in solution are not detected. This has the advantage of only 
observing the protein-bound metal, so pH dependence and stoichiometrics are readily obtained. Optical activity in 
transition metal ion complexes have been attributed to configurational, conformational and the vicinal effects. 
Klewpatinond and Viles (2007) have produced a set of empirical rules for predicting the appearance of visible CD 
spectra for Cu and Ni square-planar complexes involving histidine and main-chain coordination. 

CD gives less specific structural information than X-ray crystallography and protein NMR spectroscopy, for 
example, which both give atomic resolution data. However, CD spectroscopy is a quick method that does not require 
large amounts of proteins or extensive data processing. Thus CD can be used to survey a large number of solvent 
conditions, varying temperature, pH, salinity, and the presence of various cofactors. 

CD spectroscopy is usually used to study proteins in solution, and thus it complements methods that study the solid 
state. This is also a limitation, in that many proteins are embedded in membranes in their native state, and solutions 
containing membrane structures are often strongly scattering. CD is sometimes measured in thin films. 

Experimental limitations 

CD has also been studied in carbohydrates, but with limited success due to the experimental difficulties associated 
with measurement of CD spectra in the vacuum ultraviolet (VUV) region of the spectrum (100-200 nm), where the 
corresponding CD bands of unsubstituted carbohydrates lie. Substituted carbohydrates with bands above the VUV 
region have been successfully measured. 

Measurement of CD is also complicated by the fact that typical aqueous buffer systems often absorb in the range 
where structural features exhibit differential absorption of circularly polarized light. Phosphate, sulfate, carbonate, 
and acetate buffers are generally incompatible with CD unless made extremely dilute e.g. in the 10-50 mM range. 
The TRIS buffer system should be completely avoided when performing far-UV CD. Borate and Onium compounds 
are often used to establish the appropriate pH range for CD experiments. Some experimenters have substituted 
fluoride for chloride ion because fluoride absorbs less in the far UV, and some have worked in pure water. Another, 



Circular dichroism 269 

almost universal, technique is to minimize solvent absorption by using shorter path length cells when working in the 
far UV, 0. 1 mm path lengths are not uncommon in this work. 

In addition to measuring in aqueous systems, CD, particularly far-UV CD, can be measured in organic solvents e.g. 
ethanol, methanol, trifluoroethanol (TFE). The latter has the advantage to induce structure formation of proteins, 
inducing beta-sheets in some and alpha helices in others, which they would not show under normal aqueous 
conditions. Most common organic solvents such as acetonitrile, THF, chloroform, dichloromethane are however, 
incompatible with far-UV CD. 

It may be of interest to note that the protein CD spectra used in secondary structure estimation are related to the n to 
jt*orbital absorptions of the amide bonds linking the amino acids. These absorption bands lie partly in the so-called 
vacuum ultraviolet (wavelengths less than about 200 nm). The wavelength region of interest is actually inaccessible 
in air because of the strong absorption of light by oxygen at these wavelengths. In practice these spectra are 
measured not in vacuum but in an oxygen-free instrument (filled with pure nitrogen gas). 

Once oxygen has been eliminated, perhaps the second most important technical factor in working below 200 nm is to 
design the rest of the optical system to have low losses in this region. Critical in this regard is the use of aluminized 
mirrors whose coatings have been optimized for low loss in this region of the spectrum. 

The usual light source in these instruments is a high pressure, short-arc xenon lamp. Ordinary xenon arc lamps are 
unsuitable for use in the low UV. Instead, specially constructed lamps with envelopes made from high-purity 
synthetic fused silica must be used. 

Light from synchrotron sources has a much higher flux at short wavelengths, and has been used to record CD down 
to 160 nm. Recently the CD spectrometer at the electron storage ring facility ISA at the University of Aarhus in 

ri3i 

Denmark was used to record solid state CD spectra down to 120 nm. At the quantum mechanical level, the 
information content of circular dichroism and optical rotation are identical. 

See also 

• Circular polarization in nature 

• Dichroism 

• Linear dichroism 

• Magnetic circular dichroism 

• Optical activity 

• Optical isomerism 

• Optical rotation 

• Optical rotatory dispersion 

• Vibrational circular dichroism 

References 

[1] G. D. Fasman (1996). Plenum Press, p. 3. 

[2] P. Atkins and J. de Paula (2005). Elements of Physical Chemistry, 4th ed.. Oxford University Press. 

[3] E. I. Solomon and A. B. P. Lever (2006). 1. Wiley, p. 78. 

[4] R. W. Woody (1994). K. Nakanishi, N. Berova, R. W. Woody, ed. VCH Publishers, Inc.. p. 473. 

[5] Solomon, Neidig; A. T. Wecksler, G. Schenk, and T. R. Holman (2007). "Kinetic and Spectroscopic Studies of N694C Lipoxygenase: A 

Probe of the Substrate Activation Mechanism of a Nonheme Ferric Enzyme" (http://www.pubmedcentral.nih.gov/articlerender. 

fcgi?tool=pmcentrez&artid=2896304). JACS 129 (24): 7531-7537. doi:10.1021/ja068503d. PMID 17523638. PMC 2896304. 
[6] E. I. Solomon and A. B. P. Lever (2006). 1. Wiley, p. 78. 
[7] K. Nakanishi, N. Berova, R. W. Woody, ed (1994). VCH Publishers, Inc.. 

[8] A. Rodger and B. Norden (1997). Circular Dichroism and Linear Dichroism. Oxford University Press. 
[9] E. I. Solomon and A. B. P. Lever (2006). 1. Wiley, p. 78. 
[10] Woody,R.W.1994 



Circular dichroism 270 

[11] Whitmore L, Wallace BA (2008). "Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference 

databases". Biopolymers 89 (5): 392^00. doi:10.1002/bip.20853. PMID 17896349. 
[12] Greenfield NJ (2006). "Using circular dichroism spectra to estimate protein secondary structure" (http://www.pubmedcentral.nih.gov/ 

articlerender.fcgi?tool=pmcentrez&artid=2728378). Nature protocols 1 (6): 2876-90. doi:10.1038/nprot.2006.202. PMID 17406547. 

PMC 2728378. 
[13] U. Meierhenrich, J.J. Filippi, C. Meinert, J. H. Bredehoft, J. Takahashi, L. Nahon, N. C. Jones, S. V. Hoffmann (2010). "Circular Dichroism 

of Amino Acids in the Vacuum-Ultraviolet Region.". Angewandte Chemie Int. Ed. 49 (42): 7799-7802. doi: 10.1002/anie.201003877. 

Further reading 

1 . Alison Rodger and Bengt Norden, Circular Dichroism and Linear Dichroism (http://books.google.co.uk/ 
books?hl=en&id=THeKGC99hJcC) (1997) Oxford University Press, Oxford, UK. ISBN 019855897X. 

2. Fasman, G.D., Circular Dichroism and the Conformational Analysis of Biomolecules (1996) Plenum Press, New 
York. 

rd 

3. Hecht, E., Optics 3 Edition (1998) Addison Wesley Longman, Massachusetts. 

4. Klewpatinond, M. and Viles, J.H. (2007) Empirical rules for rationalising visible circular dichroism of Cu and 
Ni histidine complexes: Applications to the prion protein. FEBS Letters 581, 1430-1434. 

External links 

• Circular Dichroism explained (http://www.ap-lab.com/circular_dichroism.htm) 

• Circular Dichroism at UMDNJ (http://www2.umdnj.edU/cdrwjweb/index.htm#software) - a good site for 
information on structure estimation software 

• Electromagnetic waves (http://www.enzim.hu/~szia/cddemo/edemol.htm) - Animated electromagnetic 

waves. The Emanim program is a teaching resource for helping students understand the nature of electromagnetic 
waves and their interaction with birefringent and dichroic samples 

• An Introduction to Circular Dichroism Spectroscopy (http://www.photophysics.com/circulardichroism.php) - 
a very good tutorial on circular dichroism spectroscopy 



Vibrational spectroscopy 27 1 



Vibrational spectroscopy 



A molecular vibration occurs when atoms in a molecule are in periodic motion while the molecule as a whole has 
constant translational and rotational motion. The frequency of the periodic motion is known as a vibration frequency. 
The three atomic molecule have three modes of vibration irrespective of whether these are linear or nonlinear. The 
molecules with «(n must be greater than 3) atoms has 3«-6 normal modes of vibration, whereas a linear molecule 
has 3«-5 normal modes of vibration because rotation about its molecular axis is simply a rotation of the reference 
frame and cannot be observed . A diatomic molecule thus has only one normal mode of vibration. The normal 
modes of vibration of polyatomic molecules are independent of each other, each involving simultaneous vibrations 
of different parts of the molecule. 

A molecular vibration is excited when the molecule absorbs a quantum of energy, E, corresponding to the vibration's 
frequency, v, according to the relation E=hv, where h is Planck's constant. A fundamental vibration is excited when 
one such quantum of energy is absorbed by the molecule in its ground state. When two quanta are absorbed the first 
overtone is excited, and so on to higher overtones. 

To a first approximation, the motion in a normal vibration can be described as a kind of simple harmonic motion. In 
this approximation, the vibrational energy is a quadratic function (parabola) with respect to the atomic displacements 
and the first overtone has twice the frequency of the fundamental. In reality, vibrations are anharmonic and the first 
overtone has a frequency that is slightly lower than twice that of the fundamental. Excitation of the higher overtones 
involves progressively less and less additional energy and eventually leads to dissociation of the molecule, as the 
potential energy of the molecule is more like a Morse potential. 

The vibrational states of a molecule can be probed in a variety of ways. The most direct way is through infrared 
spectroscopy, as vibrational transitions typically require an amount of energy that corresponds to the infrared region 
of the spectrum. Raman spectroscopy, which typically uses visible light, can also be used to measure vibration 
frequencies directly. 

Vibrational excitation can occur in conjunction with electronic excitation (vibronic transition), giving vibrational 
fine structure to electronic transitions, particularly with molecules in the gas state. 

Simultaneous excitation of a vibration and rotations gives rise to vibration-rotation spectra. 

Vibrational coordinates 

The coordinate of a normal vibration is a combination of changes in the positions of atoms in the molecule. When 
the vibration is excited the coordinate changes sinusoidally with a frequency v, the frequency of the vibration. 

Internal coordinates 

Internal coordinates are of the following types, illustrated with reference to the planar molecule ethylene, 




Stretching: a change in the length of a bond, such as C-H or C-C 

Bending: a change in the angle between two bonds, such as the HCH angle in a methylene group 



Vibrational spectroscopy 272 

• Rocking: a change in angle between a group of atoms, such as a methylene group and the rest of the molecule. 

• Wagging: a change in angle between the plane of a group of atoms, such as a methylene group and a plane 
through the rest of the molecule, 

• Twisting: a change in the angle between the planes of two groups of atoms, such as a change in the angle between 
the two methylene groups. 

• Out-of-plane: a change in the angle between any one of the C-H bonds and the plane defined by the remaining 
atoms of the ethylene molecule. Another example is in BF when the boron atom moves in and out of the plane of 
the three fluorine atoms. 

In a rocking, wagging or twisting coordinate the bond lengths within the groups involved do not change. The angles 
do. Rocking is distinguished from wagging by the fact that the atoms in the group stay in the same plane. 

In ethene there are 12 internal coordinates: 4 C-H stretching, 1 C-C stretching, 2 H-C-H bending, 2 CH rocking, 2 
CH wagging, 1 twisting. Note that the H-C-C angles cannot be used as internal coordinates as the angles at each 
carbon atom cannot all increase at the same time. 

See infrared spectroscopy for some animated illustrations of internal coordinates. 

Symmetry-adapted coordinates 

Symmetry-adapted coordinates may be created by applying a projection operator to a set of internal coordinates. 
The projection operator is constructed with the aid of the character table of the molecular point group. For example, 
the four(un-normalised) C-H stretching coordinates of the molecule ethene are given by 

Q s4 =vw q 4 

where q - q are the internal coordinates for stretching of each of the four C-H bonds. 
Illustrations of symmetry-adapted coordinates for most small molecules can be found in Nakamoto. 

Normal coordinates 

The normal coordinates, denoted as Q, refer to the positions of atoms away from their equilibrium positions, with 
respect to a normal mode of vibration. Each normal mode is assigned a single normal coordinate, and so the normal 
coordinate refers to the "progress" along that normal mode at any given time. Formally, normal modes are 
determined by solving a secular determinant, and then the normal coordinates (over the normal modes) can be 
expressed as a summation over the cartesian cordinates (over the atom positions). The advantage of working in 
normal modes is that they diagonalize the matrix governing the molecular vibrations, so each normal mode is an 
independent molecular vibration, associated with its own spectrum of quantum mechanical states. If the molecule 
possesses symmetries, it will belong to a point group, and the normal modes will "transform as" an irreducible 
representation under that group. The normal modes can then be qualitatively determined by applying group theory 
and projecting the irreducible representation onto the cartesian coordinates. For example, when this treatment is 
applied to CO , it is found that the C=0 stretches are not independent, but rather there is a 0=C=0 symmetric stretch 
and an 0=C=0 asymmetric stretch. 

• symmetric stretching: the sum of the two C-0 stretching coordinates; the two C-0 bond lengths change by the 
same amount and the carbon atom is stationary. Q = q + q 

• asymmetric stretching: the difference of the two C-0 stretching coordinates; one C-0 bond length increases while 
the other decreases. Q = q - q 

When two or more normal coordinates belong to the same irreducible representation of the molecular point group 
(colloquially, have the same symmetry) there is "mixing" and the coefficients of the combination cannot be 



Vibrational spectroscopy 273 

determined a priori. For example, in the linear molecule hydrogen cyanide, HCN, The two stretching vibrations are 

1. principally C-H stretching with a little C-N stretching; Q = q + a q (a « 1) 

2. principally C-N stretching with a little C-H stretching; Q = b q + q (b « 1) 

The coefficients a and b are found by performing a full normal coordinate analysis by means of the Wilson GF 
method. [4] 

Newtonian mechanics 

Perhaps surprisingly, molecular vibrations can be treated using Newtonian mechanics to calculate the correct 
vibration frequencies. The basic assumption is that each vibration can be treated as though it corresponds to a spring. 
In the harmonic approximation the spring obeys Hooke's law: the force required to extend the spring is proportional 
to the extension. The proportionality constant is known as a force constant, k. The anharmonic oscillator is 
considered elsewhere. 

Force = —kQ 
By Newton's second law of motion this force is also equal to a "mass", m, times acceleration. 

l*orce = m — — 

dt 2 

Since this is one and the same force the ordinary differential equation follows. 
d 2 Q 

The solution to this equation of simple harmonic motion is 

Q(t) = AcoBpirvt); v= — u 

2ir \ m 

A is the maximum amplitude of the vibration coordinate Q. It remains to define the "mass", m. In a homonuclear 
diatomic molecule such as N , it is half the mass of one atom. In a heteronuclear diatomic molecule, AB, it is the 
reduced mass, /,< given by 

111 

[2 VTiA TUb 
The use of the reduced mass ensures that the centre of mass of the molecule is not affected by the vibration. In the 
harmonic approximation the potential energy of the molecule is a quadratic function of the normal coordinate. It 
follows that the force-constant is equal to the second derivative of the potential energy. 

_ d2V 

k ~dQ 2 

When two or more normal vibrations have the same symmetry a full normal coordinate analysis must be performed 

(see GF method). The vibration frequencies, v. are obtained from the eigenvalues,!., of the matrix product GF. G is a 

1 ' T41 

matrix of numbers derived from the masses of the atoms and the geometry of the molecule. F is a matrix derived 

from force-constant values. Details concerning the determination of the eigenvalues can be found in . 



Vibrational spectroscopy 274 

Quantum mechanics 

In the harmonic approximation the potential energy is a quadratic function of the normal coordinates. Solving the 
Schrodinger wave equation, the energy states for each normal coordinate are given by 

E " = { n+l i) h kfk- 

where n is a quantum number that can take values of 0, 1, 2 ... The difference in energy when n changes by 1 are 
therefore equal to the energy derived using classical mechanics. See quantum harmonic oscillator for graphs of the 
first 5 wave functions. Knowing the wave functions, certain selection rules can be formulated. For example, for a 
harmonic oscillator transitions are allowed only when the quantum number n changes by one, 

Ara = ±l 
but this does not apply to an anharmonic oscillator; the observation of overtones is only possible because vibrations 
are anharmonic. Another consequence of anharmonicity is that transitions such as between states n-2 and «=1 have 
slightly less energy than transitions between the ground state and first excited state. Such a transition gives rise to a 
hot band. 

Intensities 

In an infrared spectrum the intensity of an absorption band is proportional to the derivative of the molecular dipole 

T71 
moment with respect to the normal coordinate. The intensity of Raman bands depends on polarizability. 

See also 

Infrared spectroscopy 

Near infrared spectroscopy 

Raman spectroscopy 

Resonance Raman spectroscopy 

Coherent anti-Stokes Raman spectroscopy 

Eckart conditions 

FG method 

Fermi resonance 

Lennard-Jones potential 

Transition dipole moment 

References 

[1] Landau LD and Lifshitz EM (1976) Mechanics, 3rd. ed., Pergamon Press. ISBN 0-08-021022-8 (hardcover) and ISBN 0-08-029141-4 

(softcover) 

[2] F.A. Cotton Chemical applications of group theory, Wiley, 1962, 1971 

[3] K. Nakamoto Infrared and Raman spectra of inorganic and coordination compounds, 5th. edition, Part A, Wiley, 1997 

[4] E.B. Wilson, J.C. Decius and P.C. Cross, Molecular vibrations, McGraw-Hill, 1955. (Reprinted by Dover 1980) 

[5] S. Califano, Vibrational states, Wiley, 1976 

[6] P. Gans, Vibrating molecules, Chapman and Hall, 1971 

[7] D. Steele, Theory of vibrational spectroscopy, W.B. Saunders, 1971 



Vibrational spectroscopy 



275 



Further reading 

• P.M. A. Sherwood, Vibrational spectroscopy of solids, Cambridge University Press, 1972 

External links 

• Free Molecular Vibration code developed by Zs. Szabo and R. Scipioni (http://www.evtsz.bme.hu/web/staff/ 
szabo/web_molecular_vibration/molec_vib_code.html) 

• Molecular vibration and absorption (http://www.lsbu.ac.uk/water/vibrat.html) 

• small explanation of vibrational spectra and a table including force constants (http://hyperphysics.phy-astr.gsu. 
edu/Hbase/molecule/ vibspe.html). 

• Character tables for chemically important point groups (http://symmetry.jacobs-university.de/) 

Vibrational circular dichroism 



Vibrational circular dichroism (VCD) is a spectroscopic technique which detects differences in attenuation of left 
and right circularly polarized light passing through a sample. It is basically circular dichroism spectroscopy in the 
infrared and near infrared ranges . 

Because VCD is sensitive to the mutual orientation of distinct groups in a molecule, it provides three-dimensional 
structural information. Thus, it is a powerful technique as VCD spectra of enantiomers can be simulated using ab 
initio calculations, thereby allowing the identification of absolute configurations of small molecules in solution from 
VCD spectra. Among such quantum computations of VCD spectra resulting from the chiral properties of small 
organic molecules are those based on density functional theory (DFT) and gauge-invariant atomic orbitals (GIAO). 
As a simple example of the experimental results that were obtained by VCD are the spectral data obtained within the 
carbon-hydrogen (C-H) stretching region of 21 amino acids in heavy water solutions. Measurements of vibrational 
optical activity (VOA) have thus numerous applications, not only for small molecules, but also for large and 
complex biopolymers such as muscle proteins (myosin, for example) and DNA. 



Vibrational modes 



\f 



*, 



** 



Kfi 



\f 



V 




Vibrational circular dichroism 



276 



Theory of VCD 

While the fundamental quantity associated with the infrared absorption is the dipole strength, the differential 
absorption is proportional also to the rotational strength, a quantity which depends on both the electric and magnetic 
dipole transition moments. Sensitivity of the handedness of a molecule toward circularly polarized light results from 
the form of the rotational strength. 



VCD of peptides and proteins 

Extensive VCD studies have been reported for both polypeptides and several proteins in solution ; several 

recent reviews were also compiled . An extensive but not comprehensive VCD publications list is also 

provided in the "References" section. The published reports over the last 22 years have established VCD as a 
powerful technique with improved results over those previously obtained by visible/UV circular dichroism (CD) or 
optical rotatory dispersion (ORD) for proteins and nucleic acids. 



Amino acid and polypeptide structures 






+ H 3 N X 








A" 


-CH, 

V 


.0 




- 


Kf- 





Na + 














■•'■'! 










Vibrational circular dichroism 



277 



Bcl-2 Family 







^^!3| 


mm 1 1 ms-\* ^*ttifm 


wvm&rtik$r. *Zm 


Mz'jft^r""' '<9 




MjS*.£lm 


Ml*''"'3 



VCD of nucleic acids 

VCD spectra of nucleotides, synthetic polynucleotides and several nucleic acids, including DNA, have been reported 
and assigned in terms of the type and number of helices present in A- , B-, and Z- DNA. 



VCD Instrumentation 

For biopolymers such as proteins and nucleic acids, the difference in absorbance between the levo- and dextro- 
configurations is five orders of magnitude smaller than the corresonding (unpolarized) absorbance. Therefore, VCD 
of biopolymers requires the use of very sensitive, specially built instrumentation as well as time-averaging over 
relatively long intervals of time even with such sensitive VCD spectrometers. Most CD instruments produce left- and 
right- circularly polarized light which is then either sine-wave or square-wave modulated, with subsequent 
phase-sensitive detection and lock-in amplification of the detected signal. In the case of FT- VCD, a photo-elastic 
modulator (PEM) is employed in conjunction with an FT-IR interferometer set-up. An example is that of a Bomem 
model MB -100 FT-IR interferometer equipped with additional polarizing optics/ accessories needed for recording 
VCD spectra. A parallel beam emerges through a side port of the interferometer which passes first through a wire 
grid linear polarizer and then through an octagonal-shaped ZnSe crystal PEM which modulates the polarized beam at 
a fixed, lower frequency such as 37.5 kHz. A mechanically stressed crystal such as ZnSe exhibits birefringence when 
stressed by an adjacent piezoelectric transducer. The linear polarizer is positioned close to, and at 45 degrees, with 
respect to the ZnSe crystal axis. The polarized radiation focused onto the detector is doubly modulated, both by the 
PEM and by the interferometer setup. A very low noise detector, such as MCT (HgCdTe), is also selected for the 
VCD signal phase-sensitive detection. The first dedicated VCD spectrometer brought to market was the ChirallR 
from Bomem/BioTools, Inc. in 1997. Today, Thermo-Electron, Bruker, Jasco and BioTools offer either VCD 
accessories or stand-alone instrumentation. To prevent detector saturation an appropriate, long wave pass filter is 
placed before the very low noise MCT detector, which allows only radiation below 1750 cm - to reach the MCT 
detector; the latter however measures radiation only down to 750 cm - . FT- VCD spectra accumulation of the 
selected sample solution is then carried out, digitized and stored by an in-line computer. Published reviews that 
compare various VCD methods are also available. 



Vibrational circular dichroism 



278 







run 








^fip^ 



Jrc ^ |ii .J^i 1^1 




BjNj 7759 A 



H 



90.48° 




Magnetic VCD 

VCD spectra have also been reported in the presence of an applied external magnetic field 1 
enhance the VCD spectral resolution for small molecules 



[12] 



This method can 



Raman optical activity (ROA) 



-i 



ROA is a technique complementary to VCD especially useful in the 50 — 1600 cm spectral region; it is considered 
as the technique of choice for determining optical activity for photon energies less than 600 cm - . 



Notes 

[I] http://planetphysics.org/?op=getobj;from=objects;id=410 Principles of IR and NIR Spectroscopy 

[2] *"Vibrational Circular Dichroism of Polypeptides XII. Re-evaluation of the Fourier Transform Vibrational Circular Dichroism of 

Poly-gamma-Benzyl-L-Glutamate," P. Malon, R. Kobrinskaya, T. A. Keiderling, Biopolymers 27, 733-746 (1988). 
[3] ""'Vibrational Circular Dichroism of Biopolymers," T. A. Keiderling, S. C. Yasui, U. Narayanan, A. Annamalai, P. Malon, R. Kobrinskaya, 

L. Yang, in Spectroscopy of Biological Molecules New Advances ed. E. D. Schmid, F. W. Schneider, F. Siebert, p. 73-76 (1988). 
[4] *"Vibrational Circular Dichroism of Polypeptides and Proteins," S. C. Yasui, T. A. Keiderling, Mikrochimica Acta, II, 325-327, (1988). 
[5] """Vibrational Circular Dichroism of Proteins Polysaccharides and Nucleic Acids" T. A. Keiderling, Chapter 8 in Physical Chemistry of Food 

Processes, Vol. 2 Advanced Techniques, Structures and Applications., eds. I.C. Baianu, H. Pessen, T. Kumosinski, Van Norstrand— Reinhold, 

New York (1993), pp 307-337. 
[6] "Spectroscopic characterization of Unfolded peptides and proteins studied with infrared absorption and vibrational circular dichroism spectra" 

T. A. Keiderling and Qi Xu, Advances in Protein Chemistry Volume 62, [Unfolded Proteins, Dedicated to John Edsall, Ed.: George Rose, 

Academic Press:New York] (2002), pp. 111-161. 
[7] *"Protein and Peptide Secondary Structure and Conformational Determination with Vibrational Circular Dichroism " Timothy A. Keiderling, 

Current Opinions in Chemical Biology (Ed. Julie Leary and Mark Arnold) 6, 682-688 (2002). 
[8] ""Review: Conformational Studies of Peptides with Infrared Techniques. Timothy A. Keiderling and R. A. G. D. Silva, in Synthesis of 

Peptides and Peptidomimetics, Ed. M. Goodman and G. Herrman, Houben-Weyl, Vol 22Eb, Georg Thiem Verlag, New York (2002) pp. 

715-738, (written and accepted in 2000). 
[9] "Vibrational Circular Dichroism: A New Tool for the Solution-State Determination of the Structure and Absolute Configuration of Chiral 

Natural Product Molecules" Laurence A. Nafie, Natural Product Communications 3(3), 451-466 (2008) 
[10] "Polarization Modulation Fourier Transform Infrared Spectroscopy with Digital SignalProcessing: Comparison of Vibrational Circular 

Dichroism Methods." Jovencio Hilario, DavidDrapcho, Raul Curbelo, Timothy A. Keiderling, Applied Spectroscopy 55, 1435-1447(2001)— 

[II] "Vibrational circular dichroism of biopolymers. Summary of methods and applications.", Timothy A. Keiderling, Jan Kubelka, Jovencio 
Hilario, in Vibrational spectroscopy of polymers and biological systems, Ed. Mark Braiman, Vasilis Gregoriou, Taylor&Francis, Atlanta 
(CRC Press, Boca Raton, FL) (2006) pp. 253-324 (originally written in 2000, updated in 2003) 

[12] "Observation of Magnetic Vibrational Circular Dichroism," T. A. Keiderling, Journal of Chemical Physics, 75, 3639-41 (1981). 



Vibrational circular dichroism 279 

[13] "Vibrational Spectral Assignment and Enhanced Resolution Using Magnetic Vibrational Circular Dichroism," T. R. Devine and T. A. 

Keiderling, Spectrochimica Acta, 43 A, 627-629 (1987). 
[14] "Magnetic Vibrational Circular Dichroism with an FTIR" P. V. Croatto, R. K. Yoo, T. A. Keiderling, SPIE Proceedings 1 145 (7th 

International Conference on FTS, ed. D. G. Cameron) 152-153 (1989). 
[15] "Direct Measurement of the Rotational g- Value in the Ground State of Acetylene by Magnetic Vibrational Circular Dichroism." C. N. Tarn 

and T. A. Keiderling, Chemical Physics Letters, 243, 55-58 (1995). 
[16] . "Ab initio calculation of the vibrational magnetic dipole moment" P. Bour, C. N. Tam, T. A. Keiderling, Journal of Physical Chemistry 99, 

17810-17813 (1995) 
[17] "Rotationally Resolved Magnetic Vibrational Circular Dichroism. Experimental Spectra and Theoretical Simulation for Diamagnetic 

Molecules." P. Bour, C. N. Tam, B. Wang, T. A. Keiderling, Molecular Physics 87, 299-318, (1996). 

References 
Peptides and proteins 

• Huang R, Wu L, McElheny D, Bour P, Roy A, Keiderling TA. Cross-Strand Coupling and Site-Specific 
Unfolding Thermodynamics of a Trpzip beta-Hairpin Peptide Using (13)C Isotopic Labeling and IR 
Spectroscopy. The journal of physical chemistry. B. 2009 Apr;113(16):5661-74. 

• "Vibrational Circular Dichroism of Poly alpha-Benzyl-L-Glutamate," R. D. Singh, and T. A. Keiderling, 
Biopolymers, 20, 237-40 (1981). 

• "Vibrational Circular Dichroism of Polypeptides II. Solution Amide II and Deuteration Results," A. C. Sen and T. 
A. Keiderling, Biopolymers, 23, 1519-32 (1984). 

• "Vibrational Circular Dichroism of Polypeptides III. Film Studies of Several alpha-Helical and 6-Sheet 
Polypeptides," A. C. Sen and T. A. Keiderling, Biopolymers, 23, 1533-46 (1984). 

• "Vibrational Circular Dichroism of Polypeptides IV. Film Studies of L-Alanine Homo Oligopeptides," U. 
Narayanan, T. A. Keiderling, G. M. Bonora, and C. Toniolo, Biopolymers 24, 1257-63 (1985). 

• "Vibrational Circular Dichroism of Polypeptides, T. A. Keiderling, S. C. Yasui, A. C. Sen, C. Toniolo, G. M. 
Bonora, in Peptides Structure and Function, Proceedings of the 9th American Peptide Symposium," ed. C. M. 
Deber, K. Kopple, V. Hruby; Pie rce Chemical: Rockford, IL; 167-172 (1985). 

• "Vibrational Circular Dichroism of Polypeptides V. A Study of 310 Helical -Octapeptides" S. C. Yasui, T. A. 
Keiderling, G M. Bonora, C. Toniolo, Biopolymers 25, 79-89 (1986). 

• "Vibrational Circular Dichroism of Polypeptides VI. Polytyrosine alpha-helical and Random Coil Results," S. C. 
Yasui and T. A. Keiderling, Biopolymers 25, 5-15 (1986). 

• "Vibrational Circular Dichroism of Polypeptides VII. Film and Solution Studies of alpha-forming 
Homo-Oligopeptides," U. Narayanan, T. A. Keiderling, G M. Bonora, C. Toniolo, Journal of the American 
Chemical Society, 108, 2431-2437 (1986). 

• "Vibrational Circular Dichroism of Polypeptides VIII. Poly Lysine Conformations as a Function of pH in 
Aqueous Solution," S. C. Yasui, T. A. Keiderling, Journal of the American Chemical Society, 108, 5576-5581 
(1986). 

• "Vibrational Circular Dichroism of Polypeptides IX. A Study of Chain Length Dependence for 3 10-Helix 
Formation in Solution." S. C. Yasui, T. A. Keiderling, F. Formaggio, G M. Bonora, C. Toniolo, Journal of the 
American Chemical Society 108, 4988-499 3 (1986). 

• "Vibrational Circular Dichroism of Biopolymers." T. A. Keiderling, Nature, 322, 851-852 (1986). 

• "Vibrational Circular Dichroism of Polypeptides X. A Study of alpha-Helical Oligopeptides in Solution." S. C. 
Yasui, T. A. Keiderling, R. Katachai, Biopolymers 26, 1407-1412 (1987). 

• "Vibrational Circular Dichroism of Polypeptides XL Conformation of 

Poly (L-Ly sine(Z)-L-Lysine(Z)-L- 1 -Pyrenylalanine) and Poly (L-Lysine(Z)-L-Ly sine(Z)-L- 1 -Napthylala-nine) in 
Solution" S. C. Yasui, T. A. Keiderling, and M. Sisido, Macromolecules 20, 2 403-2406 (1987). 

• "Vibrational Circular Dichroism of Biopolymers" T. A. Keiderling, S. C. Yasui, A. C. Sen, U. Narayanan, A. 
Annamalai, P. Malon, R. Kobrinskaya, L. Yang, in "F.E.C.S. Second International Conference on Circular 



Vibrational circular dichroism 280 

Dichroism, Conference Proceedings," ed. M. Kajtar, L. Eotvos Univ., Budapest, 1987, p. 155-161. 

• "Vibrational Circular Dichroism of Poly-L-Proline and Other Helical Poly-peptides," R. Kobrinskaya, S. C. 
Yasui, T. A. Keiderling, in "Peptides: Chemistry and Biology, Proceedings of the 10th American Peptide 
Symposium," ed. G. R. Marshall, ESCOM, L eiden, 1988, p. 65-67. 

• "Vibrational Circular Dichroism of Polypeptides with Aromatic Side Chains," S. C. Yasui, T. A. Keiderling, in 
"Peptides: Chemistry and Biology, Proceedings of the 10th American Peptide Symposium," ed. G. R. Marshall, 
ESCOM, Leiden, 1988, p. 90-92. 

• "Vibrational Circular Dichroism of Polypeptides XII. Re-evaluation of the Fourier Transform Vibrational Circular 
Dichroism of Poly-gamma-Benzyl-L-Glutamate," P. Malon, R. Kobrinskaya, T. A. Keiderling, Biopolymers 27, 
733-746 (1988). 

• "Vibrational Circular Dichroism of Biopolymers," T. A. Keiderling, S. C. Yasui, U. Narayanan, A. Annamalai, P. 
Malon, R. Kobrinskaya, L. Yang, in Spectroscopy of Biological Molecules New Advances ed. E. D. Schmid, F. W. 
Schneider, F. Siebert, p. 73-76 (1988). 

• "Vibrational Circular Dichroism of Polypeptides and Proteins," S. C. Yasui, T. A. Keiderling, Mikrochimica Acta, 
II, 325-327, (1988). 

• "(lR,7R)-7-Methyl-6,9, -Diazatricyclo[6, 3,0,01, 6]Tridecanne-5,10-Dione, A Tricyclic Spirodilactam Containing 
Non-planar Amide Groups: Synthesis, NMR, Crystal Structure, Absolute Configuration, Electronic and 
Vibrational Circular Dichroism" P. Malon, C . L. Barness, M. Budesinsky, R. K. Dukor, D. van der Helm, T. A. 
Keiderling, Z. Koblicova, F. Pavlikova, M. Tichy, K. Blaha, Collections of Czechoslovak Chemical 
Communications 53, 2447-2472 (1988). 

• "Vibrational Circular Dichroism of Poly Glutamic Acid" R. K. Dukor, T. A. Keiderling, in Peptides 1988 (ed. G 
Jung, E. Bayer) Walter de Gruyter, Berlin (1989) pp 519-521. 

• "Biopolymer Conformational Studies with Vibrational Circular Dichroism" T. A. Keiderling, S. C. Yasui, P. 
Pancoska, R. K. Dukor, L. Yang, SPIE Proceeding 1057, ("Biomolecular Spectroscopy," ed. H. H. Mantsch, R. R. 
Birge) 7-14 (1989). 

• "Vibrational Circular Dichroism. Comparison of Techniques and Practical Considerations" T. A. Keiderling, in 
"Practical Fourier Transform Infrared Spectroscopy. Industrial and Laboratory Chemical Analysis," ed. J. R. 
Ferraro, K. Krishnan (Academic Press, San Diego, 1990) p. 203-284. 

• "Vibrational Circular Dichroism Study of Unblocked Proline Oligomers," R. K. Dukor, T. A. Keiderling, V. Gut, 
International Journal of Peptide and Protein Research, 38, 198-203 (1991). 

• "Reassessment of the Random Coil Conformation. Vibrational CD Study of Proline Oligopeptides and Related 
Polypeptides" R. K. Dukor and T. A. Keiderling, Biopolymers 31 1747-1761 (1991). 

• "Vibrational CD of the Amide II band in Some Model Polypeptides and Proteins" V. P. Gupta, T. A. Keiderling, 
Biopolymers 32 239-248 (1992). 

• "Vibrational Circular Dichroism of Proteins Polysaccharides and Nucleic Acids" T. A. Keiderling, Chapter 8 in 
Physical Chemistry of Food Processes, Vol. 2 Advanced Techniques, Structures and Applications., eds. I.C. 
Baianu, H. Pessen, T. Kumosinski, Van Norstrand — Reinhold, New York (1993), pp 307—337. 

• "Structural Studies of Biological Macromolecules using Vibrational Circular Dichroism" T. A. Keiderling, P. 
Pancoska, Chapter 6 in Advances in Spectroscopy Vol. 21, Biomolecular Spectroscopy Part B eds. R. E. Hester, 
R. J. H. Clarke, John W iley Chichester (1993) pp 267-315. 

• "Ab Initio Simulations of the Vibrational Circular Dichroism of Coupled Peptides" P. Bour and T. A. Keiderling, 
Journal of the American Chemical Society 115 9602-9607 (1993). 

• "Ab initio Simulations of Coupled Peptide Vibrational Circular Dichroism" P. Bour, T. A. Keiderling in "Fifth 
International Conference on The Spectroscopy of Biological Molecules" Th. Theophanides, J. Anastassopoulou, 
N. Fotopoulos (Eds), Kluwen Aca demic Publ., Dortrecht, 1993, p. 29-30. 

• "Vibrational Circular Dichroism Spectroscopy of Peptides and Proteins" T. A. Keiderling, in "Circular Dichroism 
Interpretations and Applications," K. Nakanishi, N. Berova, R. Woody, Eds., VCH Publishers, New York, (1994) 



Vibrational circular dichroism 28 1 

pp 497-521. 

• "Conformational Study of Sequential Lys-Leu Based Polymers and Oligomers using Vibrational and Electronic 
Circular Dichroism Spectra" V. Baumruk, D. Huo, R. K. Dukor, T. A. Keiderling, D. LeLeivre and A. Brack 
Biopolymers 34, 1115-1121 (1994). 

• "Vibrational Optical Activity of Oligopeptides" T. B. Freedman, L. A. Nafie, T. A. Keiderling Biopolymers 
(Peptide Science) 37 (ed. C. Toniolo) 265-279 (1995). 

• "Characterization of 6-bend ribbon spiral forming peptides using electronic and vibrational circular dichroism" G. 
Yoder, T. A. Keiderling, F. Formaggio, M. Crisma, C. Toniolo Biopolymers 35, 103-111 (1995). 

• "Vibrational Circular Dichroism as a Tool for Determination of Peptide Secondary Structure" P. Bour, T. A. 
Keiderling, P. Malon, in "Peptides 1994 (Proceedings of the 23rd European Peptide Symposium, 1994," (H.L.S. 
Maia, ed.), Escom, Le iden 1995, p. 517-518. 

• "Helical Screw Sense of homo-oligopeptides of C-alpha-methylated alpha-amino acids as Determined with 
Vibrational Circular Dichroism." G. Yoder, T. A. Keiderling, M. Crisma, F. Formaggio, C. Toniolo, J. Kamphuis, 
Tetrahedron Asymmetry 6, 687 -690 (1995). 

• "Conformational Study of Linear Alternating and Mixed D- and L-Proline Oligomers Using Electronic and 
Vibrational CD and Fourier Transform IR." W. M&#228stle, R. K. Dukor, G Yoder, T. A. Keiderling 
Biopolymers 36, 623-631 (1995). 

• Review: "Vibrational Circular Dichroism Applications to Conformational Analysis of Biomolecules" T. A. 
Keiderling in Circular Dichroism and the Conformational Analysis of Biomolecules ed. G D. Fasman, Plenum, 
New York (1996) p. 555-585. 

• "Mutarotation studies of Poly L-Proline using FT-IR, Electronic and Vibrational Circular Dichroism" R. K. 
Dukor, T. A. Keiderling, Biospectroscopy 2, 83-100 (1996). 

• "Vibrational Circular Dichroism Applications in Proteins and Peptides" T. A. Keiderling, Proceedings of the 
NATO ASI in Biomolecular Structure and Dynamics, Loutrakii Greece, May 1996, Ed. G Vergoten (delayed 
second volume to 1998). 

• "Transfer of Molecular Property Tensors in Cartesian Coordinates: A new algorithm for simulation of vibrational 
spectra" Petr Bour, Jana Sopkova, Lucie Bednarova, Petr Malon, T. A. Keiderling, Journal of Computational 
Chemistry 18, 6 46-659 (1997). 

• "Vibrational Circular Dichroism Characterization of Alanine-Rich Peptides." Gorm Yoder and Timothy A. 
Keiderling, "Spectroscopy of Biological Molecules: Modern Trends," Ed. P. Carmona, R. Navarro, A. Hernanz, 
Kluwer Acad. Pub., Netherlands (1997) p p. 27-28. 

• "Ionic strength effect on the thermal unfolding of alpha-spectrin peptides." D. Lusitani, N. Menhart, T.A. 
Keiderling and L. W. M. Fung. Biochemistry 37(1998)16546-16554. 

• "In search of the earliest events of hCGb folding: structural studies of the 60-87 peptide fragment" S. Sherman, L. 
Kirnarsky, O. Prakash, H. M. Rogers, R.A.G.D. Silva, T.A. Keiderling, D. Smith, A.M. Hanly, F. Perini, and 
R.W. Ruddon, American Pep tide Symposium Proceedings, 1997. 

• "Cold Denaturation Studies of (LKELPKEL)n Peptide Using Vibrational Circular Dichroism and FT-IR". R. A. 
G D. Silva, Vladimir Baumruk, Petr Pancoska, T. A. Keiderling, Eric Lacassie, and Yves Trudelle, American 
Peptide Symposium Proceedings, 1997. 

• "Simulations of oligopeptide vibrational CD. Effects of isotopic labeling." Petr Bour, Jan Kubelka,T. A. 
Keiderling Biopolymers 53, 380-395 (2000). 

• "Site specific conformational determination in thermal unfolding studies of helical peptides using vibrational 
circular dichroism with isotopic substitution" R. A. G D. Silva, Jan Kubelka, Petr Bour, Sean M. Decatur, 
Timothy A. Keiderling, Proceedings of the National Academy of Sciences (PNAS:USA) 97, 8318-8323 (2000). 

• "Folding studies on the human chorionic gonadotropin b -subunit using optical spectroscopy of peptide 
fragments" R. A. G D. Silva, S. A. Sherman, F. Perini, E. Bedows, T. A. Keiderling, Journal of the American 
Chemical Society, 122, 8623-8630 (2000). 



Vibrational circular dichroism 282 

• "Peptide and Protein Conformational Studies with Vibrational Circular Dichroism and Related Spectroscopies", 
Timothy A. Keiderling, (Revised and Expanded Chapter) In Circular Dichroism: Principles and Applications, 2nd 
Edition. (Eds. K. Nakanishi, N. Berova and R. A. Woody, John Wiley & Sons, New York (2000) p. 621-666. 

• "Conformation studies with Optical Spectroscopy of peptides taken from hairpin sequences in the Human 
Chorionic Gonadotropin " R. A. G. D. Silva, S. A. Sherman, E. Bedows, T. A. Keiderling, Peptides for the New 
Millenium [sic?], Proceedings of the 16th American Peptide Symposium, (June, 1999 Minneapolis, MN) Ed.G 
B. Fields, J. P. Tarn, G Barany, Kluwer Acad. Pub., Dordrecht,(2000) p. 325-326. 

• "Analysis of Local Conformation within Helical Peptides via Isotope-Edited Vibrational Spectroscopy." S. M. 
Decatur, T. A. Keiderling, R. A. G D. Silva, and P. Bour, Peptides for the New Millenium [sic?], Proceedings of 
the 16th American Peptide Symposium, (June, 1999 Minneapolis, MN) Ed. Ed.G. B. Fields, J. P. Tam, G Barany, 
Kluwer Acad. Pub., Dordrecht, (2000) p. 414-416. 

• "The anomalous infrared amide I intensity distribution in C-13 isotopically labeled peptide beta-sheets comes 
from extended, multiple stranded structures. An Ab Initio study." Jan Kubelka and T. A. Keiderling , Journal of 
the American Chemical Society. 123, 6142-6150 (2001). 

• "Vibrational Circular Dichroism of Peptides and Proteins: Survey of Techniques, Qualitative and Quantitative 
Analyses, and Applications" Timothy A. Keiderling, Chapter in Infrared and Raman Spectroscopy of Biological 
Materials, Ed. Bing Yan and H.-U. Gremlich, Marcel Dekker, New York (2001) p. 55-100. 

• "Chirality in peptide vibrations. Ab initio computational studies of length, solvation, hydrogen bond, dipole 
coupling and isotope effects on vibrational CD. " Jan Kubelka, Petr Bour, R. A. Gangani D. Silva, Sean M. 
Decatur, Timothy A. Keiderling, ACS Symposium Series 810, ["Chirality: Physical Chemistry," (Ed. Janice 
Hicks) American Chemical Society, Washington, DC] (2002), pp. 50—64. 

• "Spectroscopic Characterization of Selected b-Sheet Hairpin Models", J. Hilario, J. Kubelka, F. A. Syud, S. H. 
Gellman, and T. A. Keiderling. Biopolymers (Biospectroscopy) 67: 233-236 (2002) 

• " Discrimination between peptide 3 - and alpha-helices. Theoretical analysis of the impact of alpha-methyl 
substitution on experimental spectra " Jan Kubelka, R. A. Gangani D. Silva, and T. A. Keiderling, Journal of the 
American Chemical Society, 124, 5325-5332 (2002). 

• "Ab Initio Quantum Mechanical Models of Peptide Helices and their Vibrational Spectra" Petr Bour, Jan Kubelka 
and T. A. Keiderling, Biopolymers 65, 45-59 (2002). 

• "Discriminating 3 - from alpha-helices. Vibrational and electronic CD and IR Absorption study of related 
Aib-containing oligopeptides" R. A. Gangani D. Silva, Sritana Yasui, Jan Kubelka, Fernando Formaggio, Marco 
Crisma, Claudio Toniolo, and Timothy A. Keiderling, Biopolymers 65, 229-243 (2002). 

• "Spectroscopic characterization of Unfolded peptides and proteins studied with infrared absorption and 
vibrational circular dichroism spectra" T. A. Keiderling and Qi Xu, Advances in Protein Chemistry Volume 62, 
[Unfolded Proteins, Dedicated to John Edsall, Ed.: George Rose, Academic Press:New York] (2002), 

pp. 111-161. 

• "Protein and Peptide Secondary Structure and Conformational Determination with Vibrational Circular Dichroism 
" Timothy A. Keiderling, Current Opinions in Chemical Biology (Ed. Julie Leary and Mark Arnold) 6, 682-688 
(2002). 

• Review: Conformational Studies of Peptides with Infrared Techniques. Timothy A. Keiderling and R. A. G D. 
Silva, in Synthesis of Peptides and Pep tidomime tics, Ed. M. Goodman and G Herrman, Houben-Weyl, Vol 22Eb, 
Georg Thiem Verlag, New York (2002) pp. 715—738, (written and accepted in 2000). 

• "Spectroscopic Studies of Structural Changes in Two beta-Sheet Forming Peptides Show an Ensemble of 
Structures That Unfold Non-Cooperatively" Serguei V. Kuznetsov, Jovencio Hilario, T. A. Keiderling, Anjum 
Ansari, Biochemistry, 42 :4321-4332, (2003). 

• "Optical spectroscopic investigations of model beta-sheet hairpins in aqueous solution" Jovencio Hilario, Jan 
Kubelka, T. A. Keiderling, Journal of the American Chemical Society 125, 1562-151 A (2003). 



Vibrational circular dichroism 283 

• "Synthesis and conformational study of homopeptides based on (S)-Bin, a C2-symmetric binapthyl-derived 
Caa-disubstituted glycine with only axial chirality" J. -P. Mazaleyrat, K. Wright, A. Gaucher, M. Wakselman, S. 
Oancea, F. Formaggio, C. Toniolo, V. Setnicka, J. Kapitan, T. A. Keiderling, Tetrahedron Asymmetry, 14, 
1879-1893 (2003). 

• "Empirical modeling of the peptide amide I band IR intensity in water solution," Petr Bour, Timothy A. 
Keiderling, Journal of Chemical Physics, 119, 11253-11262 (2003) 

• "The Nature of Vibrational Coupling in Helical Peptides: An Isotope Labeling Study" by R. Huang, J. Kubelka, 
W. Barber- Armstrong, R. A. G. D Silva, S. M. Decatur, and T. A. Keiderling, Journal of the American Chemical 
Society, 126, 2346-2354 (2004). 

• "The Complete Chirospectroscopic Signature of the Peptide 3 Helix in Aqueous Solution" Claudio Toniolo, 
Fernando Formaggio, Sabrina Tognon, Quirinus B. Broxterman, Bernard Kaptein, Rong Huang, Vladimir 
Setnicka, Timothy A. Keiderling, Iain H. McColl, Lutz Hecht, Laurence D. Barron, Biopolymers 75, 32-45 
(2004). 

• "Induced axial chirality in the biphenyl core for the Ca-tetrasubstituted a-amino acid residue Bip and subsequent 
propagation of chirality in (Bip)n/Val oligopeptides" J. -P. Mazaleyrat, K. Wright, A. Gaucher, N. Toulemonde, 
M. Wakselman, S. Oancea, C. Peggion, F. Formaggio, V. Setnicka, T. A. Keiderling, C. Toniolo, Journal of the 
American Chemical Society 126; 12874-12879 (2004). 

• Ab initio modeling of amide I coupling in anti -parallel b-sheets and the effect of the 13C isotopic labeling on 
vibrational spectra" Petr Bour, Timothy A. Keiderling, Journal of Physical Chemistry B, 109, 5348-5357 (2005) 

• Solvent Effects on IR And VCD Spectra of Helical Peptides: Insights from Ab Initio Spectral Simulations with 
Explicit Water" Jan Kubelka and Timothy A. Keiderling, Journal of Physical Chemistry B 109, 8231-8243 (2005) 

• IR Study of Cross-Strand Coupling in a beta-Hairpin Peptide Using Isotopic Labels., Vladimir Setnicka, Rong 
Huang, Catherine L. Thomas, Marcus A. Etienne, Jan Kubelka, Robert P. Hammer, Timothy A. Keiderling 
Journal of the American Chemical Society 127, 4992-4993 (2005). 

• Vibrational spectral simulation for peptides of mixed secondary structure: Method comparisons with the trpzip 
model hairpin. Petr Bour and Timothy A. Keiderling, Journal of Physical Chemistry B 109, 232687-23697 
(2005). 

• Isotopically labeled peptides provide site-resolved structural data with infrared spectra. Probing the structural 
limit of optical spectroscopy, Timothy A. Keiderling, Rong Huang, Jan Kubelka, Petr Bour, Vladimir Setnicka, 
Robert P. Hammer, Marcus *A. Etienne, R. A. Gangani D. Silva, Sean M. Decatur Collections Symposium 
Series, 8, 42-49 (2005)— ["Biologically Active Peptides" IXth Conference, Prague Czech Republic, April 20-22, 
2005. 

Nucleic acids and polynucleotides 

• "Application of Vibrational Circular Dichroism to Synthetic Polypeptides and Polynucleic Acids" T. A. 
Keiderling, S. C. Yasui, R. K. Dukor, L. Yang, Polymer Preprints 30, 423-424 (1989). 

• "Vibrational Circular Dichroism of Polyribonucleic Acids. A Comparative Study in Aqueous Solution." A. 
Annamalai and T. A. Keiderling, Journal of the American Chemical Society, 109, 3125-3132 (1987). 

• "Conformational phase transitions (A-B and B-Z) of DNA and models using vibrational circular dichroism" L. 
Wang, L. Yang, T. A. Keiderling in Spectroscopy of Biological Molecules., eds. R. E. Hester, R. B. Girling, 
Special Publication 94 Roya 1 Society of Chemistry, Cambridge (1991) p. 137-38. 

• "Vibrational Circular Dichroism of Proteins Polysaccharides and Nucleic Acids" T. A. Keiderling, Chapter 8 in 
Physical Chemistry of Food Processes, Vol. 2 Advanced Techniques, Structures and Applications eds. I. C. 
Baianu, H. Pessen, T. Kumosinski, Van Norstrand — Reinhold, New York (1993) pp. 307—337. 

• "Structural Studies of Biological Macromolecules using Vibrational Circular Dichroism" T. A. Keiderling, P. 
Pancoska, Chapter 6 in Advances in Spectroscopy Vol. 21, "Biomolecular Spectroscopy Part B" ed. R. E. Hester, 
R. J. H. Clarke, John W iley Chichester (1993) pp 267-315. 



Vibrational circular dichroism 284 

• "Detection of Triple Helical Nucleic Acids with Vibrational Circular Dichroism," L. Wang, P. Pancoska, T. A. 
Keiderling in "Fifth International Conference on The Spectroscopy of Biological Molecules" Th. Theophanides, J. 
Anastassopoulou, N. Fotopoul os (Eds), Kluwen Academic Publ., Dortrecht, 1993, p. 81-82. 

• "Helical Nature of Poly (dl-dC) ♦ Poly (dl-dC). Vibrational Circular Dichroism Results" L. Wang and T. A. 
Keiderling Nucleic Acids Research 21 4127-4132 (1993). 

• "Detection and Characterization of Triple Helical Pyrimidine-Purine-Pyrimidine Nucleic Acids with Vibrational 
Circular Dichroism" L. Wang, P. Pancoska, T. A. Keiderling, Biochemistry 33 8428-8435 (1994). 

• "Vibrational Circular Dichroism of A-, B- and Z- form Nucleic Acids in the P02- Stretching Region" L. Wang, L. 
Yang, T. A. Keiderling, Biophysical Journal 67, 2460-2467 (1994). 

• "Studies of multiple stranded RNA and DNA with FTIR, vibrational and electronic circular dichroism," Zhihua 
Huang, Lijiang Wang and Timothy A. Keiderling, in Spectrosopy of Biological Molecules, Ed. J. C. Merlin, 
Kluwer Acad. Pub., Dordrecht, 1995, pp . 321-322. 

• "Vibrational Circular Dichroism Applications to Conformational Analysis of Biomolecules" T. A. Keiderling in 
"Circular Dichroism and the Conformational Analysis of Biomolecules" ed G. D. Fasman, Plenum, New York 
(1996) pp. 555-598. 

• "Vibrational Circular Dichroism Techniques and Application to Nucleic Acids" T. A. Keiderling, In 
"Biomolecular Structure and Dynamics", NATO ASI series, Series E: Applied Sciences- Vol.342, Eds: G. 
Vergoten and T. Theophanides, Kluwer Academ ic Publishers, Dordrecht, The Netherlands, pp. 299—317 (1997). 

See also 

Circular dichroism 

Birefringence 

Optical rotatory dispersion 

IR spectroscopy 

Polarization 

Proteins 

Nucleic Acids 

DNA 

Molecular models of DNA 

DNA structure 

Protein structure 

Amino acids 

Density functional theory 

Quantum chemistry 

Raman optical activity (ROA) 



Raman spectroscopy 



285 



Raman spectroscopy 



Raman spectroscopy (named after C. 
V. Raman, pronounced /rCCmen/) is a 
spectroscopic technique used to study 
vibrational, rotational, and other 
low-frequency modes in a system. It 
relies on inelastic scattering, or Raman 
scattering, of monochromatic light, 
usually from a laser in the visible, near 
infrared, or near ultraviolet range. The 
laser light interacts with molecular 
vibrations, phonons or other 
excitations in the system, resulting in 
the energy of the laser photons being 
shifted up or down. The shift in energy 
gives information about the phonon 
modes in the system. Infrared 
spectroscopy yields similar, but 
complementary, information. 



Virtual 

energy 

states 



J 



Vibrational 
energy states 



+ 



A 



I 



¥ 



1 — 

Infrared Rayleigh Stokes Anti-Stokes 

absorption scattering Raman Raman 

scattering scattering 

Energy level diagram showing the states involved in Raman signal. The line thickness is 
roughly proportional to the signal strength from the different transitions. 



Typically, a sample is illuminated with a laser beam. Light from the illuminated spot is collected with a lens and sent 
through a monochromator. Wavelengths close to the laser line, due to elastic Rayleigh scattering, are filtered out 
while the rest of the collected light is dispersed onto a detector. 

Spontaneous Raman scattering is typically very weak, and as a result the main difficulty of Raman spectroscopy is 
separating the weak inelastically scattered light from the intense Rayleigh scattered laser light. Historically, Raman 
spectrometers used holographic gratings and multiple dispersion stages to achieve a high degree of laser rejection. In 
the past, photomultipliers were the detectors of choice for dispersive Raman setups, which resulted in long 
acquisition times. However, modern instrumentation almost universally employs notch or edge filters for laser 
rejection and spectrographs (either axial transmissive (AT), Czerny-Turner (CT) monochromator) or FT (Fourier 
transform spectroscopy based), and CCD detectors. 

There are a number of advanced types of Raman spectroscopy, including surface-enhanced Raman, resonance 
Raman, tip-enhanced Raman, polarised Raman, stimulated Raman (analogous to stimulated emission), transmission 
Raman, spatially-offset Raman, and hyper Raman. 



Basic theory 

The Raman effect occurs when light impinges upon a molecule and interacts with the electron cloud and the bonds of 
that molecule. For the spontaneous Raman effect, which is a form of scattering, a photon excites the molecule from 
the ground state to a virtual energy state. When the molecule relaxes it emits a photon and it returns to a different 
rotational or vibrational state. The difference in energy between the original state and this new state leads to a shift in 
the emitted photon's frequency away from the excitation wavelength. The Raman effect, which is a light scattering 
phenomenon, should not be confused with absorption (as with fluorescence) where the molecule is excited to a 
discrete (not virtual) energy level. 

If the final vibrational state of the molecule is more energetic than the initial state, then the emitted photon will be 
shifted to a lower frequency in order for the total energy of the system to remain balanced. This shift in frequency is 
designated as a Stokes shift. If the final vibrational state is less energetic than the initial state, then the emitted 



Raman spectroscopy 286 

photon will be shifted to a higher frequency, and this is designated as an Anti-Stokes shift. Raman scattering is an 
example of inelastic scattering because of the energy transfer between the photons and the molecules during their 
interaction. 

A change in the molecular polarization potential — or amount of deformation of the electron cloud — with respect 
to the vibrational coordinate is required for a molecule to exhibit a Raman effect. The amount of the polarizability 
change will determine the Raman scattering intensity. The pattern of shifted frequencies is determined by the 
rotational and vibrational states of the sample. 

History 

Although the inelastic scattering of light was predicted by Adolf Smekal in 1923, it is not until 1928 that it was 
observed in practice. The Raman effect was named after one of its discoverers, the Indian scientist Sir C. V. Raman 
who observed the effect by means of sunlight (1928, together with K. S. Krishnan and independently by Grigory 
Landsberg and Leonid Mandelstam). Raman won the Nobel Prize in Physics in 1930 for this discovery 
accomplished using sunlight, a narrow band photographic filter to create monochromatic light and a "crossed" filter 
to block this monochromatic light. He found that light of changed frequency passed through the "crossed" filter. 

Systematic pioneering theory of the Raman effect was developed by Czechoslovak physicist George Placzek 
between 1930 and 1934. The mercury arc became the principal light source, first with photographic detection and 
then with spectrophotometric detection. At the present time, lasers are used as light sources. 

Raman spectra 

Raman spectra are typically expressed in wavenumbers, which have units of inverse length. In order to convert 
between spectral wavelength and wavenumbers of shift in the Raman spectrum, the following formula can be used: 

where AlL>i s the Raman shift expressed in wavenumber, X is the excitation wavelength, and X is the Raman 
spectrum wavelength. Most commonly, the units chosen for expressing wavenumber in Raman spectra is inverse 
centimeters (cm - ). Since wavelength is often expressed in units of nanometers (nm), the formula above can scale 
for this units conversion explicitly, giving 

1 s / 1 1 \ t (nm) 

AiufcnT 1 ) = f — x 10 7V 



Ao(nm) Ai(nm)/ (cm) 



Applications 



Raman spectroscopy is commonly used in chemistry, since vibrational information is specific to the chemical bonds 
and symmetry of molecules. Therefore, it provides a fingerprint by which the molecule can be identified. For 
instance, the vibrational frequencies of SiO, Si O , and Si O were identified and assigned on the basis of normal 
coordinate analyses using infrared and Raman spectra. The fingerprint region of organic molecules is in the 
(wavenumber) range 500—2000 cm - . Another way that the technique is used to study changes in chemical bonding, 
e.g., when a substrate is added to an enzyme. 

Raman gas analyzers have many practical applications. For instance, they are used in medicine for real-time 
monitoring of anaesthetic and respiratory gas mixtures during surgery. 

In solid state physics, spontaneous Raman spectroscopy is used to, among other things, characterize materials, 
measure temperature, and find the crystallographic orientation of a sample. As with single molecules, a given solid 
material has characteristic phonon modes that can help an experimenter identify it. In addition, Raman spectroscopy 
can be used to observe other low frequency excitations of the solid, such as plasmons, magnons, and 



Raman spectroscopy 287 

superconducting gap excitations. The spontaneous Raman signal gives information on the population of a given 
phonon mode in the ratio between the Stokes (downshifted) intensity and anti-Stokes (upshifted) intensity. 

Raman scattering by an anisotropic crystal gives information on the crystal orientation. The polarization of the 
Raman scattered light with respect to the crystal and the polarization of the laser light can be used to find the 
orientation of the crystal, if the crystal structure (to be specific, its point group) is known. 

Raman active fibers, such as aramid and carbon, have vibrational modes that show a shift in Raman frequency with 
applied stress. Polypropylene fibers also exhibit similar shifts. The radial breathing mode is a commonly used 
technique to evaluate the diameter of carbon nanotubes. In nanotechnology, a Raman microscope can be used to 
analyze nanowires to better understand the composition of the structures. 

Spatially-offset Raman spectroscopy (SORS), which is less sensitive to surface layers than conventional Raman, can 
be used to discover counterfeit drugs without opening their internal packaging, and for non-invasive monitoring of 

Ml 

biological tissue. Raman spectroscopy can be used to investigate the chemical composition of historical documents 



such as the Book of Kells and contribute to knowledge of the social and economic conditions at the time the 
documents were produced. This is especially helpful because Raman spectroscopy offers a non-invasive way to 
determine the best course of preservation or conservation treatment for such materials. 

Raman spectroscopy is being investigated as a means to detect explosives for airport security. 

Raman spectroscopy has also been used to confirm the prediction of existence of low-frequency phonons in 

proteins and DNA (see, e.g., greatly stimulating the studies of low-frequency collective motion in 

ri2i ri3i 
proteins and DNA and their biological functions. 

Microspectroscopy 

Raman spectroscopy offers several advantages for microscopic analysis. Since it is a scattering technique, specimens 
do not need to be fixed or sectioned. Raman spectra can be collected from a very small volume (< 1 pm in diameter); 
these spectra allow the identification of species present in that volume. Water does not generally interfere with 
Raman spectral analysis. Thus, Raman spectroscopy is suitable for the microscopic examination of minerals, 
materials such as polymers and ceramics, cells and proteins. A Raman microscope begins with a standard optical 
microscope, and adds an excitation laser, a monochromator, and a sensitive detector (such as a charge-coupled 
device (CCD), or photomultiplier tube (PMT)). FT-Raman has also been used with microscopes. 

In direct imaging, the whole field of view is examined for scattering over a small range of wavenumbers (Raman 
shifts). For instance, a wavenumber characteristic for cholesterol could be used to record the distribution of 
cholesterol within a cell culture. 

The other approach is hyperspectral imaging or chemical imaging, in which thousands of Raman spectra are 
acquired from all over the field of view. The data can then be used to generate images showing the location and 
amount of different components. Taking the cell culture example, a hyperspectral image could show the distribution 
of cholesterol, as well as proteins, nucleic acids, and fatty acids. Sophisticated signal- and image-processing 
techniques can be used to ignore the presence of water, culture media, buffers, and other interferents. 

Raman microscopy, and in particular confocal microscopy, has very high spatial resolution. For example, the lateral 
and depth resolutions were 250 nm and 1.7 pm, respectively, using a confocal Raman microspectrometer with the 
632.8 nm line from a Helium-Neon laser with a pinhole of 100 |im diameter. Since the objective lenses of 
microscopes focus the laser beam to several micrometres in diameter, the resulting photon flux is much higher than 
achieved in conventional Raman setups. This has the added benefit of enhanced fluorescence quenching. However, 
the high photon flux can also cause sample degradation, and for this reason some setups require a thermally 
conducting substrate (which acts as a heat sink) in order to mitigate this process. 

By using Raman microspectroscopy, in vivo time- and space-resolved Raman spectra of microscopic regions of 
samples can be measured. As a result, the fluorescence of water, media, and buffers can be removed. Consequently 



Raman spectroscopy 288 

in vivo time- and space-resolved Raman spectroscopy is suitable to examine proteins, cells and organs. 

Raman microscopy for biological and medical specimens generally uses near-infrared (NIR) lasers (785 nm diodes 
and 1064 nm Nd:YAG are especially common). This reduces the risk of damaging the specimen by applying higher 

4 

energy wavelengths. However, the intensity of NIR Raman is low (owing to the m dependence of Raman scattering 
intensity), and most detectors required very long collection times. Recently, more sensitive detectors have become 

available, making the technique better suited to general use. Raman microscopy of inorganic specimens, such as 

ri4i 
rocks and ceramics and polymers, can use a broader range of excitation wavelengths. 

Polarized analysis 

The polarization of the Raman scattered light also contains useful information. This property can be measured using 
(plane) polarized laser excitation and a polarization analyzer. Spectra acquired with the analyzer set at both 
perpendicular and parallel to the excitation plane can be used to calculate the depolarization ratio. Study of the 
technique is useful in teaching the connections between group theory, symmetry, Raman activity, and peaks in the 
corresponding Raman spectra. 

The spectral information arising from this analysis gives insight into molecular orientation and vibrational symmetry. 
In essence, it allows the user to obtain valuable information relating to the molecular shape, for example in synthetic 
chemistry or polymorph analysis. It is often used to understand macromolecular orientation in crystal lattices, liquid 
crystals or polymer samples. 

Variations 

Several variations of Raman spectroscopy have been developed. The usual purpose is to enhance the sensitivity (e.g., 
surface-enhanced Raman), to improve the spatial resolution (Raman microscopy), or to acquire very specific 
information (resonance Raman). 

• Surface Enhanced Raman Spectroscopy (SERS) - Normally done in a silver or gold colloid or a substrate 
containing silver or gold. Surface plasmons of silver and gold are excited by the laser, resulting in an increase in 
the electric fields surrounding the metal. Given that Raman intensities are proportional to the electric field, there 
is large increase in the measured signal (by up to 10 ). This effect was originally observed by Martin 
Fleischmann but the prevailing explanation was proposed by Van Duyne in 1977. A comprehensive theory of 
the effect is that given by Lombardi and Birke in 2008 called the a Unified Approach to Surface-Enhanced Raman 
Spectroscopy. 

• Resonance Raman spectroscopy - The excitation wavelength is matched to an electronic transition of the 
molecule or crystal, so that vibrational modes associated with the excited electronic state are greatly enhanced. 
This is useful for studying large molecules such as polypeptides, which might show hundreds of bands in 
"conventional" Raman spectra. It is also useful for associating normal modes with their observed frequency 
shifts.™ 

• Surface-Enhanced Resonance Raman Spectroscopy (SERRS) - A combination of SERS and resonance Raman 
spectroscopy that uses proximity to a surface to increase Raman intensity, and excitation wavelength matched to 
the maximum absorbance of the molecule being analysed. 

• Hyper Raman - A non-linear effect in which the vibrational modes interact with the second harmonic of the 
excitation beam. This requires very high power, but allows the observation of vibrational modes that are normally 

ri9i 

"silent". It frequently relies on SERS-type enhancement to boost the sensitivity. 

• Spontaneous Raman Spectroscopy - Used to study the temperature dependence of the Raman spectra of 
molecules. 

• Optical Tweezers Raman Spectroscopy (OTRS) - Used to study individual particles, and even biochemical 
processes in single cells trapped by optical tweezers. 



Raman spectroscopy 289 

• Stimulated Raman Spectroscopy - A spatially coincident, two color pulse (with polarization either parallel or 
perpendicular) transfers the population from ground to a rovibrationally excited state, if the difference in energy 
corresponds to an allowed Raman transition, and if neither frequency corresponds to an electronic resonance. Two 
photon UV ionization, applied after the population transfer but before relaxation, allows the intra-molecular or 
inter-molecular Raman spectrum of a gas or molecular cluster (indeed, a given conformation of molecular cluster) 
to be collected. This is a useful molecular dynamics technique. 

• Spatially Offset Raman Spectroscopy (SORS) - The Raman scatter is collected from regions laterally offset 
away from the excitation laser spot, leading to significantly lower contributions from the surface layer than with 
traditional Raman spectroscopy. 

• Coherent anti-Stokes Raman spectroscopy (CARS) - Two laser beams are used to generate a coherent 
anti-Stokes frequency beam, which can be enhanced by resonance. 

• Raman optical activity (ROA) - Measures vibrational optical activity by means of a small difference in the 

intensity of Raman scattering from chiral molecules in right- and left-circularly polarized incident light or, 

[211 
equivalently, a small circularly polarized component in the scattered light. 

• Transmission Raman - Allows probing of a significant bulk of a turbid material, such as powders, capsules, 

T221 
living tissue, etc. It was largely ignored following investigations in the late 1960s but was rediscovered in 

T231 
2006 as a means of rapid assay of pharmaceutical dosage forms. There are also medical diagnostic 

applications. 

• Inverse Raman spectroscopy. 

• Tip-Enhanced Raman Spectroscopy (TERS) - Uses a metallic (usually silver-/gold-coated AFM or STM) tip to 
enhance the Raman signals of molecules situated in its vicinity. The spatial resolution is approximately the size of 
the tip apex (20-30 nm). TERS has been shown to have sensitivity down to the single molecule level. 

References 

[I] Gardiner, DJ. (1989). Practical Raman spectroscopy. Springer- Verlag. ISBN 978-0387502540. 

[2] Placzek G.: "Rayleigh Streeung und Raman Effekt", In: Hdb. der Radiologie, Vol. VI., 2, 1934, p. 209 

[3] Khanna, R.K. (1981). "Raman-spectroscopy of oligomeric SiO species isolated in solid methane". Journal of Chemical Physics 74: 2108. 

doi: 10.1063/1.441393. 
[4] "Fake drugs caught inside the pack" (http://news.bbc.co.Uk/2/hi/health/6314287.stm). BBC News. 2007-01-31. . Retrieved 2008-12-08. 
[5] Irish classic is still a hit (in calfskin, not paperback) - New York Times (http://www.nytimes.com/2007/05/28/world/europe/28kells. 

html), nytimes.com 
[6] Ben Vogel (29 August 2008). "Raman spectroscopy portends well for standoff explosives detection" (http://www.janes.com/news/ 

transport/business/jar/jar080829_l_n.shtml). Jane's. . Retrieved 2008-08-29. 
[7] Kuo-Chen Chou and Nian-Yi Chen (1977) The biological functions of low-frequency phonons. Scientia Sinica, 20, 447-457. 
[8] Urabe, H., Tominaga, Y. and Kubota, K. (1983) Experimental evidence of collective vibrations in DNA double helix Raman spectroscopy. 

Journal of Chemical Physics, 78, 5937-5939. 
[9] Chou, K.C. (1983) Identification of low-frequency modes in protein molecules. Biochemical Journal, 215, 465-469. 
[10] Chou, K.C. (1984) Low-frequency vibration of DNA molecules. Biochemical Journal, 221, 27-31. 

[II] Urabe, H., Sugawara, Y., Ataka, M. and Rupprecht, A. (1998) Low-frequency Raman spectra of lysozyme crystals and oriented DNA films: 
dynamics of crystal water. Biophys J, 74, 1533-1540. 

[12] Kuo-Chen Chou (1988) Review: Low-frequency collective motion in biomacromolecules and its biological functions. Biophysical 

Chemistry, 30, 3-48. 
[13] Chou, K.C. (1989) Low-frequency resonance and cooperativity of hemoglobin. Trends in Biochemical Sciences, 14, 212. 
[14] Ellis DI, Goodacre R (August 2006). "Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman 

spectroscopy". Analyst 131 (8): 875-85. doi:10.1039/b602376m. PMID 17028718. 
[15] Khanna, R.K. (1957). Evidence of ion-pairing in the polarized Raman spectra of a Ba2+CrO doped KI single crystal. John Wiley & Sons, 

Ltd. doi:10.1002/jrs.l250040104. 
[16] Jeanmaire DL, van Duyne RP (1977). "Surface Raman Electrochemistry Part I. Heterocyclic, Aromatic and Aliphatic Amines Adsorbed on 

the Anodized Silver Electrode". Journal of Electroanalytical Chemistry: (Elsevier Sequouia S.A.) 84: 1—20. 

doi:10.1016/S0022-0728(77)80224-6. 
[17] Lombardi JR, Birke RL (2008). "A Unified Approach to Surface-Enhanced Raman Spectroscopy". [Journal of Physical Chemistry C] 

(American Chemical Society) 112: 5605-5617. doi:10.1021/jp800167 CCC. 



Raman spectroscopy 290 

[18] Chao RS, Khanna RK, Lippincott ER (1974). "Theoretical and experimental resonance Raman intensities for the manganate ion". J Raman 

Spectroscopy 3: 121. doi:10.1002/jrs.l250030203. 
[19] Kneipp K, et al. (1999). "Surface-Enhanced Non-Linear Raman Scattering at the Single Molecule Level". Chem. Phys. 247: 155—162. 

doi:10.1016/S0301-0104(99)00165-2. 
[20] Matousek P, Clark IP, Draper ERC, et al. (2005). "Subsurface Probing in Diffusely Scattering Media using Spatially Offset Raman 

Spectroscopy". Applied Spectroscopy 59 (12): 393. doi: 10. 1366/000370205775 142548. PMID 16390587. 
[21] Barron LD, Hecht L, McColl IH, Blanch EW (2004). "Raman optical activity comes of age". Molec. Phys. 102 (8): 73 1-744. 

doi: 10.1080/00268970410001704399. 
[22] B. Schrader, G. Bergmann, Fresenius. Z. (1967). Anal. Chem.: 225-230. 
[23] P. Matousek, A. W. Parker (2006). "Bulk Raman Analysis of Pharmaceutical Tablets". Applied Spectroscopy 60 (12): 1353-1357. 

doi: 10.1366/000370206779321463. PMID 17217583. 
[24] P. Matousek, N. Stone (2007). "Prospects for the diagnosis of breast cancer by noninvasive probing of calcifications using transmission 

Raman spectroscopy". Journal of Biomedical Optics 12 (2): 024008. doi:10.1 117/1.2718934. PMID 17477723. 

External links 

• Raman FAQs (frequently asked questions) (http://www.horiba.com/scientific/products/raman-spectroscopy/ 
tutorial-faqs/raman-faqs/), horiba.com 

• Raman on SiGe Superlattice (http://content.piacton.com/Uploads/Princeton/Documents/Library/ 
UpdatedLibrary/Raman_on_SiGe_Superlattice_Using_TriVista.pdf), princetoninstruments.com 

• An introduction to Raman spectroscopy (http://www.horiba.com/scientific/products/raman-spectroscopy/ 
raman-resource/raman-tutorial/), horiba.com 

• Raman Application examples (http://www.horiba.com/scientific/products/raman-spectroscopy/ 
application-notes/), horiba.com 

• An introduction on Raman Scattering (http://www.d3technologies.co.uk/en/10371.aspx), 
d3technologies.co.uk 

• Raman Spectroscopy Applications (http://www.renishaw.com/en/raman-spectroscopy-applications— 6259), 
renishaw.com 

• Raman Data Search and Storage (http://ramandata.sourceforge.net) - The free application with a wonderful 
database of Raman data (vibrations, assignment) with storage function and Raman spectra (discussions) with 
search function, ramandata.sourceforge.net 

• Romanian Database of Raman Spectroscopy (http://rdrs.uaic.ro) - This database contains mineral species 
(natural and synthetic) with description of crystal structure, sample image, number of sample, origin, Raman 
spectrum and vibrations, Raman discussion and references. Also, this site contains artefacts sample with sample 
image and pigment spectrum; black, red, white or blue pigment, rdrs.uaic.ro 

• Chemical Imaging Without Dyeing (http://witec.de/en/download/Raman/ImagingMicroscopy04.pdf), 
witec.de 

• DoITPoMS Teaching and Learning Package - Raman Spectroscopy (http://www.doitpoms.ac.uk/tlplib/raman/ 
index. php) - an introduction, aimed at undergraduate level, doitpoms.ac.uk 

• Raman Spectroscopy Tutorial (http://161.58. 205. 25/Raman_Spectroscopy/rtr-ramantutorial.php?ss=800) - A 
detailed explanation of Raman Spectroscopy including Resonance-Enhanced Raman Scattering and 
Surface-Enhanced Raman Scattering. 161.25.205.25 

• The Science Show, ABC Radio National (http://www.abc.net.au/rn/science/ss/stories/sl581469.htm) - 
Interview with Scientist on NASA funded project to build Raman Spectrometer for the 2009 Mars mission: a 
cellular phone size device to detect almost any substance known, with commercial <USD$5000 commercial 
spin-off, prototyped by June 2006. abc.net.au/rn 

• Raman spectroscopy for medical diagnosis (http://pubs.acs.org/subscribe/journals/ancham/79/il 1/pdf/ 
0607feature_griffiths.pdf) from the June 1, 2007 issue of Analytical Chemistry (http://pubs3.acs.org/acs/ 
journals/toe. page?incoden=ancham&indecade=0&involume=79&inissue= 11), pubs.acs.org 



Raman spectroscopy 291 

• Spontaneous Raman Scattering (SRS) (http://www.lavision.de/en/techniques/raman-scattering.php), 
lavision.de 

• Painless laser device could spot early signs of disease (http://www.bbc.co.uk/news/ 
science-environment- 1 1390951), BBC News, 2010-09-26 



Microscope image processing 



Microscope image processing is a broad term that covers the use of digital image processing techniques to process, 
analyze and present images obtained from a microscope. Such processing is now commonplace in a number of 
diverse fields such as medicine, biological research, cancer research, drug testing, metallurgy, etc. A number of 
manufacturers of microscopes now specifically design in features that allow the microscopes to interface to an image 
processing system. 

Image acquisition 

Until the early 1990s, most image acquisition in video microscopy applications was typically done with an analog 
video camera, often simply closed circuit TV cameras. While this required the use of a frame grabber to digitize the 
images, video cameras provided images at full video frame rate (25-30 frames per second) allowing live video 
recording and processing. While the advent of solid state detectors yielded several advantages, the real-time video 
camera was actually superior in many respects. 

Today, acquisition is usually done using a CCD camera mounted in the optical path of the microscope. The camera 
may be full colour or monochrome. Very often, very high resolution cameras are employed to gain as much direct 
information as possible. Cryogenic cooling is also common, to minimise noise. Often digital cameras used for this 
application provide pixel intensity data to a resolution of 12-16 bits, much higher than is used in consumer imaging 
products. 

Ironically, in recent years, much effort has been put into acquiring data at video rates, or higher (25-30 frames per 
second or higher). What was once easy with off-the-shelf video cameras now requires special, high speed electronics 
to handle the vast digital data bandwidth. 

Higher speed acquisition allows dynamic processes to be observed in real time, or stored for later playback and 
analysis. Combined with the high image resolution, this approach can generate vast quantities of raw data, which can 
be a challenge to deal with, even with a modern computer system. 

It should be observed that while current CCD detectors allow very high image resolution, often this involves a 
trade-off because, for a given chip size, as the pixel count increases, the pixel size decreases. As the pixels get 
smaller, their well depth decreases, reducing the number of electrons that can be stored. In turn, this results in a 
poorer signal to noise ratio. 

For best results, one must select an appropriate sensor for a given application. Because microscope images have an 
intrinsic limiting resolution, it often makes little sense to use a noisy, high resolution detector for image acquisition. 
A more modest detector, with larger pixels, can often produce much higher quality images because of reduced noise. 
This is especially important in low-light applications such as fluorescence microscopy. 

Moreover, one must also consider the temporal resolution requirements of the application. A lower resolution 
detector will often have a significantly higher acquisition rate, permitting the observation of faster events. 
Conversely, if the observed object is motionless, one may wish to acquire images at the highest possible spatial 
resolution without regard to the time required to acquire a single image. 



Microscope image processing 292 

2D image techniques 

Image processing for microscopy application begins with fundamental techniques intended to most accurately 
reproduce the information contained in the microscopic sample. This might include adjusting the brightness and 
contrast of the image, averaging images to reduce image noise and correcting for illumination non-uniformities. Such 
processing involves only basic arithmetic operations between images (i.e. addition, subtraction, multiplication and 
division). The vast majority of processing done on microscope image is of this nature. 

Another class of common 2D operations called image convolution are often used to reduce or enhance image details. 
Such "blurring" and "sharpening" algorithms in most programs work by altering a pixel's value based on a weighted 
sum of that and the surrounding pixels, (a more detailed description of kernel based convolution deserves an entry 
for itself). 

Other basic two dimensional techniques include operations such as image rotation, warping, color balancing etc. 

At times, advanced techniques are employed with the goal of "undoing" the distortion of the optical path of the 
microscope, thus eliminating distortions and blurring caused by the instrumentation. This process is called 
deconvolution, and a variety of algorithms have been developed, some of great mathematical complexity. The end 
result is an image far sharper and clearer than could be obtained in the optical domain alone. This is typically a 
3-dimensional operation, that analyzes a volumetric image (i.e. images taken at a variety of focal planes through the 
sample) and uses this data to reconstruct a more accurate 3-dimensional image, vaya mudura kalutha vaya 

3D image techniques 

Another common requirement is to take a series of images at a fixed position, but at different focal depths. Since 
most microscopic samples are essentially transparent, and the depth of field of the focused sample is exceptionally 
narrow, it is possible to capture images "through" a three-dimensional object using 2D equipment like confocal 
microscopes. Software is then able to reconstruct a 3D model of the original sample which may be manipulated 
appropriately. The processing turns a 2D instrument into a 3D instrument, which would not otherwise exist. In recent 
times this technique has led to a number of scientific discoveries in cell biology. 

Analysis 

Analysis of images will vary considerably according to application. Typical analysis includes determining where the 
edges of an object are, counting similar objects, calculating the area, perimeter length and other useful measurements 
of each object. A common approach is to create an image mask which only includes pixels that match certain 
criteria, then perform simpler scanning operations on the resulting mask. It is also possible to label objects and track 
their motion over a series of frames in a video sequence. 

References 

Russ, John C. (2006-12-19) [1992]. The Image Processing Handbook (5th edition ed.). CRC Press. 
ISBN 0849372542. 

• Jan-Mark Geusebroek, Color and Geometrical Structure in Images, Applications in Microscopy, ISBN 
90-5776-057-6 

• Young Ian T., Not just pretty pictures: Digital quantitative microscopy, Proc. Royal Microscopical Society, 1996, 
31(4), pp. 311-313. 

• Young Ian T., Quantitative Microscopy, IEEE Engineering in Medicine and Biology, 1996, 15(1), pp. 59-66. 

• Young Ian T., Sampling density and quantitative microscopy, Analytical and Quantitative Cytology and 
Histology, vol. 10, 1988, pp. 269-275 



Microscope image processing 



293 



See also 

• Image processing 

• Endrov 

• Gemldent 



External links 

• Quantitative Microscopy 



[l] 



[2] 



3-D Image Processing in Microscopy 

Sampling theorem - Nyquist sampling in digital microscopy 



References 

[1] http://www.ph.tn.tudelft.nl/People/young/manuscripts/QM/QM.html 
[2] http://www.med.uni-giessen.de/ipl/iplcourse.html 
[3] http://www.vanosta.be/pcrnyq.htm 



Electron microscopy 



An electron microscope is a type of microscope that produces an 
electronically-magnified image of a specimen for detailed 
observation. The electron microscope (EM) uses a particle beam of 
electrons to illuminate the specimen and create a magnified image of 
it. The microscope has a greater resolving power than a 
light-powered optical microscope, because it uses electrons that have 
wavelengths about 100,000 times shorter than visible light (photons), 
and can achieve magnifications of up to 2,000,000x, whereas 
ordinary, non-confocal light microscopes are limited to 2000x 
magnification. 




High voltage 
Electrongun 

First condenser lens 

Condenser aperture 
Second condenser [ens 
.Condenser aperture 
_ Specimen holderand air-lock 
Objective lenses and aperture 

- Electron beam 

— Fluorescent screen and camera 



Transmission Electron Microscope 

Diagram of a transmission electron microscope 



The electron microscope uses electrostatic and electromagnetic 
"lenses" to control the electron beam and focus it to form an image. 

These lenses are analogous to, but different from the glass lenses of an optical microscope that form a magnified 
image by focusing light on or through the specimen. In transmission, the electron beam is first diffracted by the 
specimen, and then, the electron microscope "lenses" re-focus the beam into a Fourier-transformed image of the 
diffraction pattern for the selected area of investigation. The real image thus formed is a highly "magnified' image by 
a factor of several million, and can be then recorded on a special photographic plate, or viewed on a detecting screen. 
Electron microscopes are used to observe a wide range of biological and inorganic specimens including 
microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, the electron microscope is 
primarily used for quality control and failure analysis in semiconductor device fabrication. 



Election microscopy 



294 



An electron microscope's advantages over X-ray crystallography are 
that the specimen need not be a single crystal or even a polycrystalline 
powder, and also that the Fourier transform reconstruction of the 
object's magnified structure occurs physically and thus avoids the need 
for solving the phase problem faced by the X-ray crystalographers after 
obatining their X-ray diffraction patterns of a single crystal or 
polycrystalline powder. The transmission electron microscope's major 
"disadvantage' is the need for extremely thin sections of the specimens, 
typically less than 10 nanometers. For biological specimens it also 
requires biological sample special "staining' with heavy atom labels in 
order to achieve the required contrast, and then chemical fixation as 
well as encasing with a polymer resin to stabilize the biological 
specimen which is thin sectioned. 




A 1973 Siemens electron microscope, Musee des 
Arts et Metiers, Paris 



History 

In 1931, the German physicist Ernst Ruska and German electrical 
engineer Max Knoll constructed the prototype electron microscope, 
capable of four-hundred-power magnification; the apparatus was a 
practical application of the principles of electron microscopy. Two 
years later, in 1933, Ruska built an electron microscope that exceeded 
the resolution attainable with an optical (lens) microscope. 
Moreover, Reinhold Rudenberg, the scientific director of 
Siemens-Schuckertwerke, obtained the patent for the electron 
microscope in May 1931. Family illness compelled the electrical 
engineer to devise an electrostatic microscope, because he wanted to 
make visible the poliomyelitis virus. 

In 1937, the Siemens company financed the development work of 
Ernst Ruska and Bodo von Borries, and employed Helmut Ruska 
(Ernst's brother) to develop applications for the microscope, especially 

with biologic specimens. Also in 1937, Manfred von Ardenne 

T31 
pioneered the scanning electron microscope. The first practical 

electron microscope was constructed in 1938, at the University of 

Toronto, by Eli Franklin Burton and students Cecil Hall, James Hillier, 

and Albert Prebus; and Siemens produced the first commercial 

Transmission Electron Microscope (TEM) in 1939. Although 

contemporary electron microscopes are capable of two million-power 

magnification, as scientific instruments, they remain based upon 

Ruska's prototype. 




Electron microscope constructed by Ernst Ruska 
in 1933 



Electron microscopy 



295 



Types 



Transmission electron microscope (TEM) 

The original form of electron microscope, the transmission electron microscope (TEM) uses a high voltage electron 
beam to create an image. The electrons are emitted by an electron gun, commonly fitted with a tungsten filament 
cathode as the electron source. The electron beam is accelerated by an anode typically at +100 keV (40 to 400 keV) 
with respect to the cathode, focused by electrostatic and electromagnetic lenses, and transmitted through the 
specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the 
specimen, the electron beam carries information about the structure of the specimen that is magnified by the 
objective lens system of the microscope. The spatial variation in this information (the "image") is viewed by 
projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator 
material such as zinc sulfide. The image can be photographically recorded by exposing a photographic film or plate 
directly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a 
fibre optic light-guide to the sensor of a CCD (charge-coupled device) camera. The image detected by the CCD may 
be displayed on a monitor or computer. 

Resolution of the TEM is limited primarily by spherical aberration, but a new generation of aberration correctors 
have been able to partially overcome spherical aberration to increase resolution. Hardware correction of spherical 
aberration for the High Resolution TEM (HRTEM) has allowed the production of images with resolution below 0.5 
Angstrom (50 picometres) at magnifications above 50 million times. The ability to determine the positions of 



atoms within materials has made the HRTEM an important tool for nano-technologies research and development 



[7] 



Scanning electron microscope (SEM) 



Unlike the TEM, where electrons of the high voltage beam carry the 
image of the specimen, the electron beam of the Scanning Electron 

ro] 

Microscope (SEM) does not at any time carry a complete image of 
the specimen. The SEM produces images by probing the specimen 
with a focused electron beam that is scanned across a rectangular area 
of the specimen (raster scanning). At each point on the specimen the 
incident electron beam loses some energy, and that lost energy is 
converted into other forms, such as heat, emission of low-energy 
secondary electrons, light emission (cathodoluminescence) or x-ray 
emission. The display of the SEM maps the varying intensity of any of 
these signals into the image in a position corresponding to the position 
of the beam on the specimen when the signal was generated. In the 
SEM image of an ant shown at right, the image was constructed from 
signals produced by a secondary electron detector, the normal or 
conventional imaging mode in most SEMs. 

Generally, the image resolution of an SEM is about an order of magnitude poorer than that of a TEM. However, 
because the SEM image relies on surface processes rather than transmission, it is able to image bulk samples up to 
many centimetres in size and (depending on instrument design and settings) has a great depth of field, and so can 
produce images that are good representations of the three-dimensional shape of the sample. 




An image of an ant in a scanning electron 
microscope 



Election microscopy 



296 



Reflection electron microscope (REM) 

In the Reflection Electron Microscope (REM) as in the TEM, an electron beam is incident on a surface, but instead 
of using the transmission (TEM) or secondary electrons (SEM), the reflected beam of elastically scattered electrons 
is detected. This technique is typically coupled with Reflection High Energy Electron Diffraction (RHEED) and 
Reflection high-energy loss spectrum (RHELS). Another variation is Spin-Polarized Low-Energy Electron 



Microscopy (SPLEEM), which is used for looking at the microstructure of magnetic domains 



[9] 



Scanning transmission electron microscope (STEM) 

The STEM rasters a focused incident probe across a specimen that (as with the TEM) has been thinned to facilitate 
detection of electrons scattered through the specimen. The high resolution of the TEM is thus possible in STEM. The 
focusing action (and aberrations) occur before the electrons hit the specimen in the STEM, but afterward in the 
TEM. The STEMs use of SEM-like beam rastering simplifies annular dark-field imaging, and other analytical 
techniques, but also means that image data is acquired in serial rather than in parallel fashion. 

Low voltage electron microscope (LVEM) 

The low voltage electron microscope (LVEM) is a combination of SEM, TEM and STEM in one instrument, which 
operates at relatively low electron accelerating voltage of 5 kV. Low voltage increases image contrast which is 
especially important for biological specimens. This increase in contrast significantly reduces, or even eliminates the 
need to stain. Sectioned samples generally need to be thinner than they would be for conventional TEM (20-65 nm). 
Resolutions of a few nm are possible in TEM, SEM and STEM modes. 



Sample preparation 



Materials to be viewed under an electron microscope may require 
processing to produce a suitable sample. The technique required varies 
depending on the specimen and the analysis required: 

• Chemical fixation for biological specimens aims to stabilize the 
specimen's mobile macromolecular structure by chemical 
crosslinking of proteins with aldehydes such as formaldehyde and 
glutaraldehyde, and lipids with osmium tetroxide. 

• Cryofixation — freezing a specimen so rapidly, to liquid nitrogen or 
even liquid helium temperatures, that the water forms vitreous 
(non-crystalline) ice. This preserves the specimen in a snapshot of 
its solution state. An entire field called cryo-electron microscopy 
has branched from this technique. With the development of 
cryo-electron microscopy of vitreous sections (CEMOVIS), it is 
now possible to observe samples from virtually any biological 
specimen close to its native state. 

• Dehydration — freeze drying, or replacement of water with organic 

solvents such as ethanol or acetone, followed by critical point drying or infiltration with embedding resins. 

• Embedding, biological specimens — after dehydration, tissue for observation in the transmission electron 
microscope is embedded so it can be sectioned ready for viewing. To do this the tissue is passed through a 
'transition solvent' such as epoxy propane and then infiltrated with a resin such as Araldite epoxy resin; tissues 
may also be embedded directly in water-miscible acrylic resin. After the resin has been polymerised (hardened) 
the sample is thin sectioned (ultrathin sections) and stained - it is then ready for viewing. 

• Embedding, materials - after embedding in resin, the specimen is usually ground and polished to a mirror-like 
finish using ultra-fine abrasives. The polishing process must be performed carefully to minimize scratches and 




An insect coated in gold for viewing with a 
scanning electron microscope. 



Electron microscopy 297 

other polishing artifacts that reduce image quality. 

• Sectioning — produces thin slices of specimen, semitransparent to electrons. These can be cut on an 
ultramicrotome with a diamond knife to produce ultrathin slices about 60-90 nm thick. Disposable glass knives 
are also used because they can be made in the lab and are much cheaper. 

• Staining — uses heavy metals such as lead, uranium or tungsten to scatter imaging electrons and thus give contrast 
between different structures, since many (especially biological) materials are nearly "transparent" to electrons 
(weak phase objects). In biology, specimens are can be stained "en bloc" before embedding and also later after 
sectioning. Typically thin sections are stained for several minutes with an aqueous or alcoholic solution of uranyl 
acetate followed by aqueous lead citrate. 

• Freeze-fracture or freeze-etch — a preparation method particularly useful for examining lipid membranes and 
their incorporated proteins in "face on" view. The fresh tissue or cell suspension is frozen rapidly (cryofixed), 
then fractured by simply breaking or by using a microtome while maintained at liquid nitrogen temperature. The 
cold fractured surface (sometimes "etched" by increasing the temperature to about —100 °C for several minutes to 
let some ice sublime) is then shadowed with evaporated platinum or gold at an average angle of 45° in a high 
vacuum evaporator. A second coat of carbon, evaporated perpendicular to the average surface plane is often 
performed to improve stability of the replica coating. The specimen is returned to room temperature and pressure, 
then the extremely fragile "pre-shadowed" metal replica of the fracture surface is released from the underlying 
biological material by careful chemical digestion with acids, hypochlorite solution or SDS detergent. The 
still-floating replica is thoroughly washed from residual chemicals, carefully fished up on fine grids, dried then 
viewed in the TEM. 

• Ion Beam Milling — thins samples until they are transparent to electrons by firing ions (typically argon) at the 
surface from an angle and sputtering material from the surface. A subclass of this is Focused ion beam milling, 
where gallium ions are used to produce an electron transparent membrane in a specific region of the sample, for 
example through a device within a microprocessor. Ion beam milling may also be used for cross-section polishing 
prior to SEM analysis of materials that are difficult to prepare using mechanical polishing. 

• Conductive Coating — an ultrathin coating of electrically-conducting material, deposited either by high vacuum 
evaporation or by low vacuum sputter coating of the sample. This is done to prevent the accumulation of static 
electric fields at the specimen due to the electron irradiation required during imaging. Such coatings include gold, 
gold/palladium, platinum, tungsten, graphite etc. and are especially important for the study of specimens with the 
scanning electron microscope. Another reason for coating, even when there is more than enough conductivity, is 
to improve contrast, a situation more common with the operation of a FESEM (field emission SEM). 



Election microscopy 



298 



Disadvantages 

Electron microscopes are expensive to build and maintain, but the 
capital and running costs of confocal light microscope systems now 
overlaps with those of basic electron microscopes. They are dynamic 
rather than static in their operation, requiring extremely stable 
high-voltage supplies, extremely stable currents to each 
electromagnetic coil/lens, continuously-pumped high- or 
ultra-high-vacuum systems, and a cooling water supply circulation 
through the lenses and pumps. As they are very sensitive to vibration 
and external magnetic fields, microscopes designed to achieve high 
resolutions must be housed in stable buildings (sometimes 
underground) with special services such as magnetic field cancelling 
systems. Some desktop low voltage electron microscopes have TEM 
capabilities at very low voltages (around 5 kV) without stringent 
voltage supply, lens coil current, cooling water or vibration isolation 
requirements and as such are much less expensive to buy and far easier 
to install and maintain, but do not have the same ultra-high (atomic 
scale) resolution capabilities as the larger instruments. 




False-color SEM image of the filter setae of an 
Antarctic krill. (Raw electron microscope images 

carry no color information.) 

Pictured: First degree filter setae with V-shaped 

second degree setae pointing towards the inside 

of the feeding basket. The purple ball is 1 um in 

diameter. 



The samples largely have to be viewed in vacuum, as the molecules 

that make up air would scatter the electrons. One exception is the environmental scanning electron microscope, 

which allows hydrated samples to be viewed in a low-pressure (up to 20 Torr/2.7 kPa), wet environment. 

Scanning electron microscopes usually image conductive or semi-conductive materials best. Non-conductive 
materials can be imaged by an environmental scanning electron microscope. A common preparation technique is to 
coat the sample with a several-nanometer layer of conductive material, such as gold, from a sputtering machine; 
however, this process has the potential to disturb delicate samples. 

Small, stable specimens such as carbon nanotubes, diatom frustules and small mineral crystals (asbestos fibres, for 
example) require no special treatment before being examined in the electron microscope. Samples of hydrated 
materials, including almost all biological specimens have to be prepared in various ways to stabilize them, reduce 
their thickness (ultrathin sectioning) and increase their electron optical contrast (staining). These processes may 
result in artifacts, but these can usually be identified by comparing the results obtained by using radically different 
specimen preparation methods. It is generally believed by scientists working in the field that as results from various 
preparation techniques have been compared and that there is no reason that they should all produce similar artifacts, 
it is reasonable to believe that electron microscopy features correspond with those of living cells. In addition, 
higher-resolution work has been directly compared to results from X-ray crystallography, providing independent 
confirmation of the validity of this technique. Since the 1980s, analysis of cryofixed, vitrified specimens has also 
become increasingly used by scientists, further confirming the validity of this technique. 



Electron microscopy 



299 



Applications 



Semiconductor and data storage 

• Circuit edit 

• Defect analysis 

• Failure analysis 

Biology and life sciences 

Diagnostic electron microscopy 

Cryobiology 

Protein localization 

Electron tomography 

Cellular tomography 

Cryo-electron microscopy 

Toxicology 

Biological production and viral load monitoring 

Particle analysis 

Pharmaceutical QC 

Structural biology 

3D tissue imaging 

Virology 

Vitrification 



Research 

• Electron beam-induced deposition 

• Materials qualification 

• Materials and sample preparation 

• Nanoprototyping 

• Nanometrology 

• Device testing and characterization 

Industry 

• High-resolution imaging 

• 2D & 3D micro-characterization 

• Macro sample to nanometer metrology 

• Particle detection and characterization 

• Direct beam-writing fabrication 

• Dynamic materials experiments 

• Sample preparation 

• Forensics 

• Mining (mineral liberation analysis) 

• Chemical/Petrochemical 



See also 

Category:Electron microscope images 

Electron energy loss spectroscopy (EELS) 

Energy filtered transmission electron microscopy (EFTEM) 

Field emission microscope 

HiRISE 

High-resolution transmission electron microscopy (HRTEM) 

Scanning tunneling microscope 

Scanning confocal electron microscopy 

Scanning electron microscope (SEM) 

Scanning transmission electron microscope (STEM) 

Transmission Electron Aberration-corrected Microscope 

Electron diffraction 

X-ray diffraction 

X-ray microscope 

X-ray crystallography 

X-ray photoelectron spectroscopy (XPS) 

Microscope image processing 

Microscopy 

Acronyms in microscopy 

Nanoscience 

Nanotechnology 

Surface science 

Ultramicroscopy (journal) 



Electron microscopy 300 

References 

[I] Ernst Ruska (1986). "Ernst Ruska Autobiography" (http://nobelprize.org/nobel_prizes/physics/laureates/1986/ruska-autobio.html). 
Nobel Foundation. . Retrieved 2010-01-31. 

[2] Kruger DH, Schneck P, Gelderblom HR (May 2000). "Helmut Ruska and the visualisation of viruses" (http://linkinghub.elsevier.com/ 

retrieve/pii/S0140673600022509). Lancet 355 (9216): 1713-7. doi:10.1016/S0140-6736(00)02250-9. PMID 10905259. . 
[3] M von Ardenne and D Beischer (1940). "Untersuchung von metalloxyd-rauchen mit dem universal-elektronenmikroskop" (in German). 

Zeitschrift Electrochemie 46: 270—277. 
[4] "James Hillier" (http://web.mit.edu/Invent/iow/hillier.html). Inventor of the Week: Archive. 2003-05-01. . Retrieved 2010-01-31. 
[5] Erni, Rolf; Rossell, MD; Kisielowski, C; Dahmen, U (2009). "Atomic-Resolution Imaging with a Sub-50-pm Electron Probe". Physical 

Review Letters 102 (9): 096101. doi:10.1103/PhysRevLett.l02.096101. PMID 19392535. 
[6] "The Scale of Things" (http://www.sc.doe.gov/bes/scale_of_things.html). Office of Basic Energy Sciences, U.S. Department of Energy. 

2006-05-26. . Retrieved 2010-01-31. 
[7] O'Keefe MA, Allard LF (pdf). Sub-Angstrom Electron Microscopy for Sub-Angstrom Nano-Metrology (http://www.osti.gov/bridge/ 

servlets/purl/821768-E3YVgN/native/821768.pdf). Information Bridge: DOE Scientific and Technical Information - Sponsored by OSTI. . 

Retrieved 2010-01-31. 
[8] McMullan D (1993). "Scanning Electron Microscopy, 1928 - 1965" (http://www-g.eng.cam.ac.uk/125/achievements/mcmullan/mcm. 

htm). . Cincinnati, OH. . Retrieved 2010-01-31. 
[9] "SPLEEM" (http://ncem.lbl.gov/frames/spleem.html). National Center for Electron Microscopy (NCEM). . Retrieved 2010-01-31. 
[10] Nebesafoval, Jana; Vancova, Marie (2007). "How to Observe Small Biological Objects in Low Voltage Electron Microscope" (http:// 

journals.cambridge.org/abstract_S143192760708124X). Microscopy and Microanalysis 13 (3): 248-249. . 

[II] Drummy, Lawrence, F.; Yang, Junyan; Martin, David C. (2004). "Low-voltage electron microscopy of polymer and organic molecular thin 
films". Ultramicroscopy 99 (4): 247-256. doi:10.1016/j.ultramic.2004.01.011. PMID 15149719. 

[12] Adrian, Marc; Dubochet, Jacques; Lepault, Jean; McDowall, Alasdair W. (1984). "Cryo-electron microscopy of viruses". Nature 308 

(5954): 32-36. doi:10.1038/308032a0. PMID 6322001. 
[13] Sabanay, I.; Arad, T.; Weiner, S.; Geiger, B. (1991). "Study of vitrified, unstained frozen tissue sections by cryoimmunoelectron 

microscopy" (http://jcs.biologists.Org/cgi/content/abstract/100/l/227). Journal of Cell Science 100 (1): 227-236. PMID 1795028. . 
[14] Kasas, S.; Dumas, G.; Dietler, G.; Catsicas, S.; Adrian, M. (2003). "Vitrification of cryoelectron microscopy specimens revealed by 

high-speed photographic imaging". Journal of Microscopy 211 (1): 48-53. doi:10.1046/j. 1365-2818.2003.01 193.x. 

External links 

• Science Aid: Electron Microscopy (http://scienceaid.co.uk/biology/cell/analysingcells.html) High School 
(GCSE, A Level) resource 

• Cell Centered Database - Electron microscopy data (http://ccdb.ucsd. edu/sand/main?typeid=4& 
event=showMPByType&start=l) 

General 

• Nanohedron.comlNano image gallery (http://www.nanohedron.com/) beautiful images generated with electron 
microscopes. 

• electron microscopy (http://www.microscopy.ethz.ch) Website of the ETH Zurich: Very good graphics and 
images, which illustrate various procedures. 

• Environmental Scanning Electron Microscope (ESEM) (http://www.danilatos.com) 

• X-ray element analysis in electron microscope (http://www.microanalyst.net/index_e.phtml) — Information 
portal with X-ray microanalysis and EDX contents 



Electron microscopy 301 

History 

• John H.L. Watson: Very early Electron Microscopy in the Department of Physics, the University of Toronto — A 
personal recollection (http://www.physics.utoronto.ca/overview/history/microsco) 

• Rubin Borasky Electron Microscopy Collection, 1930-1988 (http://americanhistory.si.edu/archives/d8452. 
htm) Archives Center, National Museum of American History, Smithsonian Institution. 

Other 

• The Royal Microscopical Society, Electron Microscopy Section (UK) (http://www.rms.org.uk/em.shtml) 

• Albert Lleal micrograph. Scanning Electron Micrograph Coloured SEM (http://www.albertlleal.com/ 
microphotography . html) 



Diagnostic electron microscopy 



The transmission electron microscope (TEM) is used as an important diagnostic tool to screen human tissues at high 
magnification (the ultrastructural level), often in conjunction with other methods, particularly light microscopy and 
immunofluorescence techniques. The TEM was first used extensively for this purpose in the 1980s, especially for 
identifying the markers of cell differentiation to identify tumours, and in renal disease. Immunolabelling techniques 
are now generally used instead of the TEM for tumour diagnosis but the technique retains a critical role in the 
diagnosis of renal disease and a range of other conditions. 

Specifically, TEM should be used for diagnostic purposes when it: (1) provides useful (complementary) information 
in the context of a carefully considered differential diagnosis; (2) provides an 'improved' diagnosis that results in 
better treatment strategies and; (3) is time & cost effective in respect to alternative techniques. For diagnostic 
purposes solid tissues are prepared for TEM in the same way as other biological tissues, they are fixed in 
glutaraldehyde and osmium tetroxide then dehydrated and embedded in epoxy resin. The epoxy resin block is 
trimmed and the target tissue is selected using a light microscope by viewing semithin sections stained with toluidine 
blue. The block is then retrimmed and the specific area for observation is ultrathin sectioned, preferably using a 
diamond knife. The ultrathin sections are collected on 3mm copper (mesh) grids and stained with uranyl acetate and 
lead citrate to make the contents of the tissue electron dense (and thus visible in the electron microscope). 

References 

[1] Woods AE, Stirling JW. 2008. Electron microscopy. In, Theory and Practice of Histological Techniques. Eds, Bancroft JD and Gamble M. 
6th edition. Churchill Livingstone: pages 601-640 

External links 

The Association of Clinical Electron Microscopists (UK) (http://www.acem.org.uk/) 



HiRISE 



302 



HiRISE 



High Resolution Imaging Science 
Experiment is a camera on board the Mars 
Reconnaissance Orbiter. The 65 kg (143 lb), 
$40 million (USD) instrument was built 
under the direction of the University of 
Arizona's Lunar and Planetary Laboratory 
by Ball Aerospace & Technologies Corp.. It 
consists of a 0.5 m (19.7 in) aperture 
reflecting telescope, the largest of any deep 
space mission, which allows it to take 
pictures of Mars with resolutions up to 0.3 
m/pixel, resolving objects below a meter 
across. 

By 2010, HiRISE mapped about 1 percent 




A worker prepares HiRISE before it is shipped for attachment to the spacecraft 



of Martian surface at this quality 



[l] 



History 




Crop of first image of Mars from the HiRISE 
camera 



In the late 1980s, Alan Delamere of Ball Aerospace began planning the 
kind of high-resolution imaging needed to support sample return and 
surface exploration of Mars. In early 2001 he teamed up with Alfred 
McEwen of the University of Arizona to propose such a camera for the 
Mars Reconnaissance Orbiter (MRO), and NASA formally accepted it 
November 9, 2001 



r-\ 



Ball Aerospace was given the responsibility to build the camera and 
they delivered HiRISE to NASA on December 6, 2004, for integration 
with the rest of the spacecraft. It was prepared for launch on board 
the MRO on August 12, 2005, to the cheers of the HiRISE team who 
were present. 



HiRISE 



303 



During the cruise phase of MRO, HiRISE took several calibration 
shots including several of the Moon and the Jewel Box cluster. These 
images helped to calibrate the camera and prepare it for taking pictures 
of Mars. 

On March 10, 2006, MRO achieved Martian orbit and primed HiRISE 
to acquire some initial images of Mars. The instrument had two 
opportunities to take pictures of Mars (the first was on March 24, 
2006) before MRO entered aerobraking, during which time the camera 
was turned off for six months. It was turned on successfully 
September 27, and took its first high-resolution pictures of Mars on 
September 29. 

On October 6, 2006 HiRISE took the first image of Victoria Crater, a 
site which is also under study by the Opportunity rover. 

In February 2007 seven detectors showed signs of degradation, with 
one IR channel almost completely degraded, and one other showing 

advanced signs of degradation. The problems appear to disappear when higher temperatures are used to take pictures 

rxi 
with the camera. As of March, the degradation appeared to have stabilized, but the underlying cause remained 

mi 

unknown. Subsequent experiments with the Engineering Model (EM) at Ball Aerospace provided definitive 
evidence for the cause: contamination in the analog-to-digital converters (ADCs) which results in flipping bits to 
create the apparent noise or bad data in the images, combined with design flaws leading to delivery of poor analog 
waveforms to the ADCs. Further work showed that the degradation can be reversed by heating the ADCs. 

On 2007-10-03, HiRISE was turned toward Earth, and took a picture of it and the Moon. In a full-resolution color 
image, Earth was 90 pixels across and the Moon was 24 pixels across from a distance of 142 million km. 

On May 25, 2008, HiRISE imaged NASA's Mars Phoenix Lander parachuting down to Mars. It was the first time 




Artist's rendition of HiRISE at Mars 



that a spacecraft imaged the final descent of another spacecraft onto a planetary body 



[11] 



On April 1, 2010, NASA released the first images under the HiWish program in which just plain folk suggested 

ri2i 
places for HiRISE to photograph. One of the eight locations was Aureum Chaos. The first image below gives a 

ri3i 

wide view of the area. The next two images are from the HiRISE image. 



HiRISE 



304 







THEMIS image 
of wide view of 

following 
HiRISE images. 
Black box shows 
approximate 
location of 
HiRISE images. 
This image is just 
a part of the vast 

area know as 

Aureum Chaos. 

Click on image to 

see more details. 




Aureum Chaos, as 
seen by HiRISE, 
under the HiWish 
program. Image is 

located in 

Margaritifer Sinus 

quadrangle. 




Close up view of previous image, 

as seen by HiRISE under HiWish 

program. Small round dots are 

boulders. 



Probable glacier as seen by 
HiRISE under HiWish program. 
Radar studies have found that it 
is made up of almost completely 
pure ice. It appears to be moving 
from the high ground (a mesa) on 
the right. Location is Ismenius 
Lacus quadrangle. 



Purpose 

The HiRISE camera is designed to view surface features of Mars in 

1141 
greater detail than has previously been possible. This allows for the 

study of the age of Martian features, looking for landing sites for future 

Mars landers, and in general, seeing the Martian surface in far greater 

detail than has previously been done from orbit. By doing so, it is 

allowing better studies of Martian channels and valleys, volcanic 

landforms, possible former lakes and oceans, and other surface 

landforms as they exist on the Martian surface. 




Comparison of resolution of MRO HiRISE 
camera with predecessor, the MOC aboard MGS 



The general public will soon be allowed to request sites for the HiRISE 

camera to capture. For this reason, and due to the unprecedented access of pictures to the general public, shortly after 

they have been received and processed, the camera has been given the philosophy, "The People's Camera". 



HiRISE 



305 



Design 

HiRISE was designed to be a High Resolution camera from the 
beginning. It consists of a large mirror, as well as a large CCD camera. 
Because of this, it achieves a resolution of 1 microradian, or 0.3 meter 

at a height of 300 km. (For comparison purposes, satellite images on 

ri7i 
Google Maps are available to 1 meter. ) It can image in three color 

bands, 400-600 nm (blue-green or B-G), 550-850 nm (red) and 



800-1,000 nm (near infrared or NIR) 



[18] 




Earth and Moon from Mars Reconnaissance 
Orbiter taken by HiRISE 



HiRISE incorporates a 0.5-meter primary mirror, the largest optical 
telescope ever sent beyond Earth's orbit. The mass of the instrument is 
64.2 kg. [19] 

Red color images are at 20,048 pixels wide (6 km in a 300 km orbit), 

and Green-Blue and NIR are at 4,048 pixels wide (1.2 km). HiRISE's 

onboard computer reads out these lines in time with the orbiter's 

ground speed, meaning the images are potentially unlimited in height. 

Practically this is limited by the onboard computer's 28 Gb memory capacity. The nominal maximum size of red 

images (compressed to 8 bits per pixel) is about 20,000 x 126,000 pixels, or 2520 megapixels and 4,000 x 126,000 

pixels (504 megapixels) for the narrower images of the B-G and NIR bands. A single uncompressed image uses up 

to 28 Gb. However, these images are transmitted compressed,with a typical max size of 11.2 Gigabits. These images 

are released to the general public on the HiRISE website via a new format called JPEG 2000 



[20] [21] 



To facilitate the mapping of potential landing sites, HiRISE can produce stereo pairs of images from which the 
topography can be measured to an accuracy of 0.25 meter. 



Images naming conventions 

HIRISE images are available to the public, so it can be useful to know how they are named. This is an excerpt from 



the official documentation 



[22]. 



Name : 

ppp_oooooo_tttt_f f f f_c . IMG 

ppp = Mission Phase: 

INT = Integration and Testing 

CAL = Calibration Observations 

ATL = ATLO Observations 

KSC = Kennedy Space Center Observations 

SVT = Sequence Verification Test 

LAU = Launch 

CRU = Cruise Observations 

APR = Mars Approach Observations 

AEB = Aerobraking Phase 

TRA = Transition Phase 

PSP = Primary Science Orbit (nov 2006-nov 200E 

REL = Relay phase 

E01 = 1st Extended Mission Phase if needed 

Exx = Additional extended Missions if needed 



HiRISE 306 

oooooo = MRO orbit number 

tttt = Target code 

ffff Filter/CCD designation: 
RED0-RED9 - Red filter CCDs 
IR10-IR11 -Near-Infrared filter CCDs 
BG12-BG13 -Blue-Green filter CCDs 

c = Channel number of CCD (0 or 1) 

The target code refers to the latitudinal position of the center of the planned observation relative to the start of orbit. 
The start of orbit is located at the equator on the descending side (night side) of the orbit. A target code of 0000 
refers to the start of orbit. The target code increases in value along the orbit track ranging from 0000 to 3595. This 
convention allows the file name ordering to be time sequential. The first three digits refers to the number of whole 
degrees from the start of orbit, the fourth digit refers to the fractional degrees rounded to the nearest 0.5 degrees. 
Values greater than 3595 identify observations as off-Mars or special observations. 

Examples of target code: 

0000 —planned observation at the equator on descending side of orbit. 

0900 —planned observation at the south pole. 

1800 —planned observation at the equator on the ascending side (day side) of the orbit. 

2700 —planned observation at the north pole. 



Off-Mars and Special Observations Values: 

4000 -Star Observation 

4001 — Phobos Observation 

4002 — Deimos Observation 

4003 —Special Calibration Observation 

Footnotes 

[I] Microsoft and NASA Bring Mars Down to Earth Through the Worldwide Telescope (07.12.10) - NASA (http://www.nasa.gov/ 
topics/nasalife/features/microsoft_ww_telescope.html) 

[2] UANews (2001-11-09). "UA-Led Team's Ultra-High Resolution Camera Selected for 2005 Launch to Mars" (http://uanews.org/cgi-bin/ 

WebObjects/UANews.woa/4/wa/MainStoryDetails?ArticleID=4493). Press release. . Retrieved 2006-06-08. 
[3] UANews (2004-12-06). "Ultra-sharp, Mars-Bound HiRISE Camera Delivered" (http://uanews.Org/cgi-bin/WebObjects/UANews.woa/4/ 

wa/MainStoryDetails?ArticleID=10192). Press release. . Retrieved 2006-06-08. 
[4] UANews (2005-08-08). "UA Team Cheers Launch of Mars Reconnaissance Orbiter, HiRISE" (http://uanews.org/cgi-bin/WebObjects/ 

UANews. woa/4/wa/MainStoryDetails?ArticleID=l 1437). Press release. . Retrieved 2006-06-08. 
[5] "Mars Reconnaissance Orbiter Successfully Enters Orbit Around Mars!" (http://web.archive.org/web/2006060306181 l/http://mars.jpl. 

nasa.gov/mro/mission/orbiter_update.html). NASA MRO website. Archived from the original (http://mars.jpl.nasa.gov/mro/mission/ 

orbiter_update.html) on 2006-06-03. . Retrieved 2006-06-08. 
[6] NASA (2006-03-24). "UA Team Cheers Launch of Mars Reconnaissance Orbiter, HiRISE" (http://mars.jpl.nasa.gov/mro/newsroom/ 

pressreleases/20060324a.html). Press release. . Retrieved 2006-06-08. 
[7] HiRISE I Victoria Crater at Meridiani Planum (TRA_000873_1780) (http://hiroc.lpl.arizona.edu/images/TRA/TRA_000873_1780/) 
[8] NASA (2007-02-07). "Spacecraft Set to Reach Milestone, Reports Technical Glitches" (http://www.jpl.nasa.gov/news/news. 

cfm?release=2007-013). Press release. . Retrieved 2007-03-06. 
[9] Shiga, David (16 March 2007). "Ailing Mars camera is stable — for now" (http://space.newscientist.com/article/ 

dnll402-ailing-mars-camera-is-stable--for-now.html). NewScientist.com news service. . Retrieved 2007-03-18. 
[10] "Earth and Moon as Seen from Mars" (http://www.nasa.gov/mission_pages/MRO/multimedia/mro20080303earth.html). NASA. 

2008-03-03. . Retrieved 2008-06-21. 

[II] "Camera on Mars Orbiter Snaps Phoenix During Landing" (http://www.jpl. nasa.gov/news/phoenix/release. php?ArticleID=1714). JPL 
website. . Retrieved 2008-05-28. 



HiRISE 307 

[12] http://uahirise.org/releases/hiwish-captions.php 
[13] http://hirise.lpl.arizona.edu/ESP_016869_1775 
[14] Alan Delamere (2003) (PDF). MRO HiRISE: Instrument Development (http://marsoweb.nas.nasa.gov/HiRISE/papers/ 

6th_int_mars_coni7Delamere_HiRISE_InstDev.pdf). 6th International Mars Conference. . Retrieved 2008-05-25. 
[15] "Science Goals" (http://hirise.lpl.arizona.edu). Lunar and Planetary Laboratory, University of Arizona. . Retrieved June 7, 2006. 
[16] "HiRISE" (http://hirise.lpl.arizona.edu). Lunar and Planetary Laboratory, University of Arizona. . Retrieved 19 March 2006. 
[17] " Google Earth FAQ (http://earth.google.eom/faq.html#4)" Google Earth Website. 
[18] "MRO HiRISE Camera Specifications" (http://marsoweb.nas.nasa.gOv/HiRISE/instrument.html#components). HiRISE website. . 

Retrieved 2 January 2006. 
[19] Mission to Mars: the HiRISE camera on-board MRO (http://cat.inist.fr/?aModele=afficheN&cpsidt=20649610), Focal plane arrays for 

space telescopes III, 27—28 August 2007, San Diego, California, USA 
[20] "HiRISE: Instrument Development" (http://marsoweb.nas.nasa.gov/HiRISE/papers/6th_int_mars_conf/Delamere_HiRISE_InstDev. 

pdf) (PDF). NASA Ames Research Center website. . Retrieved 7 February 2006. 
[21] "Fact Sheet: HiRISE" (http://www.nasm.si.edu/research/ceps/cepsicons/highlights/fact_sheet_front.pdf) (PDF). National Air and 

Space Museum. . Retrieved 18 February 2006. 
[22] http://hirise.lpl.arizona.edu/pdf/HiRISE_EDR_SIS_2007_03_15.pdf 

External links 

• HiRISE official website (http://hirise.lpl.arizona.edu/) 

• HiBlog, the official HiRISE blog (http://hirise.lpl.arizona.edu/HiBlog/) 

• Help NASA categorize images taken by HiRISE (http://clickworkers.arc.nasa.gov/hirise) 

• Patterns of Mars - 12 High Resolution Photos by HiRISE on www.time.com (http://feeds.feedburner.com/~r/ 
time/topstories/~3/365740126/0,29307,1827176,00.html) 

• Browse Map of Images (http://global-data.mars.asu.edu/bin/hirise.pl) from ASU. 

Scanning confocal electron microscopy 

Scanning confocal electron microscopy (SCEM) is an electron microscopy technique analogous to scanning 
confocal optical microscopy (SCOM). In this technique, the studied sample is illuminated by a focussed electron 
beam, as in other scanning microscopy techniques, such as scanning transmission electron microscopy or scanning 
electron microscopy. However, in SCEM, the collection optics is arranged symmetrically to the illumination optics 
to gather only the electrons that pass the beam focus. This results in superior depth resolution of the imaging. The 
technique is relatively new and is being actively developed. 

History 

The idea of SCEM logically follows from SCOM and thus is rather old. However, practical design and construction 
of scanning confocal electron microscope is a complex problem first solved by Nestor J. Zaluzec. His first 

scanning confocal electron microscope demonstrated the 3D properties of the SCEM, but have not realized the 
sub-nanometer lateral spatial resolution achievable with high-energy electrons (lateral resolution of only -80 nm has 
been demonstrated). Several groups are currently working on construction of atomic resolution SCEM. In 
particular, atomically resolved SCEM images have already been obtained 



Scanning confocal electron microscopy 



308 



Operation 

The sample is illuminated by a focussed electron beam, 
and the beam is re-focussed on the detector, thus 
collecting only electrons passing through the focus. In 
order to produce an image, the beam should be laterally 
scanned. In the original design, this was achieved by 
placing synchronized scanning and descanning 
deflectors. Such design is complex and only a few 
custom-built setups exist. Another approach is to use 
stationary illumination and collection, but perform scan 
by moving the sample with a high-precision 
piezo-controlled holder. Such holders are readily 
available and can fit into most commercial electron 
microscopes thereby realizing the SCEM mode. As a 
practical demonstration, atomically resolved SCEM 



images have been recorded 



[6] [7] 



Advantages of SCEM 

High energies of incident particles (200 keV electrons 
vs. 2 eV photons) result in much higher spatial 
resolution of SCEM as compared to SCOM (lateral 
resolution <1 nm vs. >400 nm). 



Source |T] 



Objective lens 



Upper deflector XI 




Sample 



Lower deflector g| 



Collector lens 



Detector | 

Schematic of SCEM 



As compared to conventional electron microscopy (TEM, STEM, SEM), SCEM offers 3-dimensional imaging. 3D 
imaging in SCEM was expected from the confocal geometry of SCEM, and it has recently been confirmed by 

ro] 

theoretical modeling. In particular, it is predicted that a heavy layer (gold) can be identified in light matrix 
(aluminum) with -10 nm precision in depth; this depth resolution is limited by the convergence angle of the electron 
beam and could be improved to a few nanometers in next-generation electron microscopes equipped with two 
fifth-order spherical aberration correctors 



[9] [10] 



References 

[1] N.J. Zaluzec, US Patent # 6,548,810 -0, 2003 

[2] NJ. Zaluzec (2003). "The Scanning Confocal Electron Microscope" (http://www.amc.anl.gov/Docs/ANL/SCEM/ 

MToday-SCEM-Article.pdf). Microsc. Today 6. 8. . 
[3] NJ. Zaluzec (2007). "Scanning Confocal Electron Microscopy". Microsc. Microanal. 13: 1560. doi:10.1017/S1431927607074004. 
[4] S.P. Frigo, Z.H. Levine, NJ. Zaluzec (2002). "Submicron imaging of buried integrated circuit structures using scanning confocal electron 

microscopy" (http://link.aip.Org/link/7APPLAB/81/2112/l). Appl. Phys. Lett. 81: 2112. doi: 10. 1063/1. 1506010. . 
[5] "Dr. Peter Nellist" (http://www.materials.ox.ac.uk/peoplepages/nellist.html). . Retrieved 2009-06-06. 
[6] A. Hashimoto et al. (2008). "Development of Stage-scanning System for Confocal Scanning Transmission Electron Microscopy" (http:// 

www.jstage.jst.go.jp/article/ejssnt/6/0/lll/_pdf). E-J. Surf. Set Nanotech. 6: 111-114. doi:10.1380/ejssnt.2008.111. . 
[7] M. Takeguchi et al. (2008). /. Elec. Microscopy 57: 123. 
[8] K. Mitsuishi et al. (2008). "Bloch wave-based calculation of imaging properties of high-resolution scanning confocal electron microscopy". 

Ultramicroscopy 108 (9): 981. doi:10.1016/j.ultramic.2008.04.005. PMID 18519159. 
[9] P. D. Nellist et al. (=2006). "Confocal operation of a transmission electron microscope with two aberration correctors" (http://link.aip.org/ 

link/?APPLAB/89/124105/l). Appl. Phys. Lett. 89: 124105. . 
[10] JeolNews 39 (1). 2004. 



Scanning confocal electron microscopy 309 

See also 

• Confocal microscopy 

• Confocal laser scanning microscopy 

• Electron microscopy 

• Scanning electron microscope 

• Scanning transmission electron microscopy 

• Transmission electron microscopy 

Acronyms in microscopy 

List of materials analysis methods: 

Contents: Top-O-9-ABCDEFGHIJKLMNOPQRSTUVWXYZ 

• uSR - see Muon spin spectroscopy 

• x " see Magnetic susceptibility 



Analytical ultracentrifugation - Analytical ultracentrifugation 

AAS - Atomic absorption spectroscopy 

AED - Auger electron diffraction 

AES - Auger electron spectroscopy 

AFM - Atomic force microscopy 

AFS - Atomic fluorescence spectroscopy 

APFIM - Atom probe field ion microscopy 

APS - Appearance potential spectroscopy 

ARPES - Angle resolved photoemission spectroscopy 

ARUPS - Angle resolved ultraviolet photoemission spectroscopy 

ATR - Attenuated total reflectance 



B 



BET - BET surface area measurement (BET from Brunauer, Emmett, Teller) 
BiFC - Bimolecular fluorescence complementation 
BKD - Backscatter Kikuchi diffraction, see EBSD 
BRET - Bioluminescence resonance energy transfer 
BSED - Back scattered electron diffraction, see EBSD 



Acronyms in microscopy 310 



CAICISS - Coaxial impact collision ion scattering spectroscopy 

CARS - Coherent anti-Stokes Raman spectroscopy 

CBED - Convergent beam electron diffraction 

CCM - Charge collection microscopy 

CDI - Coherent diffraction imaging 

CE - Capillary electrophoresis 

CET - Cryo-electron tomography 

CL - Cathodoluminescence 

CLSM - Confocal laser scanning microscopy 

COSY - Correlation spectroscopy 

Cryo-EM - Cryo-electron microscopy 

CV - Cyclic voltammetry 



D 



DE(T)A - Dielectric thermal analysis 

dHvA - De Haas-van Alphen effect 

DIC - Differential interference contrast microscopy 

Dielectric spectroscopy - Dielectric spectroscopy 

DLS - Dynamic light scattering 

DLTS - Deep-level transient spectroscopy 

DMA - Dynamic mechanical analysis 

DPI - Dual polarisation interferometry 

DRS - Differential reflectance spectroscopy 

DSC - Differential scanning calorimetry 

DTA - Differential thermal analysis 

DVS - Dynamic vapour sorption 



E 



EBIC - Electron beam induced current (and see IBIC: ion beam induced charge) 

EBS - Elastic (non-Rutherford) backscattering spectrometry (see RBS) 

EBSD - Electron backscatter diffraction 

ECOSY - Exclusive correlation spectroscopy 

ECT - Electrical capacitance tomography 

EDAX - Energy-dispersive analysis of x-rays 

EDMR - Electrically Detected Magnetic Resonance, see ESR or EPR 

EDS - Energy Dispersive Spectroscopy 

EDX - Energy dispersive X-ray spectroscopy 

EELS - Electron energy loss spectroscopy 

EFTEM - Energy filtered transmission electron microscopy 

EID - Electron induced desorption 

EIT and ERT - Electrical impedance tomography and Electrical resistivity tomography 

EL - Electroluminescence 

Electron crystallography - Electron crystallography 

ELS - Electrophoretic light scattering 

ENDOR - Electron nuclear double resonance, see ESR or EPR 



Acronyms in microscopy 311 

EPMA - Electron probe microanalysis 

EPR - Electron paramagnetic resonance spectroscopy 

ERD or ERDA - Elastic recoil detection or Elastic recoil detection analysis 

ESCA - Electron spectroscopy for chemical analysis* see XPS 

ESD - Electron stimulated desorption 

ESEM - Environmental scanning electron microscopy 

ESI-MS or ES-MS - Electrospray ionization mass spectrometry or Electrospray mass spectrometry 

ESR - Electron spin resonance spectroscopy 

ESTM - Electrochemical scanning tunneling microscopy 

EXAFS - Extended X-ray absorption fine structure 

EXSY - Exchange spectroscopy 



FCS - Fluorescence correlation spectroscopy 

FCCS - Fluorescence cross-correlation spectroscopy 

FEM - Field emission microscopy 

FIB - Focused ion beam microscopy 

FIM-AP - Field ion microscopy— atom probe 

Flow birefringence - Flow birefringence 

Fluorescence anisotropy - Fluorescence anisotropy 

FLIM - Fluorescence lifetime imaging 

Fluorescence microscopy - Fluorescence microscopy 

FRET - Fluorescence resonance energy transfer 

FRS - Forward Recoil Spectrometry, a synonym of ERD 

FTICR or FT-MS - Fourier transform ion cyclotron resonance or Fourier transform mass spectrometry 

FTIR - Fourier transform infrared spectroscopy 



GC-MS - Gas chromatography-mass spectrometry 

GDMS - Glow discharge mass spectrometry 

GDOS - Glow discharge optical spectroscopy 

GISAXS - Grazing incidence small angle X-ray scattering 

GIXD - Grazing incidence X-ray diffraction 

GIXR - Grazing incidence X-ray reflectivity 

GLC - Gas-liquid chromatography 



Acronyms in microscopy 312 

H 

HAADF - high angle annular dark-field imaging 

HAS - Helium atom scattering 

HPLC - High performance liquid chromatography 

HREELS - High resolution electron energy loss spectroscopy 

HREM - High-resolution electron microscopy 

HRTEM - High-resolution transmission electron microscopy 



IAES - Ion induced Auger electron spectroscopy 

IBA - Ion beam analysis 

IBIC - Ion beam induced charge microscopy 

ICP-AES - Inductively_coupled_plasma_atomic_emission_spectroscopy 

ICP-MS - Inductively coupled plasma mass spectrometry 

Immunofluorescence - Immunofluorescence 

ICR - Ion cyclotron resonance 

IETS - Inelastic electron tunneling spectroscopy 

IGA - Intelligent gravimetric analysis 

IIX - Ion induced X-ray analysis: See Particle induced X-ray emission 

INS - Ion neutralization spectroscopy 

Inelastic neutron scattering 

IRS - Infrared spectroscopy 

ISS - Ion scattering spectroscopy 

ITC - Isothermal titration calorimetry 

IVEM - Intermediate voltage electron microscopy 



List of materials analysis methods (deliberate self-link) 

LALLS - Low-angle laser light scattering 

LC-MS - Liquid chromatography-mass spectrometry 

LEED - Low-energy electron diffraction 

LEEM - Low-energy electron microscopy 

LEIS - Low-energy ion scattering 

LIBS - Laser induced breakdown spectroscopy 

LOES - Laser optical emission spectroscopy 

LS - Light (Raman) scattering 



Acronyms in microscopy 313 



M 



MALDI - Matrix-assisted laser desorption/ionization 

MBE - Molecular beam epitaxy 

MEIS - Medium energy ion scattering 

MFM - Magnetic force microscopy 

MIT - Magnetic induction tomography 

MPM - Multiphoton fluorescence microscopy 

MRFM - Magnetic resonance force microscopy 

MRI - Magnetic resonance imaging 

MS - Mass spectrometry 

MS/MS - Tandem mass spectrometry 

Mossbauer spectroscopy - Mossbauer spectroscopy 

MTA - Microthermal analysis 



N 



NAA - Neutron activation analysis 

Nanovid microscopy - Nanovid microscopy 

ND - Neutron diffraction 

NDP - Neutron depth profiling 

NEXAFS - Near edge X-ray absorption fine structure 

NIS - Nuclear inelastic scattering/absorption 

NMR - Nuclear magnetic resonance spectroscopy 

NOESY - Nuclear Overhauser effect spectroscopy 

NRA - Nuclear reaction analysis 

NSOM - Near-field optical microscopy 



o 



OBIC - Optical beam induced current 

ODNMR - Optically detected magnetic resonance, see ESR or EPR 

OES - Optical emission spectroscopy 

Osmometry - Osmometry 



PAS - Positron annihilation spectroscopy 

Photoacoustic spectroscopy - Photoacoustic spectroscopy 

PAT or PACT - Photoacoustic tomography or photoacoustic computed tomography 

PAX - Photoemission of adsorbed xenon 

PC or PCS - Photocurrent spectroscopy 

Phase contrast microscopy - Phase contrast microscopy 

PhD - Photoelectron diffraction 

PD - Photodesorption 

PDEIS - Potentiodynamic electrochemical impedance spectroscopy 

PDS - Photothermal deflection spectroscopy 

PED - Photoelectron diffraction 

PEELS - parallel electron energy loss spectroscopy 



Acronyms in microscopy 314 

PES - Photoelectron spectroscopy 

PINEM - photon-induced near-field electron microscopy 

PIGE - Particle (or proton) induced gamma-ray spectroscopy, see Nuclear reaction analysis 

PIXE - Particle (or proton) induced X-ray spectroscopy 

PL - Photoluminescence 

Porosimetry - Porosimetry 

Powder diffraction - Powder diffraction 

PTMS - Photothermal microspectroscopy 

PTS - Photothermal spectroscopy 

Q 

• QENS - Quasi-elastic neutron scattering 



R 



Raman - Raman spectroscopy 

RAXRS - Resonant anomalous X-ray scattering 

RBS - Rutherford backscattering spectrometry 

REM - Reflection electron microscopy 

RDS - Reflectance Difference Spectroscopy 

RHEED - Reflection high energy electron diffraction 

RIXS - Resonant inelastic X-ray scattering 

RR spectroscopy - Resonance Raman spectroscopy 



SAD - Selected area diffraction 

SAED - Selected area electron diffraction 

SAM - Scanning Auger microscopy 

SANS - Small angle neutron scattering 

SAXS - Small angle X-ray scattering 

SCANIIR - Surface composition by analysis of neutral species and ion-impact radiation 

SCEM - Scanning confocal electron microscopy 

SE - Spectroscopic ellipsometry 

SEC - Size exclusion chromatography 

SEIRA - Surface enhanced infrared absorption spectroscopy 

SEM - Scanning electron microscopy 

SERS - Surface enhanced Raman spectroscopy 

SERRS - Surface enhanced resonance Raman spectroscopy 

SEXAFS - Surface extended X-ray absorption fine structure 

SICM - Scanning ion-conductance microscopy 

SIL - Solid immersion lens 

SIM - Solid immersion mirror 

SIMS - Secondary ion mass spectrometry 

SNMS - Sputtered neutral species mass spectroscopy 

SNOM - Scanning near-field optical microscopy 

SPECT - Single photon emission computed tomography 

SPM - Scanning probe microscopy 



Acronyms in microscopy 315 

SRM-CE/MS - Selected-reaction-monitoring capillary-electrophoresis mass-spectrometry 

SSNMR - Solid-state nuclear magnetic resonance 

Stark spectroscopy - Stark spectroscopy 

STED - Stimulated Emission Depletion microscopy 

STEM - Scanning transmission electron microscopy 

STM - Scanning tunneling microscopy 

STS - Scanning tunneling spectroscopy 

SXRD - Surface X-ray Diffraction (SXRD) 



TAT or TACT - Thermoacoustic tomography or thermoacoustic computed tomography (see also photoacoustic 

tomography - PAT) 

TEM - transmission electron microscope/microscopy 

TGA - Thermogravimetric analysis 

TIKA - Transmitting ion kinetic analysis 

TIRFM - Total internal reflection fluorescence microscopy 

TLS - Photothermal lens spectroscopy, a type of Photothermal spectroscopy 

TMA - Thermomechanical analysis 

TOF-MS - Time-of- flight mass spectrometry 

Two-photon excitation microscopy - Two-photon excitation microscopy 

TXRF - Total reflection X-ray fluorescence analysis 



u 

• Ultrasound attenuation spectroscopy - Ultrasound attenuation spectroscopy 

• Ultrasonic testing - Ultrasonic testing 

• UPS - UV-photoelectron spectroscopy 



VEDIC - Video-enhanced differential interference contrast microscopy 
Voltammetry - Voltammetry 



w 



WAXS - Wide angle X-ray scattering 

WDX or WDS - Wavelength dispersive X-ray spectroscopy 



XAES - X-ray induced Auger electron spectroscopy 

XANES - XANES, synonymous with NEXAFS (Near edge X-ray absorption fine structure) 

XAS - X-ray absorption spectroscopy 

X-CTR - X-ray crystal truncation rod scattering 

X-ray crystallography - X-ray crystallography 

XDS - X-ray diffuse scattering 

XPEEM - X-ray photoelectron emission microscopy 

XPS - X-ray photoelectron spectroscopy 

XRD - X-ray diffraction 



Acronyms in microscopy 



316 



XRES - X-ray resonant exchange scattering 

XRF - X-ray fluorescence analysis 

XRR - X-ray reflectivity 

XRS - X-ray Raman scattering 

XSW - X-ray standing wave technique 



References 

• Callister, WD (2000). Materials Science and Engineering - An Introduction. John Wiley and Sons : London. 
ISBN 0-471-32013-7. 

• Yao, N, ed (2007). Focused Ion Beam Systems: Basics and Applications. Cambridge University Press : 
Cambridge, UK. ISBN 978-052183-1994. 



Nanoscience 



Part of a series of articles on 



History 

Implications 

Applications 

Regulation 

Organizations 

Popular culture 

List of topics 




Fullerene 
Carbon Nanotubes 
Nanoparticles 



Nanomedicine 



Nanotoxicology 
Nanosensor 



Molecular self-assembly 



Self-assembled monolayer 
Supramolecular assembly 
DNA nanotechnology 



Nanoelectronics 



Molecular electronics 
Nanolithography 



Scanning probe microscopy 



Atomic force microscope 
Scanning tunneling microscope 



Molecular nanotechnology 



Molecular assembler 

Nanorobotics 

Mechanosynthesis 



Nanotechnology Portal 



Nanoscience 



317 



Nanotechnology, shortened to "nanotech", is the study of manipulating matter on an atomic and molecular scale. 
Generally nanotechnology deals with structures sized between 1 to 100 nanometer in at least one dimension, and 
involves developing materials or devices within that size. Quantum mechanical effects are very important at this 
scale. 

Nanotechnology is very diverse, ranging from extensions of conventional device physics to completely new 
approaches based upon molecular self-assembly, from developing new materials with dimensions on the nanoscale 
to investigating whether we can directly control matter on the atomic scale. 

There is much debate on the future implications of nanotechnology. Nanotechnology may be able to create many 
new materials and devices with a vast range of applications, such as in medicine, electronics, biomaterials and 
energy production. On the other hand, nanotechnology raises many of the same issues as any new technology, 
including concerns about the toxicity and environmental impact of nanomaterials, and their potential effects on 
global economics, as well as speculation about various doomsday scenarios. These concerns have led to a debate 
among advocacy groups and governments on whether special regulation of nanotechnology is warranted. 




Origins 

The first use of the concepts found in 'nano-technology' (but pre-dating 
use of that name) was in "There's Plenty of Room at the Bottom", a talk 
given by physicist Richard Feynman at an American Physical Society 
meeting at Caltech on December 29, 1959. Feynman described a 
process by which the ability to manipulate individual atoms and 
molecules might be developed, using one set of precise tools to build 
and operate another proportionally smaller set, and so on down to the 
needed scale. In the course of this, he noted, scaling issues would arise 
from the changing magnitude of various physical phenomena: gravity 
would become less important, surface tension and van der Waals 
attraction would become increasingly more significant, etc. This basic 
idea appeared plausible, and exponential assembly enhances it with 
parallelism to produce a useful quantity of end products. The term 
"nanotechnology" was defined by Tokyo Science University Professor 
Norio Taniguchi in a 1974 paper as follows: "'Nano-technology' 
mainly consists of the processing of, separation, consolidation, and 
deformation of materials by one atom or by one molecule." In the 1980s 
the basic idea of this definition was explored in much more depth by Dr. K. Eric Drexler, who promoted the 
technological significance of nano-scale phenomena and devices through speeches and the books Engines of 
Creation: The Coming Era of Nanotechnology (1986) and Nanosystems: Molecular Machinery, Manufacturing, and 
Computation, and so the term acquired its current sense. Engines of Creation: The Coming Era of Nanotechnology 
is considered the first book on the topic of nanotechnology. Nanotechnology and nanoscience got started in the early 
1980s with two major developments; the birth of cluster science and the invention of the scanning tunneling 
microscope (STM). This development led to the discovery of fullerenes in 1985 and carbon nanotubes a few years 
later. In another development, the synthesis and properties of semiconductor nanocrystals was studied; this led to a 
fast increasing number of metal and metal oxide nanoparticles and quantum dots. The atomic force microscope 
(AFM or SFM) was invented six years after the STM was invented. In 2000, the United States National 
Nanotechnology Initiative was founded to coordinate Federal nanotechnology research and development and is 
evaluated by the President's Council of Advisors on Science and Technology. 



Buckminsterfullerene C , also known as the 

60 

buckyball, is a representative member of the 

carbon structures known as fullerenes. Members 

of the fullerene family are a major subject of 

research falling under the nanotechnology 

umbrella. 



Nanoscience 



318 



Fundamental concepts 



,-9 



One nanometer (nm) is one billionth, or 10~ , of a meter. By comparison, typical carbon-carbon bond lengths, or the 
spacing between these atoms in a molecule, are in the range 0.12—0.15 nm, and a DNA double-helix has a diameter 
around 2 nm. On the other hand, the smallest cellular life-forms, the bacteria of the genus Mycoplasma, are around 
200 nm in length. 

To put that scale in another context, the comparative size of a nanometer to a meter is the same as that of a marble to 

Ml 

the size of the earth. Or another way of putting it: a nanometer is the amount an average man's beard grows in the 



time it takes him to raise the razor to his face 



[4] 



Two main approaches are used in nanotechnology. In the "bottom-up" approach, materials and devices are built from 
molecular components which assemble themselves chemically by principles of molecular recognition. In the 
"top-down" approach, nano-objects are constructed from larger entities without atomic-level control. 

Areas of physics such as nanoelectronics, nanomechanics, nanophotonics and nanoionics have evolved during the 
last few decades to provide a basic scientific foundation of nanotechnology. 



Larger to smaller: a materials perspective 

A number of physical phenomena become pronounced as the size of 
the system decreases. These include statistical mechanical effects, as 
well as quantum mechanical effects, for example the "quantum size 
effect" where the electronic properties of solids are altered with great 
reductions in particle size. This effect does not come into play by going 
from macro to micro dimensions. However, quantum effects become 
dominant when the nanometer size range is reached, typically at 
distances of 100 nanometers or less, the so called quantum realm. 
Additionally, a number of physical (mechanical, electrical, optical, 
etc.) properties change when compared to macroscopic systems. One 
example is the increase in surface area to volume ratio altering 
mechanical, thermal and catalytic properties of materials. Diffusion 
and reactions at nanoscale, nanostructures materials and nanodevices 
with fast ion transport are generally referred to nanoionics. Mechanical 
properties of nanosystems are of interest in the nanomechanics 
research. The catalytic activity of nanomaterials also opens potential risks in their interaction with biomaterials 




Image of reconstruction on a clean Gold(100) 

surface, as visualized using scanning tunneling 

microscopy. The positions of the individual 

atoms composing the surface are visible. 



[6] 



Materials reduced to the nanoscale can show different properties compared to what they exhibit on a macroscale, 
enabling unique applications. For instance, opaque substances become transparent (copper); stable materials turn 
combustible (aluminum); insoluble materials become soluble (gold). A material such as gold, which is chemically 
inert at normal scales, can serve as a potent chemical catalyst at nanoscales. Much of the fascination with 



nanotechnology stems from these quantum and surface phenomena that matter exhibits at the nanoscale 



[7] 



Simple to complex: a molecular perspective 

Modern synthetic chemistry has reached the point where it is possible to prepare small molecules to almost any 
structure. These methods are used today to manufacture a wide variety of useful chemicals such as pharmaceuticals 
or commercial polymers. This ability raises the question of extending this kind of control to the next-larger level, 
seeking methods to assemble these single molecules into supramolecular assemblies consisting of many molecules 
arranged in a well defined manner. 

These approaches utilize the concepts of molecular self-assembly and/or supramolecular chemistry to automatically 
arrange themselves into some useful conformation through a bottom-up approach. The concept of molecular 



Nanoscience 319 

recognition is especially important: molecules can be designed so that a specific configuration or arrangement is 
favored due to non-covalent intermolecular forces. The Watson— Crick basepairing rules are a direct result of this, as 
is the specificity of an enzyme being targeted to a single substrate, or the specific folding of the protein itself. Thus, 
two or more components can be designed to be complementary and mutually attractive so that they make a more 
complex and useful whole. 

Such bottom-up approaches should be capable of producing devices in parallel and be much cheaper than top-down 
methods, but could potentially be overwhelmed as the size and complexity of the desired assembly increases. Most 
useful structures require complex and thermodynamically unlikely arrangements of atoms. Nevertheless, there are 
many examples of self-assembly based on molecular recognition in biology, most notably Watson— Crick basepairing 
and enzyme-substrate interactions. The challenge for nanotechnology is whether these principles can be used to 
engineer new constructs in addition to natural ones. 

Molecular nanotechnology: a long-term view 

Molecular nanotechnology, sometimes called molecular manufacturing, describes engineered nanosystems 
(nanoscale machines) operating on the molecular scale. Molecular nanotechnology is especially associated with the 
molecular assembler, a machine that can produce a desired structure or device atom-by-atom using the principles of 
mechanosynthesis. Manufacturing in the context of productive nanosystems is not related to, and should be clearly 
distinguished from, the conventional technologies used to manufacture nanomaterials such as carbon nanotubes and 
nanoparticles. 

When the term "nanotechnology" was independently coined and popularized by Eric Drexler (who at the time was 
unaware of an earlier usage by Norio Taniguchi) it referred to a future manufacturing technology based on molecular 
machine systems. The premise was that molecular scale biological analogies of traditional machine components 
demonstrated molecular machines were possible: by the countless examples found in biology, it is known that 
sophisticated, stochastically optimised biological machines can be produced. 

It is hoped that developments in nanotechnology will make possible their construction by some other means, perhaps 

ro] 

using biomimetic principles. However, Drexler and other researchers have proposed that advanced 
nanotechnology, although perhaps initially implemented by biomimetic means, ultimately could be based on 
mechanical engineering principles, namely, a manufacturing technology based on the mechanical functionality of 
these components (such as gears, bearings, motors, and structural members) that would enable programmable, 
positional assembly to atomic specification. The physics and engineering performance of exemplar designs were 
analyzed in Drexler's book Nanosystems. 

In general it is very difficult to assemble devices on the atomic scale, as all one has to position atoms on other atoms 
of comparable size and stickiness. Another view, put forth by Carlo Montemagno, is that future nanosystems will 
be hybrids of silicon technology and biological molecular machines. Yet another view, put forward by the late 
Richard Smalley, is that mechanosynthesis is impossible due to the difficulties in mechanically manipulating 
individual molecules. 

This led to an exchange of letters in the ACS publication Chemical & Engineering News in 2003. Though biology 
clearly demonstrates that molecular machine systems are possible, non-biological molecular machines are today only 
in their infancy. Leaders in research on non-biological molecular machines are Dr. Alex Zettl and his colleagues at 

Lawrence Berkeley Laboratories and UC Berkeley. They have constructed at least three distinct molecular devices 

ri2i 
whose motion is controlled from the desktop with changing voltage: a nanotube nanomotor, a molecular actuator, 

ri3i 

and a nanoelectromechanical relaxation oscillator. 

An experiment indicating that positional molecular assembly is possible was performed by Ho and Lee at Cornell 
University in 1999. They used a scanning tunneling microscope to move an individual carbon monoxide molecule 
(CO) to an individual iron atom (Fe) sitting on a flat silver crystal, and chemically bound the CO to the Fe by 
applying a voltage. 



Nanoscience 



320 



Current research 



Nanomaterials 

The nanomaterials field includes subfields which develop or study 
materials having unique properties arising from their nanoscale 
dimensions. 

• Interface and colloid science has given rise to many materials which 
may be useful in nanotechnology, such as carbon nanotubes and 
other fullerenes, and various nanoparticles and nanorods. 
Nanomaterials with fast ion transport are related also to nanoionics 
and nanoelectronics. 

• Nanoscale materials can also be used for bulk applications; most 
present commercial applications of nanotechnology are of this 
flavor. 

• Progress has been made in using these materials for medical 
applications; see Nanomedicine. 

• Nanoscale materials are sometimes used in solar cells which 
combats the cost of traditional Silicon solar cells 

• Development of applications incorporating semiconductor 
nanoparticles to be used in the next generation of products, such as 
display technology, lighting, solar cells and biological imaging; see 
quantum dots. 




Macrocycle 

Dumbbell shaped molecule 

Graphical representation of a rotaxane, useful as a 
molecular switch. 




Sarfus image of a DNA biochip elaborated by 
bottom-up approach. 



Bottom-up approaches 

These seek to arrange smaller components into more complex 
assemblies. 

• DNA nanotechnology utilizes the specificity of Watson— Crick 
basepairing to construct well-defined structures out of DNA and 
other nucleic acids. 

• Approaches from the field of "classical" chemical synthesis also aim 
at designing molecules with well-defined shape (e.g. bis-peptides 

)■ 

• More generally, molecular self-assembly seeks to use concepts of 
supramolecular chemistry, and molecular recognition in particular, 
to cause single-molecule components to automatically arrange 
themselves into some useful conformation. 

• Atomic force microscope tips can be used as a nanoscale "write 
head" to deposit a chemical upon a surface in a desired pattern in a 
process called dip pen nanolithography. This technique fits into the 
larger subfield of nanolithography. 




This device transfers energy from nano-thin 
layers of quantum wells to nanocrystals above 
them, causing the nanocrystals to emit visible 
light. 



Top-down approaches 



Nanoscience 321 

These seek to create smaller devices by using larger ones to direct their assembly. 

• Many technologies that descended from conventional solid-state silicon methods for fabricating microprocessors 

are now capable of creating features smaller than 100 nm, falling under the definition of nano technology. Giant 

ri7i 
magnetoresistance-based hard drives already on the market fit this description, as do atomic layer deposition 

(ALD) techniques. Peter Griinberg and Albert Fert received the Nobel Prize in Physics in 2007 for their discovery 

n si 
of Giant magnetoresistance and contributions to the field of spintronics. 

• Solid-state techniques can also be used to create devices known as nanoelectromechanical systems or NEMS, 
which are related to microelectromechanical systems or MEMS. 

• Focused ion beams can directly remove material, or even deposit material when suitable pre-cursor gasses are 
applied at the same time. For example, this technique is used routinely to create sub-100 nm sections of material 
for analysis in Transmission electron microscopy. 

• Atomic force microscope tips can be used as a nanoscale "write head" to deposit a resist, which is then followed 
by a etching process to remove material in a top-down method. 

Functional approaches 

These seek to develop components of a desired functionality without regard to how they might be assembled. 

• Molecular electronics seeks to develop molecules with useful electronic properties. These could then be used as 
single-molecule components in a nanoelectronic device. For an example see rotaxane. 

• Synthetic chemical methods can also be used to create synthetic molecular motors, such as in a so-called nanocar. 

Biomimetic approaches 

• Bionics or biomimicry seeks to apply biological methods and systems found in nature, to the study and design of 
engineering systems and modern technology. Biomineralization is one example of the systems studied. 

• Bionano technology the use of biomolecules for applications in nanotechnology, including use of viruses. 

Speculative 

These subfields seek to anticipate what inventions nanotechnology might yield, or attempt to propose an agenda 
along which inquiry might progress. These often take a big-picture view of nanotechnology, with more emphasis on 
its societal implications than the details of how such inventions could actually be created. 

• Molecular nanotechnology is a proposed approach which involves manipulating single molecules in finely 
controlled, deterministic ways. This is more theoretical than the other subfields and is beyond current capabilities. 

• Nanorobotics centers on self-sufficient machines of some functionality operating at the nanoscale. There are 

[21] [221 T231 

hopes for applying nanorobots in medicine, but it may not be easy to do such a thing because of several 

T241 
drawbacks of such devices. Nevertheless, progress on innovative materials and methodologies has been 

demonstrated with some patents granted about new nanomanufacturing devices for future commercial 

applications, which also progressively helps in the development towards nanorobots with the use of embedded 

nanobioelectronics concepts. 

• Productive nanosystems are "systems of nanosystems" which will be complex nanosystems that produce 
atomically precise parts for other nanosystems, not necessarily using novel nanoscale-emergent properties, but 
well-understood fundamentals of manufacturing. Because of the discrete (i.e. atomic) nature of matter and the 
possibility of exponential growth, this stage is seen as the basis of another industrial revolution. Mihail Roco, one 
of the architects of the USA's National Nanotechnology Initiative, has proposed four states of nanotechnology that 

seem to parallel the technical progress of the Industrial Revolution, progressing from passive nanostructures to 

T271 
active nanodevices to complex nanomachines and ultimately to productive nanosystems. 

• Programmable matter seeks to design materials whose properties can be easily, reversibly and externally 
controlled though a fusion of information science and materials science. 



Nanoscience 



322 



• Due to the popularity and media exposure of the term nanotechnology, the words picotechnology and 
femtotechnology have been coined in analogy to it, although these are only used rarely and informally. 

Tools and techniques 



4 quadrant 
photo detector 



There are several important modern 

developments. The atomic force microscope 

(AFM) and the Scanning Tunneling 

Microscope (STM) are two early versions of 

scanning probes that launched 

nanotechnology. There are other types of 

scanning probe microscopy, all flowing from 

the ideas of the scanning confocal 

microscope developed by Marvin Minsky in 

1961 and the scanning acoustic microscope 

(SAM) developed by Calvin Quate and 

coworkers in the 1970s, that made it possible 

to see structures at the nanoscale. The tip of a 

scanning probe can also be used to 

manipulate nanostructures (a process called 

positional assembly). Feature-oriented 

scanning-positioning methodology suggested 

by Rostislav Lapshin appears to be a 

promising way to implement these 

nanomanipulations in automatic mode. 

However, this is still a slow process because of low scanning velocity of the microscope. Various techniques of 

nanolithography such as optical lithography, X-ray lithography dip pen nanolithography, electron beam lithography 

or nanoimprint lithography were also developed. Lithography is a top-down fabrication technique where a bulk 

material is reduced in size to nanoscale pattern. 




Typical AFM setup. A microfabricated cantilever with a sharp tip is deflected by 
features on a sample surface, much like in a phonograph but on a much smaller 

scale. A laser beam reflects off the backside of the cantilever into a set of 

photodetectors, allowing the deflection to be measured and assembled into an 

image of the surface. 



Another group of nanotechnological techniques include those used for fabrication of nanowires, those used in 
semiconductor fabrication such as deep ultraviolet lithography, electron beam lithography, focused ion beam 
machining, nanoimprint lithography, atomic layer deposition, and molecular vapor deposition, and further including 
molecular self-assembly techniques such as those employing di-block copolymers. However, all of these techniques 
preceded the nanotech era, and are extensions in the development of scientific advancements rather than techniques 
which were devised with the sole purpose of creating nanotechnology and which were results of nanotechnology 
research. 

The top-down approach anticipates nanodevices that must be built piece by piece in stages, much as manufactured 
items are made. Scanning probe microscopy is an important technique both for characterization and synthesis of 
nanomaterials. Atomic force microscopes and scanning tunneling microscopes can be used to look at surfaces and to 
move atoms around. By designing different tips for these microscopes, they can be used for carving out structures on 
surfaces and to help guide self-assembling structures. By using, for example, feature-oriented scanning-positioning 
approach, atoms can be moved around on a surface with scanning probe microscopy techniques. At present, it is 
expensive and time-consuming for mass production but very suitable for laboratory experimentation. 

In contrast, bottom-up techniques build or grow larger structures atom by atom or molecule by molecule. These 
techniques include chemical synthesis, self-assembly and positional assembly. Dual polarisation interferometry is 
one tool suitable for characterisation of self assembled thin films. Another variation of the bottom-up approach is 



Nanoscience 



323 



molecular beam epitaxy or MBE. Researchers at Bell Telephone Laboratories like John R. Arthur. Alfred Y. Cho, 
and Art C. Gossard developed and implemented MBE as a research tool in the late 1960s and 1970s. Samples made 
by MBE were key to the discovery of the fractional quantum Hall effect for which the 1998 Nobel Prize in Physics 
was awarded. MBE allows scientists to lay down atomically precise layers of atoms and, in the process, build up 
complex structures. Important for research on semiconductors, MBE is also widely used to make samples and 
devices for the newly emerging field of spintronics. 

However, new therapeutic products, based on responsive nanomaterials, such as the ultradeformable, stress-sensitive 
Transfersome vesicles, are under development and already approved for human use in some countries. 



Result: Id-Vg Characteristics 



~| Result: |3D electron density for Vd-0.6 



Applications 

As of August 21, 2008, the Project on Emerging Nanotechnologies estimates that over 800 manufacturer-identified 

nanotech products are publicly available, with new ones hitting the market at a pace of 3—4 per week. The project 

[29] 
lists all of the products in a publicly accessible online. Most applications are limited to the use of "first 

generation" passive nanomaterials which includes titanium dioxide in sunscreen, cosmetics and some food products; 

Carbon allotropes used to produce gecko tape; silver in food packaging, clothing, disinfectants and household 

appliances; zinc oxide in sunscreens and cosmetics, surface coatings, paints and outdoor furniture varnishes; and 

cerium oxide as a fuel catalyst. 

The National Science Foundation (a 
major distributor for nanotechnology 
research in the United States) funded 
researcher David Berube to study the 
field of nanotechnology. His findings 
are published in the monograph 

Nano-Hype: The Truth Behind the 

1311 
Nanotechnology Buzz. This study 

concludes that much of what is sold as 

"nanotechnology" is in fact a recasting 

of straightforward materials science, 

which is leading to a "nanotech 

industry built solely on selling 

nanotubes, nanowires, and the like" which will "end up with a few suppliers selling low margin products in huge 

volumes." Further applications which require actual manipulation or arrangement of nanoscale components await 

further research. Though technologies branded with the term 'nano' are sometimes little related to and fall far short of 

the most ambitious and transformative technological goals of the sort in molecular manufacturing proposals, the term 

still connotes such ideas. According to Berube, there may be a danger that a "nano bubble" will form, or is forming 

already, from the use of the term by scientists and entrepreneurs to garner funding, regardless of interest in the 

1321 
transformative possibilities of more ambitious and far-sighted work. 



1E-5 -m 




1E-6 -s 




1E-7 -^ 










1E-8-. 










lE-9-i 
















E-10-i 










E-11 -^ 










E-12 -= 










E-13 Z 








E-14 J 








VgM 



electron density for - 1-Vd=0.GV-Vg=OV 

■ D 



n o t - 



One of the major application of nanotechnology is in the area of nanoelectronics with 
MOSFET's being made of small nanowires -10 nm in length. Here is a simulation of such 

a nanowire 



Nanoscience 324 

Implications 

Because of the far-ranging claims that have been made about potential applications of nanotechnology, a number of 
serious concerns have been raised about what effects these will have on our society if realized, and what action if any 
is appropriate to mitigate these risks. 

There are possible dangers that arise with the development of nanotechnology. The Center for Responsible 
Nanotechnology suggests that new developments could result, among other things, in untraceable weapons of mass 
destruction, networked cameras for use by the government, and weapons developments fast enough to destabilize 
arms races ("Nanotechnology Basics"). 

One area of concern is the effect that industrial-scale manufacturing and use of nanomaterials would have on human 
health and the environment, as suggested by nanotoxicology research. Groups such as the Center for Responsible 
Nanotechnology have advocated that nanotechnology should be specially regulated by governments for these 
reasons. Others counter that overregulation would stifle scientific research and the development of innovations 
which could greatly benefit mankind. 

Other experts, including director of the Woodrow Wilson Center's Project on Emerging Nanotechnologies David 

[331 
Rejeski, have testified that successful commercialization depends on adequate oversight, risk research strategy, 

and public engagement. Berkeley, California is currently the only city in the United States to regulate 

[341 [351 

nanotechnology; Cambridge, Massachusetts in 2008 considered enacting a similar law, but ultimately rejected 
this.™ 

Health and environmental concerns 

Some of the recently developed nanoparticle products may have unintended consequences. Researchers have 

discovered that silver nanoparticles used in socks only to reduce foot odor are being released in the wash with 

[371 
possible negative consequences. Silver nanoparticles, which are bacteriostatic, may then destroy beneficial 

T381 

bacteria which are important for breaking down organic matter in waste treatment plants or farms. 

A study at the University of Rochester found that when rats breathed in nanoparticles, the particles settled in the 
brain and lungs, which led to significant increases in biomarkers for inflammation and stress response. A study in 
China indicated that nanoparticles induce skin aging through oxidative stress in hairless mice. 

A two-year study at UCLA's School of Public Health found lab mice consuming nano-titanium dioxide showed 
DNA and chromosome damage to a degree "linked to all the big killers of man, namely cancer, heart disease, 
neurological disease and aging". 

A major study published more recently in Nature Nanotechnology suggests some forms of carbon nanotubes — a 
poster child for the "nanotechnology revolution" — could be as harmful as asbestos if inhaled in sufficient quantities. 
Anthony Seaton of the Institute of Occupational Medicine in Edinburgh, Scotland, who contributed to the article on 
carbon nanotubes said "We know that some of them probably have the potential to cause mesothelioma. So those 
sorts of materials need to be handled very carefully." In the absence of specific nano-regulation forthcoming from 
governments, Paull and Lyons (2008) have called for an exclusion of engineered nanoparticles from organic food. 
A newspaper article reports that workers in a paint factory developed serious lung disease and nanoparticles were 
found in their lungs. 

Regulation 

Calls for tighter regulation of nanotechnology have occurred alongside a growing debate related to the human health 
and safety risks associated with nanotechnology. Furthermore, there is significant debate about who is responsible 
for the regulation of nanotechnology. While some non-nanotechnology specific regulatory agencies currently cover 
some products and processes (to varying degrees) — by "bolting on" nanotechnology to existing regulations — there 
are clear gaps in these regimes. In "Nanotechnology Oversight: An Agenda for the Next Administration," 



Nanoscience 325 

former EPA deputy administrator J. Clarence (Terry) Davies lays out a clear regulatory roadmap for the next 
presidential administration and describes the immediate and longer term steps necessary to deal with the current 
shortcomings of nanotechnology oversight. 

Stakeholders concerned by the lack of a regulatory framework to assess and control risks associated with the release 

of nanoparticles and nanotubes have drawn parallels with bovine spongiform encephalopathy ('mad cow's disease), 

[491 
thalidomide, genetically modified food, nuclear energy, reproductive technologies, biotechnology, and asbestosis. 

Dr. Andrew Maynard, chief science advisor to the Woodrow Wilson Center's Project on Emerging 

Nano technologies, concludes (among others) that there is insufficient funding for human health and safety rese