The scanning electron microscope (SEM) is a type ofelectron microscope that images the sample surface by scanning it with a high-energy beam of electrons in a raster scan pattern. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography, composition and other properties such as electrical conductivity. The types of signals produced by an SEM includesecondary electrons, back-scattered electrons (BSE),characteristic X-rays, light (cathodoluminescence), specimen current and transmitted electrons. Secondary electron detectors are common in all SEMs, but it is rare that a single machine would have detectors for all possible signals. The signals result from interactions of the electron beam with atoms at or near the surface of the sample. In the most common or standard detection mode, secondary electron imaging or SEI, the SEM can produce very high-resolution images of a sample surface, revealing details about less than 1 to 5 nm in size. Due to the very narrow electron beam, SEM micrographs have a large depth of fieldyielding a characteristic three-dimensional appearance useful for understanding the surface structure of a sample. This is exemplified by the micrograph of pollen shown to the right. A wide range of magnifications is possible, from about 10 times (about equivalent to that of a powerful hand-lens) to more than 500,000 times, about 250 times the magnification limit of the best light microscopes. Backscattered electrons (BSE) are beam electrons that are reflected from the sample by elastic scattering. BSE are often used in analytical SEM along with the spectra made from the characteristic X-rays. Because the intensity of the BSE signal is strongly related to the atomic number (Z) of the specimen, BSE images can provide information about the distribution of different elements in the sample. For the same reason, BSE imaging can image colloidal goldimmuno-labels of 5 or 10 nm diameter which would otherwise be difficult or impossible to detect in secondary electron images in biological specimens. Characteristic X-rays are emitted when the electron beam removes an inner shell electron from the sample, causing a higher energy electron to fill the shell and release energy. These characteristic X-rays are used to identify the composition and measure the abundance of elements in the sample.  
Contents
[hide]

 

1 History 2 Scanning process and image formation

 

2.1 Magnification

3 Sample preparation

 

3.1 Biological samples 3.2 Materials

             

3.3 ESEM

4 Detection of secondary electrons 5 Detection of backscattered electrons 6 Beam-injection analysis of semiconductors 7 Cathodoluminescence 8 X-ray microanalysis 9 Resolution of the SEM 10 Environmental SEM 11 3D in SEM 12 Gallery of SEM images 13 See also 14 Terminology 15 References 16 External links

    

16.1 General 16.2 History 16.3 Images

[edit]History The first SEM image was obtained by Max Knoll, who in 1935 obtained an image of silicon steelshowing electron channeling contrast.[1] Further pioneering work on the physical principles of the SEM and beam specimen interactions was performed by Manfred von Ardenne in 1937,[2][3] who produced a British patent[4] but never made a practical instrument. The SEM was further developed by Professor Sir Charles Oatley and his postgraduate student Gary Stewart and was first marketed in 1965 by the Cambridge Instrument Company as the "Stereoscan". The first instrument was delivered to DuPont. [edit]Scanning

process and image formation

  
Schematic diagram of an SEM.

In a typical SEM, an electron beam is thermionicallyemitted from an electron gun fitted with a tungsten filamentcathode. Tungsten is normally used in thermionic electron guns because it has the highest melting point and lowest vapour pressure of all metals, thereby allowing it to be heated for electron emission, and because of its low cost. Other types of electron emitters include lanthanum hexaboride (LaB6) cathodes, which can be used in a standard tungsten filament SEM if the vacuum system is upgraded and field emission guns (FEG), which may be of the cold-cathode type using tungsten single crystal emitters or the thermallyassisted Schottky type, using emitters of zirconium oxide. The electron beam, which typically has an energy ranging from 0.5 keV to 40 keV, is focused by one or two condenser lenses to a spot about 0.4 nm to 5 nm in diameter. The beam passes through pairs of scanning coils or pairs of deflector plates in the electron column, typically in the final lens, which deflect the beam in the x and y axes so that it scans in a raster fashion over a rectangular area of the sample surface. When the primary electron beam interacts with the sample, the electrons lose energy by repeated random scattering and absorption within a teardrop-shaped volume of the specimen known as the interaction volume, which extends from less than 100 nm to around 5 m into the surface. The size of the interaction volume depends on the electron's landing energy, the atomic number of the specimen and the specimen's density. The energy exchange between the electron beam and the sample results in the reflection of high-energy electrons by elastic scattering, emission of secondary electrons byinelastic scattering and the emission of electromagnetic radiation, each of which can be detected by specialized detectors. The beam current absorbed by the specimen can also be detected and used to create images of the distribution of specimen current. Electronic amplifiers of various types are used to amplify the signals which are displayed as variations in brightness on a cathode ray tube. The raster scanning of the CRT display is synchronised with that of the beam on the specimen in the microscope, and the resulting image is therefore a distribution map of the intensity of the signal being emitted from the scanned area of the specimen. The image may be captured by photography from a high resolution cathode ray tube, but in modern machines is digitally captured and displayed on acomputer monitor and saved to a computer's hard disk.

   
Low-temperature SEM magnification series for a snowcrystal. The crystals are captured, stored, and sputter coated with platinum at cryo-temperatures for imaging.

[edit]Magnification

   
An SEM micrograph of a house fly compound eye surface at 450 magnification.

Magnification in a SEM can be controlled over a range of up to 6 orders of magnitude from about 10 to 500,000 times. Unlike optical and transmission electron microscopes, image magnification in the SEM is not a function of the power of theobjective lens. SEMs may have condenser and objective lenses, but their function is to focus the beam to a spot, and not to image the specimen. Provided the electron gun can generate a beam with sufficiently small diameter, a SEM could in principle work entirely without condenser or objective lenses, although it might not be very versatile or achieve very high resolution. In a SEM, as in scanning probe microscopy, magnification results from the ratio of the dimensions of the raster on the specimen and the raster on the display device. Assuming that the display screen has a fixed size, higher magnification results from reducing the size of the raster on the specimen, and vice versa. Magnification is therefore controlled by the current supplied to the x, y scanning coils, or the voltage supplied to the x, y deflector plates, and not by objective lens power. [edit]Sample

preparation

   
A spider coated in gold, having been prepared for viewing with a scanning electron microscope.

All samples must also be of an appropriate size to fit in the specimen chamber and are generally mounted rigidly on a specimen holder called a specimen stub. Several models of SEM can examine any part of a 6-inch (15 cm) semiconductor wafer, and some can tilt an object of that size to 45. For conventional imaging in the SEM, specimens must be electrically conductive, at least at the surface, and electrically grounded to prevent the accumulation ofelectrostatic charge at the

surface. Metal objects require little special preparation for SEM except for cleaning and mounting on a specimen stub. Nonconductive specimens tend to charge when scanned by the electron beam, and especially in secondary electron imaging mode, this causes scanning faults and other image artifacts. They are therefore usually coated with an ultrathin coating of electricallyconducting material, commonly gold, deposited on the sample either by low vacuumsputter coating or by high vacuum evaporation. Conductive materials in current use for specimen coating include gold, gold/palladium alloy, platinum, osmium, iridium, tungsten, chromium andgraphite. Coating prevents the accumulation of static electric charge on the specimen during electron irradiation.  Two reasons for coating, even when there is enough specimen conductivity to prevent charging, are to increase signal and surface resolution, especially with samples of low atomic number (Z). The improvement in resolution arises because backscattering and secondary electron emission near the surface are enhanced and thus an image of the surface is formed.  An alternative to coating for some biological samples is to increase the bulk conductivity of the material by impregnation with osmium using variants of the OTO staining method (O-osmium, Tthiocarbohydrazide, O-osmium).[6][7] Nonconducting specimens may be imaged uncoated using specialized SEM instrumentation such as the "Environmental SEM" (ESEM) or field emission gun (FEG) SEMs operated at low voltage. Environmental SEM instruments place the specimen in a relatively high pressure chamber where the working distance is short and the electron optical column is differentially pumped to keep vacuum adequately low at the electron gun. The high pressure region around the sample in the ESEM neutralizes charge and provides an amplification of the secondary electron signal. Low voltage SEM of non-conducting specimens can be operationally difficult to accomplish in a conventional SEM and is typically a research application for specimens that are sensitive to the process of applying conductive coatings. Low-voltage SEM is typically conducted in an FEG-SEM because the FEG is capable of producing high primary electron brightness even at low accelerating potentials. Operating conditions must be adjusted such that the local space charge is at or near neutral with adequate low voltage secondary electrons being available to neutralize any positively charged surface sites. This requires that the primary electron beam's potential and current be tuned to the characteristics of the sample specimen.  Embedding in a resin with further polishing to a mirror-like finish can be used for both biological and materials specimens when imaging in backscattered electrons or when doing quantitative X  ray microanalysis. [edit]Biological
[5]

samples

For SEM, a specimen is normally required to be completely dry, since the specimen chamber is at high vacuum. Hard, dry materials such as wood, bone, feathers, dried insects or shells can be

examined with little further treatment, but living cells and tissues and whole, soft-bodied organisms usually require chemical fixation to preserve and stabilize their structure. Fixation is usually performed by incubation in a solution of a buffered chemical fixative, such as glutaraldehyde, sometimes in combination with formaldehyde[8][9][10] and other fixatives, optionally followed by postfixation with osmium tetroxide.
[8] [11]

and

The fixed tissue is then dehydrated.

Because air-drying causes collapse and shrinkage, this is commonly achieved by critical point drying, which involves replacement of water in the cells with organic solvents such as ethanol or acetone, and replacement of these solvents in turn with a transitional fluid such as liquid carbon dioxide at high pressure. The carbon dioxide is finally removed while in a supercritical state, so that no gas-liquid interface is present within the sample during drying. The dry specimen is usually mounted on a specimen stub using an adhesive such as epoxy resin or electrically-conductive double-sided adhesive tape, and sputter coated with gold or gold/palladium alloy before examination in the microscope.  If the SEM is equipped with a cold stage for cryo-microscopy, cryofixation may be used and lowtemperature scanning electron microscopy performed on the cryogenically fixed specimens.[8] Cryo-fixed specimens may be cryo-fractured under vacuum in a special apparatus to reveal internal structure, sputter coated and transferred onto the SEM cryo-stage while still frozen.[12] Low-temperature scanning electron microscopy is also applicable to the imaging of temperature-sensitive materials such as ice[13][14] (see e.g. illustration at right) and fats.[15]  Freeze-fracturing, freeze-etch or freeze-and-break is a preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view. The preparation method reveals the proteins embedded in the lipid bilayer.  Gold has a high atomic number and sputter coating with gold produces high topographic contrast and resolution. However, the coating has a thickness of a few nanometers, and can obscure the underlying fine detail of the specimen at very high magnification. Low-vacuum SEMs with differential pumping apertures allow samples to be imaged without such coating and without the loss of natural contrast caused by the coating, but are unable to achieve the resolution attainable   by conventional SEMs with coated specimens.[16] [edit]Materials Back scattered electron imaging, quantitative X-ray analysis, and X-ray mapping of geological specimens and metals requires that the surfaces be ground and polished to an ultra smooth surface. Geological specimens that undergo WDS or EDS analysis are often carbon coated. Metals are not generally coated prior to imaging in the SEM because they are conductive and provide their own pathway to ground.

Fractography is the study of fractured surfaces that can be done on a light microscope or commonly, on an SEM. The fractured surface is cut to a suitable size, cleaned of any organic residues, and mounted on a specimen holder for viewing in the SEM. Integrated circuits may be cut with a focused ion beam (FIB) or other ion beam milling instrument for viewing in the SEM. The SEM in the first case may be incorporated into the FIB. Metals, geological specimens, and integrated circuits all may also be chemically polished for viewing in the SEM. Special high resolution coating techniques are required for high magnification imaging of inorganic thin films. [edit]ESEM The accumulation of electric charge on the surfaces of non-metallic specimens can be avoided by using environmental SEM in which the specimen is placed in an internal chamber at higher pressure, rather than the vacuum in the electron optical column. Positively charged ions generated by beam interactions with the gas help to neutralize the negative charge on the specimen surface. The pressure of gas in the chamber can be controlled, and the type of gas used can be varied according to need. Coating is thus unnecessary, and X-ray analysis unhindered. [edit]Detection

  

 

of secondary electrons

The most common imaging mode collects low-energy (<50 eV) secondary electrons that are ejected from the k-orbitals of the specimen atoms by inelastic scattering interactions with beam electrons. Due to their low energy, these electrons originate within a few nanometers from the sample surface.[17]The electrons are detected by an Everhart-Thornley detector
[18]

which is a type

ofscintillator-photomultiplier system. The secondary electrons are first collected by attracting them towards an electrically-biased grid at about +400 V, and then further accelerated towards a phosphor or scintillator positively biased to about +2,000 V. The accelerated secondary electrons are now sufficiently energetic to cause the scintillator to emit flashes of light (cathodoluminescence) which are conducted to a photomultiplier outside the SEM column via a light pipe and a window in the wall of the specimen chamber. The amplified electrical signal output by the photomultiplier is displayed as a two-dimensional intensity distribution that can be viewed and photographed on an analogue video display, or subjected to analog-to-digital conversion and displayed and saved as a digital image. This process relies on a raster-scanned primary beam. The brightness of the signal depends on the number of secondary electrons reaching the detector. If the beam enters the sample perpendicular to the surface, then the activated region is uniform about the axis of the beam and a certain number of electrons "escape" from within the sample. As the angle of incidence increases, the "escape" distance of one side of the beam will decrease, and more secondary electrons will be emitted.

Thus steep surfaces and edges tend to be brighter than flat surfaces, which results in images with a well-defined, three-dimensional appearance. Using this technique, image resolution less than 0.5 nm is possible.  [edit]Detection

of backscattered electrons

  
Comparison of SEM techniques: Top: backscattered electron analysis - composition Bottom: secondary electron analysis - topography

Backscattered electrons (BSE) consist of high-energy electrons originating in the electron beam, that are reflected or back-scattered out of the specimen interaction volume by elastic scattering interactions with specimen atoms. Since heavy elements (high atomic number) backscatter electrons more strongly than light elements (low atomic number), and thus appear brighter in the image, BSE are used to detect contrast between areas with different chemical compositions.[17] The Everhart-Thornley detector, which is normally positioned to one side of the specimen, is inefficient for the detection of backscattered electrons because few such electrons are emitted in the solid angle subtended by the detector, and because the positively biased detection grid has little ability to attract the higher energy BSE electrons. Dedicated backscattered electron detectors are positioned above the sample in a "doughnut" type arrangement, concentric with the electron beam, maximising the solid angle of collection. BSE detectors are usually either of scintillator or semiconductor types. When all parts of the detector are used to collect electrons symmetrically about the beam, atomic number contrast is produced. However, strong topographic contrast is produced by collecting back-scattered electrons from one side above the specimen using an asymmetrical, directional BSE detector; the resulting contrast appears as illumination of the topography from that side. Semiconductor detectors can be made in radial segments that can be switched in or out to control the type of contrast produced and its directionality. Backscattered electrons can also be used to form an electron backscatter diffraction (EBSD) image that can be used to determine the crystallographic structure of the specimen. [edit]Beam-injection

 

analysis of semiconductors

The nature of the SEM's probe, energetic electrons, makes it uniquely suited to examining the optical and electronic properties of semiconductor materials. The high-energy electrons from the SEM beam will inject charge carriers into the semiconductor. Thus, beam electrons lose energy by promoting electrons from the valence band into the conduction band, leaving behind holes. In a direct bandgap material, recombination of these electron-hole pairs will result incathodoluminescence; if the sample contains an internal electric field, such as is present at a pn junction, the SEM beam injection of carriers will cause electron beam induced current (EBIC) to flow. Cathodoluminescence and EBIC are referred to as "beam-injection" techniques, and are very powerful probes of the optoelectronic behavior of semiconductors, particularly for studying nanoscale features and defects. [edit]Cathodoluminescence

 

Cathodoluminescence, the emission of light when atoms excited by high-energy electrons return to their ground state, is analogous to UV-induced fluorescence, and some materials such as zinc sulfide and some fluorescent dyes, exhibit both phenomena. Cathodoluminescence is most commonly experienced in everyday life as the light emission from the inner surface of the cathode ray tube in television sets and computer CRT monitors. In the SEM, CL detectors either collect all light emitted by the specimen, or can analyse the wavelengths emitted by the specimen and display an emissionspectrum or an image of the distribution of cathodoluminescence emitted by the specimen in real colour. [edit]X-ray

 

microanalysis

X-rays, which are also produced by the interaction of electrons with the sample, may also be detected in an SEM equipped for energy-dispersive X-ray spectroscopy or wavelength dispersive X-ray spectroscopy. [edit]Resolution

of the SEM

   
A video illustrating a typical practical magnification range of a scanning electron microscope designed for biological specimens. The video starts at 25x, about 6 mm across the whole field of view, and zooms in to 12000x, about

12 macross the whole field of view. The spherical objects are glass beads with a diameter of 10 m, similar in diameter to ared blood cell.

The spatial resolution of the SEM depends on the size of the electron spot, which in turn depends on both the wavelength of the electrons and the electron-optical system which produces the scanning beam. The resolution is also limited by the size of the interaction volume, or the extent to which the material interacts with the electron beam. The spot size and the interaction volume are both large compared to the distances between atoms, so the resolution of the SEM is not high enough to image individual atoms, as is possible in the shorter wavelength (i.e. higher energy) transmission electron microscope(TEM). The SEM has compensating advantages, though, including the ability to image a comparatively large area of the specimen; the ability to image bulk materials (not just thin films or foils); and the variety of analytical modes available for measuring the composition and properties of the specimen. Depending on the instrument, the resolution can fall somewhere between less than 1 nm and 20 nm. By 2009, The world's highest SEM resolution at high beam energies (0.4 nm at 30 kV) is obtained with the Hitachi S-5500. At low beam energies, the best resolution (by 2009) is achieved by the Magellan system from FEI Company (0.9 nm at 1 kV). [edit]Environmental

  

SEM

Main article: ESEM Conventional SEM requires samples to be imaged under vacuum, because a gas atmosphere rapidly spreads and attenuates electron beams. Consequently, samples that produce a significant amount ofvapour, e.g. wet biological samples or oil-bearing rock need to be either dried or cryogenically frozen. Processes involving phase transitions, such as the drying of adhesives or melting of alloys, liquid transport, chemical reactions, solid-air-gas systems in general cannot be observed. Some observation of living insects has been possible [19] The first commercial development of the Environmental SEM (ESEM) in the late 1980s [20][21] allowed samples to be observed in low-pressure gaseous environments (e.g. 150 Torr) and high relativehumidity (up to 100%). This was made possible by the development of a secondary-electron detector[22][23] capable of operating in the presence of water vapour and by the use of pressure-limiting apertures with differential pumping in the path of the electron beam to separate the vacuum region (around the gun and lenses) from the sample chamber. The first commercial ESEMs were produced by the ElectroScan Corporation in USA in 1988. ElectroScan were later taken over by Philips (who later sold their electron-optics division to FEI Company) in 1996 [24]. ESEM is especially useful for non-metallic and biological materials because coating with carbon or gold is unnecessary. Uncoated Plastics and Elastomers can be routinely examined, as can uncoated biological samples. Coating can be difficult to reverse, may conceal small features on

the surface of the sample and may reduce the value of the results obtained. X-ray analysis is difficult with a coating of a heavy metal, so carbon coatings are routinely used in conventional SEMs, but ESEM makes it possible to perform X-ray microanalysis on uncoated non-conductive specimens. ESEM may be the preferred for electron microscopy of unique samples from criminal or civil actions, where forensic analysis may need to be repeated by several different experts.         [edit]3D

in SEM

3D data can be measured in the SEM with different methods such as: photogrammetry (2 or 3 images from tilted specimen) photometric stereo (use of 4 images from BSE detector) inverse reconstruction using electron-material interactive models[25][26] Possible applications are roughness measurement, measurement of fractal dimension, corrosion measurement and height step measurement. [edit]Gallery

of SEM images

The following are examples of images taken using a scanning electron microscope.

 
Coloured SEM image of soybean cyst nematode and egg. The colour makes the image easier for nonspecialists to view and understand the structures and surfaces revealed in micrographs.

 
Compound eye of Antarctic krillEuphausia superba. Arthropod eyes are a common subject in SEM micrographs due to the depth of focus that an SEM image can capture.

 
Ommatidia of Antarc tic krill eye, a higher magnification of the krill's eye. SEMs cover a range from light microscopy up to the magnifications available with aTEM.

 
SEM image of normal circulating human blood. This is an older and noisy micrograph of a common subject for SEM micrographs: red blood cells.

SEM image of a hederelloid from the Devonian of Michigan (largest tube diameter is 0.75 mm). The SEM is used extensively for capturing detailed images of micro and macro fossils.

Backscattered Electron (BSE) image of an Antimony rich region in a fragment of ancient glass. Museums use SEMs for studying valuable artifacts in a nondestructive manner. Many BSE images are taken at atmospheric rather than destructive high vacuum conditions.

SEM image of the corrosion layer on the surface of an ancient glass fragment; note the laminar structure of the corrosion layer.

SEM image of a photoresist layer used in semiconductorma nufacturing taken on a field emission SEM at 1000 volts, a very low accelerating voltage for an SEM, but achievable with field emission SEMsthis one taken with a Schottky fieldemission gun. These SEMs are important in the semiconductor industry for their high resolution capabilities.

[edit]See

also

Wikibooks has a book on the topic of

 

Nanotechnology Wikimedia Commons has media related to: Scanning

electron microscopic images

AFM probe

        

Atomic Force Microscope (AFM) Forensic engineering Forensic science List of surface analysis methods SEM-EDX Transmission electron microscopy (TEM) [edit]Terminology DR-SEM  Defect Review - Scanning Electron Microscopy CD-SEM        Critical Dimension Measurement - Scanning Electron Microscopy FIB-SEM Focused Ion Beam - Scanning Electron Microscopy (used mainly for cutting samples)  SBFSEM Serial Block-Face Scanning Electron Microscopy  ESEM Environmental Scanning Electron Microscopy  LV SEM Low Vacuum Scanning Electron Microscopy  FESEM Field emission gun Scanning Electron Microscopy

Transmission electron microscopy (TEM) is amicroscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen; the image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer ofphotographic film, or to be detected by a sensor such as aCCD camera.

TEMs are capable of imaging at a significantly higherresolution than light microscopes, owing to the small de Broglie wavelength of electrons. This enables the instrument's user to examine fine detaileven as small as a single column of atoms, which is tens of thousands times smaller than the smallest resolvable object in a light microscope. TEM forms a major analysis method in a range of scientific fields, in both physical and biological sciences. TEMs find application in cancer research, virology, materials science as well as pollution and semiconductor research.

At smaller magnifications TEM image contrast is due to absorption of electrons in the material, due to the thickness and composition of the material. At higher magnifications complex wave interactions modulate the intensity of the image, requiring expert analysis of observed images.

Alternate modes of use allow for the TEM to observe modulations in chemical identity, crystal orientation, electronic structure and sample induced electron phase shift as well as the regular absorption based imaging.  The first TEM was built by Max Knoll and Ernst Ruska in 1931, with this group developing the first TEM with resolving power greater than that of light in 1933 and the first commercial TEM in 1939.   
1 History Contents
[hide]

   

Initial development Improving resolution Further research

Background

    

Electrons Source formation Optics Display

Components

     

Vacuum system Specimen stage Electron gun Electron lens Apertures

Imaging methods

   

Contrast formation Diffraction Three dimensional imaging

Sample preparation

     

Tissue sectioning Sample staining Mechanical milling Chemical etching Ion etching

Modifications

Low voltage electron microscope (LVEM)

Limitations

     
See also

Resolution limits

References 10 External links

[edit]History [edit]Initial

development

  
The first practical TEM, Originally installed at I. G Farben-Werke and now on display at the Deutsches Museum in Munich, Germany

  
Sketch of first electron microscope, originally from Ruska's notebook in 1931, capable of only 16 times magnification

Ernst Abbeoriginally proposed that the ability to resolve detail in an object waslimited by thewavelength of the light used in imaging, thus limiting the useful obtainable magnification from an optical microscope to a few micrometers. Developments into ultraviolet(UV) microscopes, led by Khler and Rohr, allowed for an increase in resolving power of about a factor of two.[2] However this required more expensive quartz optical components, due to the absorption of UV by glass. At this point it was believed that obtaining an image with sub-micrometer information was simply impossible due to this wavelength constraint.[3]

It had earlier been recognized by Plcker in 1858 that the deflection of "cathode rays" (electrons) was possible by the use of magnetic fields.[4] This effect had been utilised to build primitive cathode ray oscilloscopes (CROs) as early as 1897 by Ferdinand Braun, intended as a measurement device.[5]Indeed in 1891 it was recognized by Riecke that the cathode rays could be focused by these magnetic fields, allowing for simple lens designs. Later this theory was extended by Hans Busch in his work published in 1926, who showed that the lens maker's equation, could under appropriate assumptions, be applicable to electrons.[6]

In 1928, at the Technological University of Berlin Adolf Matthias, Professor of High voltage Technology and Electrical Installations, appointed Max Knoll to lead a team of researchers to advance the CRO design. The team consisted of several PhD students including Ernst Ruska and Bodo von Borries. This team of researchers concerned themselves with lens design and

CRO column placement, which they attempted to obtain the parameters that could be optimised to allow for construction of better CROs, as well as the development of electron optical components which could be used to generate low magnification (nearly 1:1) images. In 1931 the group successfully generated magnified images of mesh grids placed over the anode aperture. The device used two magnetic lenses to achieve higher magnifications, arguably the first electron microscope. In that same year, Reinhold Rudenberg, the scientific director of the Siemens company, had patented an electrostatic lens electron microscope.   [edit]Improving
[3][7]

resolution

At this time the wave nature of electrons, which were considered charged matter particles, had not been fully realised until the publication of the De Broglie hypothesis in 1927.[8] The group was unaware of this publication until 1932, where it was quickly realized that the De Broglie wavelength of electrons was many orders of magnitude smaller than that for light, theoretically allowing for imaging at atomic scales. In April 1932, Ruska suggested the construction of a new electron microscope for direct imaging of specimens inserted into the microscope, rather than simple mesh grids or images of apertures. With this device successful diffraction and normal imaging of aluminium sheet was achieved, however exceeding the magnification achievable with light microscopy had still not been successfully demonstrated. This goal was achieved in September 1933, using images of cotton fibers, which were quickly acquired before being damaged by the electron beam.[3]

At this time, interest in the electron microscope had increased, with other groups, such as Albert Prebus and James Hillier at the University of Toronto who constructed the first TEM in north America in 1938,[9] continually advancing TEM design.

Research continued on the electron microscope at Siemens in 1936, the aim of the research was the development improvement of TEM imaging properties, particularly with regard to biological specimens. At this time electron microscopes were being fabricated for specific groups, such as the "EM1" device used at the UK National Physical Laboratory.[10] In 1939 the first commercial electron microscope, pictured, was installed in the Physics department of I. G Farben-Werke. Further work on the electron microscope was hampered by the destruction of a new laboratory constructed at Siemens by an air-raid, as well as the death of two of the researchers, Heinz Mller and Friedrick Krause during World War II.[11]

 

[edit]Further

research
[11]

After World War II, Ruska resumed work at Siemens, where he continued to develop the electron microscope, producing the first microscope with 100k magnification. The fundamental structure of this microscope design, with multi-stage beam preparation optics, is still used in modern

microscopes. The worldwide electron microscopy community advanced with electron microscopes being manufactured in Manchester UK, the USA (RCA), Germany (Siemens) and Japan . The first international conference in electron microscopy was in Delft in 1942, with more than one hundred attendees.
[10]

Later conferences included the "First" international conference in

Paris, 1950 and then in London in 1954.  With the development of TEM, the associated technique of scanning transmission electron microscopy (STEM) was re-investigated and did not become developed until the 1970s, with Albert Crewe at the University of Chicago developing the field emission gun[12] and adding a high quality objective lens to create the modern STEM. Using this design, Crewe demonstrated the ability to image atoms using annular dark-field imaging. Crewe and coworkers at the University of Chicago developed the cold field electron emission source and built a STEM able to visualize single heavy atoms on thin carbon substrates. [13]    [edit]Background [edit]Electrons Theoretically, the maximum resolution, d, that one can obtain with a light microscope has been limited by the wavelength of the photons that are being used to probe the sample, the numerical aperture of the system, NA.
[14]

and

  Early twentieth century scientists theorised ways of getting around the limitations of the relatively large wavelength of visible light (wavelengths of 400700 nanometers) by using electrons. Like all matter, electrons have both wave and particle properties (as theorized by Louis-Victor de Broglie), and their wave-like properties mean that a beam of electrons can be made to behave like a beam of electromagnetic radiation. The wavelength of electrons is found by equating the de Broglie equation to the kinetic energy of an electron. An additional correction must be made to account for relativistic effects, as in a TEM an electron's velocity approaches the speed of light, c.
[15]

  where, h is Planck's constant, m0 is the rest mass of an electron and E is the energy of the accelerated electron. Electrons are usually generated in an electron microscope by a process known as thermionic emission from a filament, usually tungsten, in the same manner as a light bulb, or alternatively by field electron emission.
[16]

The electrons are

then accelerated by an electric potential(measured in volts) and focused by electrostatic and electromagnetic lenses onto the sample. The transmitted beam contains information about electron density, phase and periodicity; this beam is used to form an image.  [edit]Source

formation

  
Layout of optical components in a basic TEM

  
Single crystal LaB6 filament

   
Hairpin style tungsten filament

From the top down, the TEM consists of an emission source, which may be atungsten filament, or a lanthanum hexaboride (LaB6) source.[17] For tungsten, this will be of the form of either a hairpin-style filament, or a small spike-shaped filament. LaB6sources utilize small single crystals. By connecting this gun to a high voltage source (typically ~100-300 kV) the gun will, given sufficient current, begin to emit electrons either by thermionic or field electron emission into the vacuum. This extraction is usually aided by the use of a Wehnelt cylinder. Once extracted, the upper lenses of the TEM allow for the formation of the electron probe to the desired size and location for later interaction with the sample.[18]

Manipulation of the electron beam is performed using two physical effects. The interaction of electrons with a magnetic field will cause electrons to move according to the right hand rule, thus allowing forelectromagnets to manipulate the electron beam. The use of magnetic fields allows for the formation of a magnetic lens of variable focusing power, the lens shape originating due to the distribution of magnetic flux. Additionally, electrostatic fields can cause the electrons to be deflected through a constant angle. Coupling of two deflections in opposing directions with a small intermediate gap allows for the formation of a shift in the beam path, this being used in TEM for beam shifting, subsequently this is extremely important to STEM. From these two effects, as well as the use of an electron imaging system, sufficient control over the beam path is possible for TEM operation[citation
needed]

. The optical configuration of a TEM

can be rapidly changed, unlike that for an optical microscope, as lenses in the beam path can be enabled, have their strength changed, or be disabled entirely simply via rapid

electrical switching, the speed of which is limited by effects such as the magnetic hysteresis of the lenses.   [edit]Optics The lenses of a TEM allow for beam convergence, with the angle of convergence as a variable parameter, giving the TEM the ability to change magnification simply by modifying the amount of current that flows through the coil, quadrupole or hexapole lenses. The quadrupole lens is an arrangement of electromagnetic coils at the vertices of the square, enabling the generation of a lensing magnetic fields, the hexapole configuration simply enhances the lens symmetry by using six, rather than four coils.  Typically a TEM consists of three stages of lensing. The stages are the condensor lenses, the objective lenses, and the projector lenses. The condensor lenses are responsible for primary beam formation, whilst the objective lenses focus the beam that comes through the sample itself (in STEM scanning mode, there are also objective lenses above the sample to make the incident electron beam convergent). The projector lenses are used to expand the beam onto the phosphor screen or other imaging device, such as film. The magnification of the TEM is due to the ratio of the distances between the specimen and the objective lens' image plane.[19] Additional quad or hexapole lenses allow for the correction of asymmetrical beam distortions, known as astigmatism. It is noted that TEM optical configurations differ significantly with implementation, with manufacturers using custom lens configurations, such as in spherical aberration corrected instruments,[18] or TEMs utilising energy filtering to correct electron chromatic aberration.   [edit]Display Imaging systems in a TEM consist of a phosphor screen, which may be made of fine (10100 m) particulate zinc sulphide, for direct observation by the operator. Optionally, an image recording system such as film based or doped YAG screen coupled CCDs.
[20]

Typically these devices can be removed or inserted into the beam path by the

operator as required.  [edit]Components

  
The electron source of the TEM is at the top, where the lensing system (4,7 and 8) focuses the beam on the specimen and then projects it onto the viewing screen (10). The beam control is on the right (13 and 14)

A TEM is composed of several components, which include a vacuum system in which the electrons travel, an electron emission source for generation of the electron stream, a series of electromagnetic lenses, as well as electrostatic plates. The latter two allow the operator to guide and manipulate the beam as required. Also required is a device to allow the insertion into, motion within, and removal of specimens from the beam path. Imaging devices are subsequently used to create an image from the electrons that exit the system.

 

[edit]Vacuum

system
4

To increase the mean free path of the electron gas interaction, a standard TEM is
[21] evacuated to low pressures, typically on the order of 10 Pa. The need for this is

twofold: first the allowance for the voltage difference between the cathode and the ground without generating an arc, and secondly to reduce the collision frequency of electrons with gas atoms to negligible levelsthis effect is characterised by the mean free path. TEM components such as specimen holders and film cartridges must be routinely inserted or replaced requiring a system with the ability to re-evacuate on a regular basis.

As such, TEMs are equipped with multiple pumping systems and airlocks and are not permanently vacuum sealed.  The vacuum system for evacuating a TEM to an operating pressure level consists of several stages. Initially a low or roughing vacuum is achieved with either a rotary vane pump or diaphragm pumpsbringing the TEM to a sufficiently low pressure to allow the operation of a turbomolecular or diffusion pump which brings the TEM to its high vacuum level necessary for operations. To allow for the low vacuum pump to not require continuous operation, while continually operating the turbomolecular pumps, the vacuum side of a low-pressure pump may be connected to chambers which accommodate the exhaust gases from the turbomolecular pump.
[22]

Sections of the TEM may be isolated by

the use of gate valves, to allow for different vacuum levels in specific areas, such as a higher vacuum of 104 to 107 Pa or higher in the electron gun in high resolution or field emission TEMs.  High-voltage TEMs require ultra high vacuums on the range of 107 to 109 Pa to prevent generation of an electrical arc, particularly at the TEM cathode.[23] As such for higher voltage TEMs a third vacuum system may operate, with the gun isolated from the main chamber either by use of gate valves or by the use of a differential pumping aperture. The differential pumping aperture is a small hole that prevents diffusion of gas molecules into the higher vacuum gun area faster than they can be pumped out. For these very low pressures either an ion pump or a getter material is used.  Poor vacuum in a TEM can cause several problems, from deposition of gas inside the TEM onto the specimen as it is being viewed through a process known as electron beam induced deposition, or in more severe cases damage to the cathode from an electrical discharge [23]. Vacuum problems due to specimen sublimation are limited by the use of a cold trap to adsorb sublimated gases in the vicinity of the specimen.[22]  [edit]Specimen

stage

 

 

TEM sample support mesh "grid", with ultramicrotomy sections

TEM specimen stage designs include airlocks to allow for insertion of the specimen holder into the vacuum with minimal increase in pressure in other areas of the microscope. The specimen holders are adapted to hold a standard size of grid upon which the sample is placed or a standard size of self-supporting specimen. Standard TEM grid sizes is a 3.05 mm diameter ring, with a thickness and mesh size ranging from a few to 100 m. The sample is placed onto the inner meshed area having diameter of approximately 2.5 mm. Usual grid materials are copper, molybdenum, gold or platinum. This grid is placed into the sample holder which is paired with the specimen stage. A wide variety of designs of stages and holders exist, depending upon the type of experiment being performed. In addition to 3.05 mm grids, 2.3 mm grids are sometimes, if rarely, used. These grids were particularly used in the mineral sciences where a large degree of tilt can be required and where specimen material may be extremely rare. Electron transparent specimens have a thickness around 100 nm, but this value depends on the accelerating voltage.

Once inserted into a TEM, the sample often has to be manipulated to present the region of interest to the beam, such as in single grain diffraction, in a specific orientation. To accommodate this, the TEM stage includes mechanisms for the translation of the sample in the XY plane of the sample, for Z height adjustment of the sample holder, and usually for at least one rotation degree of freedom for the sample. Thus a TEM stage may provide four degrees of freedom for the motion of the specimen. Most modern TEMs provide the ability for two orthogonal rotation angles of movement with specialized holder designs called double-tilt sample holders. Of note however is that some stage designs, such as top-entry or vertical insertion stages once common for high resolution TEM studies, may simply only have X-Y translation available. The design criteria of TEM stages are complex, owing to the simultaneous requirements of mechanical and electronoptical constraints and have thus generated many unique implementations.

A TEM stage is required to have the ability to hold a specimen and be manipulated to bring the region of interest into the path of the electron beam. As the TEM can operate over a wide range of magnifications, the stage must simultaneously be highly resistant to mechanical drift, with drift requirements as low as a few nm/minute while being able to move several um/minute, with repositioning accuracy on the order of nanometers.[24] Earlier designs of TEM accomplished this with a complex set of mechanical downgearing devices, allowing the operator to finely control the motion of the

stage by several rotating rods. Modern devices may use electrical stage designs, using screw gearing in concert with stepper motors, providing the operator with a computerbased stage input, such as a joystick or trackball.  Two main designs for stages in a TEM exist, the side-entry and top entry version.
[20]

Each

design must accommodate the matching holder to allow for specimen insertion without either damaging delicate TEM optics or allowing gas into TEM systems under vacuum.

  
A diagram of a single axis tilt sample holder for insertion into a TEM goniometer. Titling of the holder is achieved by rotation of the entire goniometer

The most common is the side entry holder, where the specimen is placed near the tip of a long metal (brass or stainless steel) rod, with the specimen placed flat in a small bore. Along the rod are several polymer vacuum rings to allow for the formation of a vacuum seal of sufficient quality, when inserted into the stage. The stage is thus designed to accommodate the rod, placing the sample either in between or near the objective lens, dependent upon the objective design. When inserted into the stage, the side entry holder has its tip contained within the TEM vacuum, and the base is presented to atmosphere, the airlock formed by the vacuum rings.

Insertion procedures for side entry TEM holders typically involve the rotation of the sample to triggermicro switches that initiate evacuation of the airlock before the sample is inserted into the TEM column.

The second design is the top-entry holder consists of a cartridge that is several cm long with a bore drilled down the cartridge axis. The specimen is loaded into the bore, possibly utilising a small screw ring to hold the sample in place. This cartridge is inserted into an airlock with the bore perpendicular to the TEM optic axis. When sealed, the airlock is manipulated to push the cartridge such that the cartridge falls into place, where the bore hole becomes aligned with the beam axis, such that the beam travels down the cartridge bore and into the specimen. Such designs are typically unable to be tilted without blocking the beam path or interfering with the objective lens.[20]

[edit]Electron

gun

   
Cross sectional diagram of an electron gun assembly, illustrating electron extraction

The electron gun is formed from several components: the filament, a biasing circuit, a Wehnelt cap, and an extraction anode. By connecting the filament to the negative component power supply, electrons can be "pumped" from the electron gun to the anode plate, and TEM column, thus completing the circuit. The gun is designed to create a beam of electrons exiting from the assembly at some given angle, known as the gun divergence semiangle, . By constructing the Wehnelt cylinder such that it has a higher negative charge than the filament itself, electrons that exit the filament in a diverging manner are, under proper operation, forced into a converging pattern the minimum size of which is the gun crossover diameter.

The thermionic emission current density, J, can be related to the work function of the emitting material and is a Boltzmann distribution given below, where A is a constant, the work function and T is the temperature of the material.[20] is

  This equation shows that in order to achieve sufficient current density it is necessary to heat the emitter, taking care not to cause damage by application of excessive heat, for this reason materials with either a high melting point, such as tungsten, or those with a low work function (LaB6) are required for the gun filament.
[25]

Furthermore both lanthanum hexaboride and tungsten thermionic

sources must be heated in order to achieve thermionic emission, this can be achieved by the use of a small resistive strip. To prevent thermal shock, there is often a delay enforced in the application of current to the tip, to prevent thermal

gradients from damaging the filament, the delay is usually a few seconds for LaB6, and significantly lower for tungsten[citation needed].  [edit]Electron

lens

   
Diagram of a TEM split polepiece design lens

Electron lenses are designed to act in a manner emulating that of an optical lens, by focusing parallel rays at some constant focal length. Lenses may operate electrostatically or magnetically. The majority of electron lenses for TEM utilise electromagnetic coils to generate a convex lens. For these lenses the field produced for the lens must be radially symmetric, as deviation from the radial symmetry of the magnetic lens causes aberrations such as astigmatism, and worsens spherical and chromatic aberration. Electron lenses are manufactured from iron, iron-cobalt or nickel cobalt alloys,[26] such as permalloy. These are selected for their magnetic properties, such as magnetic saturation,hysteresis and permeability.

The components include the yoke, the magnetic coil, the poles, the polepiece, and the external control circuitry. The polepiece must be manufactured in a very symmetrical manner, as this provides the boundary conditions for the magnetic field that forms the lens. Imperfections in the manufacture of the polepiece can induce severe distortions in the magnetic field symmetry, which induce distortions that will ultimately limit the lenses' ability to reproduce the object plane. The exact dimensions of the gap, pole piece internal diameter and taper, as well as the overall design of the lens is often performed by finite element analysis of the magnetic field, whilst considering the thermal and electrical constraints of the design.[26]

The coils which produce the magnetic field are located within the lens yoke. The coils can contain a variable current, but typically utilise high voltages, and

therefore require significant insulation in order to prevent short-circuiting the lens components. Thermal distributors are placed to ensure the extraction of the heat generated by the energy lost to resistance of the coil windings. The windings may be water cooled, using a chilled water supply in order to facilitate the removal of the high thermal duty.   [edit]Apertures Apertures are annular metallic plates, through which electrons that are further than a fixed distance from the optic axis may be excluded. These consist of a small metallic disc that is sufficiently thick to prevent electrons from passing through the disc, whilst permitting axial electrons. This permission of central electrons in a TEM causes two effects simultaneously: firstly, apertures decrease the beam intensity as electrons are filtered from the beam, which may be desired in the case of beam sensitive samples. Secondly, this filtering removes electrons that are scattered to high angles, which may be due to unwanted processes such as spherical or chromatic aberration, or due to diffraction from interaction within the sample.[27]  Apertures are either a fixed aperture within the column, such as at the condensor lens, or are a movable aperture, which can be inserted or withdrawn from the beam path, or moved in the plane perpendicular to the beam path. Aperture assemblies are mechanical devices which allow for the selection of different aperture sizes, which may be used by the operator to trade off intensity and the filtering effect of the aperture. Aperture assemblies are often equipped with micrometers to move the aperture, required during optical calibration.   [edit]Imaging

methods

Imaging methods in TEM utilize the information contained in the electron waves exiting from the sample to form an image. The projector lenses allow for the correct positioning of this electron wave distribution onto the viewing system. The observed intensity of the image, I, assuming sufficiently high quality of imaging device, can be approximated as proportional to the time-average amplitude of the electron wavefunctions, where the wave which form the exit beam is denoted by .[28]

Different imaging methods therefore attempt to modify the electron waves exiting the sample in a form that is useful to obtain information with regards to the sample, or beam itself. From the previous equation, it can be deduced that the observed image depends not only on the amplitude of beam, but also on the phase of the electrons, although phase effects may often be ignored at lower magnifications. Higher resolution imaging requires thinner samples and higher energies of incident electrons. Therefore the sample can no longer be considered to be absorbing electrons, via a Beer's law effect, rather the sample can be modelled as an object that does not change the amplitude of the incoming electron wavefunction. Rather the sample modifies the phase of the incoming wave; this model is known as a pure phase object, for sufficiently thin specimens phase effects dominate the image, complicating analysis of the observed intensities.[28] For example, to improve the contrast in the image the TEM may be operated at a slight defocus to enhance contrast, owing to convolution by the contrast transfer function of the TEM[29], which would normally decrease contrast if the sample was not a weak phase object.

 

[edit]Contrast

formation

Contrast formation in the TEM depends greatly on the mode of operation. Complex imaging techniques, which utilise the unique ability to change lens strength or to deactivate a lens, allow for many operating modes. These modes may be used to discern information that is of particular interest to the investigator.

 

Bright field The most common mode of operation for a TEM is the bright field imaging mode. In this mode the contrast formation, when considered classically, is formed directly by occlusion and absorption of electrons in the sample. Thicker regions of the sample, or regions with a higher atomic number will appear dark, whilst regions with no sample in the beam path will appear bright hence the term "bright field". The image is in effect assumed to be a simple two dimensional projection of the sample down the optic axis, and to a first approximation may be

modelled via Beer's law

[14]

, more complex analyses require the modelling

[28]

of the sample to include phase information.   Diffraction contrast Main article: Electron diffraction

  
Transmission electron micrograph ofdislocations in steel, which are faults in the structure of the crystal lattice at the atomic scale

Samples can exhibit diffraction contrast, whereby the electron beam undergoes Bragg scattering, which in the case of a crystalline sample, disperses electrons into discrete locations in the back focal plane. By the placement of apertures in the back focal plane, i.e. the objective aperture, the desired Bragg reflections can be selected (or excluded), thus only parts of the sample that are causing the electrons to scatter to the selected reflections will end up projected onto the imaging apparatus.

If the reflections that are selected do not include the unscattered beam (which will appear up at the focal point of the lens), then the image will appear dark wherever no sample scattering to the selected peak is present, as such a region without a specimen will appear dark. This is known as a dark-field image.

Modern TEMs are often equipped with specimen holders that allow the user to tilt the specimen to a range of angles in order to obtain specific diffraction conditions, and apertures placed above the specimen allow the user to select electrons that would otherwise be diffracted in a particular direction from entering the specimen.

Applications for this method include the identification of lattice defects in crystals. By carefully selecting the orientation of the sample, it is possible not just to determine the position of defects but also to determine the

type of defect present. If the sample is oriented so that one particular plane is only slightly tilted away from the strongest diffracting angle (known as the Bragg Angle), any distortion of the crystal plane that locally tilts the plane to the Bragg angle will produce particularly strong contrast variations. However, defects that produce only displacement of atoms that do not tilt the crystal to the Bragg angle (i. e. displacements parallel to the crystal plane) will not produce strong contrast.    Electron energy loss Main article: Electron energy loss spectroscopy Utilizing the advanced technique of EELS, for TEMs appropriately equipped electrons can be rejected based upon their voltage (which, due to constant charge is their energy), using magnetic sector based devices known as EELS spectrometers. These devices allow for the selection of particular energy values, which can be associated with the way the electron has interacted with the sample. For example different elements in a sample result in different electron energies in the beam after the sample. This normally results in chromatic aberration however this effect can, for example, be used to generate an image which provides information on elemental composition, based upon the atomic transition during electron-electron interaction.[31]  EELS spectrometers can often be operated in both spectroscopic and imaging modes, allowing for isolation or rejection of elastically scattered beams. As for many images inelastic scattering will include information that may not be of interest to the investigator thus reducing observable signals of interest, EELS imaging can be used to enhance contrast in observed images, including both bright field and diffraction, by rejecting unwanted components.    Phase contrast Main article: High-resolution transmission electron microscopy Crystal structure can also be investigated by High Resolution Transmission Electron Microscopy(HRTEM), also known as phase contrast. When utilizing a Field emission source, of uniform thickness, the images are formed due to differences in phase of electron waves,
[30]

which is caused by specimen interaction.

[29]

Image formation is given by

the complex modulus of the incoming electron beams. As such, the image is not only dependent on the number of electrons hitting the screen, making direct interpretation of phase contrast images more complex. However this effect can be used to an advantage, as it can be manipulated to provide more information about the sample, such as in complex phase retrieval techniques.   [edit]Diffraction Main article: Selected area diffraction

   
Crystalline diffraction pattern from a twinned grain of FCC Austenitic steel

As previously stated, by adjusting the magnetic lenses such that the back focal plane of the lens rather than the imaging plane is placed on the imaging apparatus a diffraction pattern can be generated. For thin crystalline samples, this produces an image that consists of a pattern of dots in the case of a single crystal, or a series of rings in the case of a polycrystalline or amorphous solidmaterial. For the single crystal case the diffraction pattern is dependent upon the orientation of the specimen and the structure of the sample illuminated by the electron beam. This image provides the investigator with information about the space groupsymmetries in the crystal and the crystal's orientation to the beam path. This is typically done without utilising any information but the position at which the diffraction spots appear and the observed image symmetries.

Diffraction patterns can have a large dynamic range, and for crystalline samples, may have intensities greater than those recordable by CCD. As

such, TEMs may still be equipped with film cartridges for the purpose of obtaining these images, as the film is a single use detector.

   
Convergent Beam Kikuchi lines from Silicon, near the [100] zone axis

Analysis of diffraction patterns beyond point-position can be complex, as the image is sensitive to a number of factors such as specimen thickness and orientation, objective lens defocus, spherical and chromatic aberration. Although quantitative interpretation of the contrast shown in lattice images is possible, it is inherently complicated and can require extensive computer simulation and analysis, such as electron multislice analysis.[32]

More complex behaviour in the diffraction plane is also possible, with phenomena such as Kikuchi lines arising from multiple diffraction within the crystalline lattice. In convergent beam electron diffraction (CBED) where a non-parallel, i.e. converging, electron wavefront is produced by concentrating the electron beam into a fine probe at the sample surface, the interaction of the convergent beam can provide information beyond structural data such as sample thickness.

[edit]Three

dimensional imaging

 

  

A three dimensional TEM image of a parapoxavirus[33]

As TEM specimen holders typically allow for the rotation of a sample by a desired angle, multiple views of the same specimen can be obtained by rotating the angle of the sample along an axis perpendicular to the beam. By taking multiple images of a single TEM sample at differing angles, typically in 1 increments, a set of images known as a "tilt series" can be collected. This methodology was proposed in the 1970s by Walter Hoppe. Under purely absorption contrast conditions, this set of images can be used to construct a three-dimensional representation of the sample.[34]

The reconstruction is accomplished by a two-step process, first images are aligned to account for errors in the positioning of a sample; such errors can occur due to vibration or mechanical drift. Alignment methods useimage registration algorithms, such as autocorrelation methods to correct these errors. Secondly, using a technique known as filtered back projection, the aligned image slices can be transformed from a set of two-dimensional images, Ij(x,y), to a single three-dimensional image, I'j(x,y,z). This three dimensional image is of particular interest when morphological information is required, further study can be undertaken using computer algorithms, such as isosurfaces and data slicing to analyse the data.

As TEM samples cannot typically be viewed at a full 180 rotation, the observed images typically suffer from a "missing wedge" of data, which when using Fourier based back projection methods decreases the range of resolvable frequencies in the three dimensional reconstruction.[34] Mechanical techniques, such as multi-axis tilting, as well as numerical techniques exist to limit the impact of this missing data on the observed specimen morphology. Variants on this method, referred to as single particle analysis, use images of multiple identical objects at different orientations to produce the image data required for three dimensional reconstruction. Assuming that objects do not have significant preferred orientations, this method does not suffer from the

missing data wedge, however it assumes that the different objects imaged can be treated as if the data was generated from a single object.   [edit]Sample

preparation

Sample preparation in TEM can be a complex procedure. TEM specimens are required to be at most hundreds of nanometers thick, as unlike neutron or X-Ray radiation the electron beam interacts readily with the sample, an effect that increases roughly with atomic number squared (z ).[14] High quality samples will have a thickness that is comparable to the mean free path of the electrons that travel through the samples, which may be only a few tens of nanometers. Preparation of TEM specimens is specific to the material under analysis and the desired information to obtain from the specimen. As such, many generic techniques have been used for the preparation of the required thin sections.
2

Materials that have dimensions small enough to be electron transparent, such as powders or nanotubes, can be quickly prepared by the deposition of a dilute sample containing the specimen onto support grids or films. In the biological sciences in order to withstand the instrument vacuum and facilitate handling, biological specimens can be fixated using either a negative staining material such as uranyl acetate or by plastic embedding. Alternately samples may be held at liquid nitrogentemperatures after embedding in vitreous ice.[35] In material science and metallurgy the specimens tend to be naturally resistant to vacuum, but still must be prepared as a thin foil, or etched so some portion of the specimen is thin enough for the beam to penetrate. Constraints on the thickness of the material may be limited by the scattering cross-section of the atoms from which the material is comprised.

  

[edit]Tissue

sectioning

Main article: Microtome By passing samples over a glass or diamond edge, small, thin sections can be readily obtained using a semi-automated method.[36] This method is used to obtain thin, minimally deformed samples that allow for the observation of tissue samples. Additionally inorganic samples have been

studied, such as aluminium, although this usage is limited owing to the heavy damage induced in the less soft samples.
[37]

To prevent charge

build-up at the sample surface, tissue samples need to be coated with a thin layer of conducting material, such as carbon, where the coating thickness is several nanometers. This may be achieved via an electric arc deposition process using a sputter coating device.  [edit]Sample

staining

  
A section of a cell of Bacillus subtilis, taken with a Tecnai T-12 TEM. The scale bar is 200 nm.

Details in light microscope samples can be enhanced bystains that absorb light; similarly TEM samples of biological tissues can utilize high atomic number stains to enhance contrast. The stain absorbs electrons or scatters part of the electron beam which otherwise is projected onto the imaging system. Compounds of heavy metals such asosmium, lead, or uranium may be used prior to TEM observation to selectively deposit electron dense atoms in or on the sample in desired cellular or protein regions, requiring an understanding of how heavy metals bind to biological tissues.

 

[edit]Mechanical

milling

Mechanical polishing may be used to prepare samples. Polishing needs to be done to a high quality, to ensure constant sample thickness across the region of interest. A diamond, or cubic boron nitridepolishing compound may be used in the final stages of polishing to remove any scratches that may cause contrast fluctuations due to varying sample thickness. Even after careful mechanical milling, additional fine methods such as ion etching may be required to perform final stage thinning.

  

[edit]Chemical

etching

Main article: Chemical etching Certain samples may be prepared by chemical etching, particularly metallic specimens. These samples are thinned using a chemical etchant, such as an acid, to prepare the sample for TEM observation. Devices to control the thinning process may allow the operator to control either the voltage or current passing through the specimen, and may include systems to detect when the sample has been thinned to a sufficient level of optical transparency.

 

[edit]Ion

etching

Main article: Sputtering

  
SEM image of a thin TEM sample milled byFIB. The thin membrane shown here is suitable for TEM examination; however, at ~300-nm thick, it would not be suitable for high-resolution TEM without further milling.

Ion etching is a sputtering process that can remove very fine quantities of material. This is used to perform a finishing polish of specimens polished by other means. Ion etching uses an inert gas passed through an electric field to generate a plasma stream that is directed to the sample surface. Acceleration energies for gases such as argon are typically a few kilovolts. The sample may be rotated to promote even polishing of the sample surface. The sputtering rate of such methods is on the order of tens of micrometers per hour, limiting the method to only extremely fine polishing.

More recently focussed ion beam methods have been used to prepare samples. FIB is a relatively new technique to prepare thin samples for TEM examination from larger specimens. Because FIB can be used to

micro-machine samples very precisely, it is possible to mill very thin membranes from a specific area of interest in a sample, such as a semiconductor or metal. Unlike inert gas ion sputtering, FIB makes use of significantly more energetic gallium ions and may alter the composition or structure of the material through gallium implantation.   [edit]Modifications The capabilities of the TEM can be further extended by additional stages and detectors, sometimes incorporated on the same microscope. An electron cryomicroscope (CryoTEM) is a TEM with a specimen holder capable of maintaining the specimen at liquid nitrogen or liquid helium temperatures. This allows imaging specimens prepared in vitreous ice, the preferred preparation technique for imaging individual molecules or macromolecular assemblies.[39]  A TEM can be modified into a scanning transmission electron microscope (STEM) by the addition of a system that rasters the beam across the sample to form the image, combined with suitable detectors. Scanning coils are used to deflect the beam, such as by an electrostatic shift of the beam, where the beam is then collected using a current detector such as a faraday cup, which acts as a direct electron counter. By correlating the electron count to the position of the scanning beam (known as the "probe"), the transmitted component of the beam may be measured. The non-transmitted components may be obtained either by beam tilting or by the use of annular dark field detectors.  In-situ experiments may also be conducted with experiments such as insitu reactions or material deformation testing.[40]  Modern research TEMs may include aberration correctors,[18] to reduce the amount of distortion in the image. Incident beam Monochromators may also be used which reduce the energy spread of the incident electron beam to less than 0.15 eV.[18] Major TEM makers include JEOL, Hitachi High-technologies, FEI Company (from merging with Philips Electron Optics), Carl Zeiss and NION.  [edit]Low voltage electron microscope (LVEM)
[38]

The low voltage electron microscope (LVEM) is a combination of SEM, TEM and STEM in one instrument, which operated 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.
[41][42]

 

[edit]Limitations There are a number of drawbacks to the TEM technique. Many materials require extensive sample preparation to produce a sample thin enough to be electron transparent, which makes TEM analysis a relatively time consuming process with a low throughput of samples. The structure of the sample may also be changed during the preparation process. Also the field of view is relatively small, raising the possibility that the region analysed may not be characteristic of the whole sample. There is potential that the sample may be damaged by the electron beam, particularly in the case of biological materials.

  

[edit]Resolution

limits

See also: Transmission Electron Aberration-corrected Microscope The limit of resolution obtainable in a TEM may be described in several ways, and is typically referred to as the information limit of the microscope. One commonly used value is a cut-off value of thecontrast transfer function, a function that is usually quoted in the frequency domain to define the reproduction of spatial frequencies of objects in the object plane by the microscope optics. A cut-off frequency, qmax, for the transfer function may be approximated with the following equation, where Cs is the spherical aberration coefficient and wavelength:
[27]

is the electron

  For a 200 kV microscope, with partly corrected spherical aberrations ("to the third order") and a Csvalue of 1 m,[43] a theoretical cut-off value

might be 1/qmax = 42 pm [27]. The same microscope without a corrector would have Cs = 0.5 mm and thus a 200-pm cut-off.
[43]

Practically, the

spherical aberrations are suppressed in the best, "aberration-corrected" microscopes. Their resolution is however limited by electron source geometry and brightness and chromatic aberrations in the objective lens system. 
[18][44]

Intriguingly, the frequency domain representation of the contrast transfer function may often have an oscillatory nature
[45]

, which can be tuned by

adjusting the focal value of the objective lens. This oscillatory nature implies that some spatial frequencies are faithfully imaged by the microscope, whilst others are suppressed. By combining multiple images with different spatial frequencies, the use of techniques such as focal series reconstruction can be used to improve the resolution of the TEM in a limited manner.[27] The contrast transfer function can, to some extent, be experimentally approximated through techniques such as Fourier transforming images of amorphous material, such as amorphous carbon.  More recently, advances in aberration corrector design have been able to reduce spherical aberrations[46] and to achieve resolution below 0.5 ngstrms (50 pm)[44] at magnifications above 50 million times.[47] Improved resolution allows for the imaging of lighter atoms that scatter electrons less efficiently, such as lithium atoms in lithium battery materials.[48] The ability to determine the position of atoms within materials has made the HRTEM an indispensable tool for nanotechnology research and development in many fields, including heterogeneous catalysis and the development ofsemiconductor devices for electronics and photonics.         [edit]See
[49]

also

Electron beam induced deposition Electron diffraction Electron energy loss spectroscopy (EELS) Electron microscope Energy filtered transmission electron microscopy (EFTEM) High-resolution transmission electron microscopy (HRTEM) Low Voltage Electron Microscopy (LVEM)

    

Scanning confocal electron microscopy Scanning electron microscope (SEM) Scanning transmission electron microscope (STEM) Transmission Electron Aberration-corrected Microscope Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very high-resolution type ofscanning 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 andHeinrich 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.  
Contents
[hide]

 

1 Basic principles 2 Imaging modes

      

2.1 Contact mode 2.2 Non-contact mode 2.3 Tapping mode

3 AFM cantilever deflection measurement 4 Force spectroscopy 5 Identification of individual surface atoms 6 Advantages and disadvantages

    

6.1 Advantages 6.2 Disadvantages

7 Piezoelectric scanners 8 See also References

  

External links 11 Further reading

[edit]Basic

principles

 
Electron micrograph of a used AFM cantilever image width ~100 micrometers...

  
and ~30 micrometers

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 aWheatstone 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-tosample 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. [edit]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 externallyoscillated at or close to its fundamental resonance frequency or a harmonic. The oscillation amplitude, phase and resonance frequency are modified by tipsample interaction forces. These changes in oscillation with respect to the external reference oscillation provide information about the sample's characteristics.

 

[edit]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.

[edit]Non-contact

mode

   
AFM - 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 (<10 nm). 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.

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 noncontact 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. 
[1]

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.

[edit]Tapping

mode

   
Single polymer chains (0.4 nm thick) recorded in a tapping mode under aqueous media with different pH.[2]

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]

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.  [edit]AFM
[2]

cantilever deflection measurement

   
AFM beam deflection detection

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 adifferential 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. [edit]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 piezoelectricdisplacement. 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. substrate.
[5] [4]

Furthermore, AFM was used to

measure in aqueous environment dispersion force due to polymer adsorbed on the 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. 
[6]

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] [edit]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.[8]

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. [edit]Advantages

and disadvantages

   
The first atomic force microscope

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.

 

[edit]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, 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 andtransmission electron microscopy.

 

[edit]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 150150 micrometers. One method of improving the scanned area size for AFM is by using parallel probes in a fashion similar to that ofmillipede 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 fast-acting designs
[11][12] [9][10]

making the AFM microscope less suited for

measuring accurate distances between topographical features on the image. However, several 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. 
[9][10] [13]

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.   [edit]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
[13]

between the two scan directions. This can be corrected by applying a non-

linear voltage to the piezo electrodes to cause linear scanner movement and calibrating the scanner accordingly. 
[13]

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 scanners 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.
[14]

Field Emission Scanning Electron Microscopy (FESEM)

   

Principle of Operation

A field-emission cathode in the electron gun of a scanning electronmicroscope provides narrower probing beams at low as well as high electronenergy, resulting in both improved spatial resolution and minimized samplecharging and damage. For applications which demand the highest magnification possible, we also offer

In-lens FESEM.

Applications include: Semiconductor device cross section analyses for gate widths, gate oxides,film thicknesses, and construction details Advanced coating thickness and structure uniformity determination Small contamination feature geometry and elemental composition measurement  Why Field Emission SEM? FESEM produces clearer, less electrostatically distorted images withspatial resolution down to 1 1/2 nm. That's 3 to 6 times better than conventional SEM. Smaller-area contamination spots can be examined at electron acceleratingvoltages compatible with Energy Dispersive X-ray Spectroscopy.


Reduced penetration of low kinetic energy electrons probes closer tothe immediate material surface. High quality, low voltage images are obtained with negligible electricalcharging of samples. (Accelerating voltages range from 0.5 to 30 kV.) Need for placing conducting coatings on insulating materials is virtuallyeliminated. For ultra-high magnification imaging, use our InlensFESEM.
A scanning tunneling microscope (STM) is an instrument for imaging surfaces at the atomic level. Its development in 1981 earned its inventors, Gerd Binnig andHeinrich Rohrer (at IBM Zrich), the Nobel Prize in Physicsin 1986.[1][2] For an STM, good resolution is considered to be 0.1 nm lateral resolution and 0.01 nm depth resolution.[3] 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.[4] 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.  
Contents
[hide]

     

1 Procedure 2 Instrumentation 3 Other STM related studies 4 Principle of operation 5 Early invention 6 See also

   

7 References 8 Further reading 9 External links

[edit]Procedure

   
A close-up of a simple scanning tunneling microscope head using a platinum-iridium stylus.

First, a voltage bias is applied and the tip is brought close to the sample by some coarse sampleto-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 range, which is the equilibrium position between attractive (3<W<10) and repulsive (W<3) interactions[4]. 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 measured itself, or the height, z, of the tip corresponding to
[4] 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[5]. 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[6]. 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
[6]

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 postprocessing 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 to the density of states far from impurities. 
[7] [3] [6]

Framerates of at least 1 Hz enable so called Video-STM (up to 50 Hz is possible).[8][9] This can be used to scan surface diffusion.[10] [edit]Instrumentation

   
Schematic view of an STM

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[5].

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] [3]

[3]

The tip is often made of tungsten or platinum-iridium, though gold is also used . Tungsten tips are usually made by electrochemical etching, and platinum-iridium tips by mechanical shearing .
[3]

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.
[4]

Maintaining the tip position with respect to the sample, scanning the sample and acquiring the data is computer controlled[5]. The computer may also be used for enhancing the image with the help ofimage processing[12][13] as well as performing quantitative measurements.[14] [edit]Other

STM related studies

  
Nanomanipulation via STM of a self-assembled organic semiconductormonolayer (here: PTCDA molecules) ongraphite, in which the logo of the Center for NanoScience (CeNS), LMU has been written.

Many other microscopy techniques have been developed based upon STM. These
[3] include photon scanning microscopy (PSTM), which uses an optical tip to tunnel photons ;

scanning tunneling potentiometry (STP), which measures electric potential across a surface[3]; spin polarized scanning tunneling microscopy (SPSTM), which uses a ferromagnetic tip to tunnel spin-polarized electrons into a magnetic sample,[15] 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 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 nickelsurface 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.  [edit]Principle
[3]

of operation
 This article may be too technical for most readers to understand. Please improve this article to make it understandable to non-experts, without removing the technical details. (September 2010)

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 theelectron, 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 1dimensional case, the energy levels equation,
n(z)

of the electrons are given by solutions to Schrdingers

  where

, is the reduced Plancks constant, z is the position, and m is the mass of an electron . If
[4]

an electron of energy E is incident upon an energy barrier of height U(z), the electron wave function is atraveling wave solution,   where ,

  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,   where ,
[4]

  quantifies the decay of the wave inside the barrier, with the barrier in the +z direction for 
[4]

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[4]. 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,

 

[4]

If the bias is small, we can let U E M in the expression for , where 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 excited[4]. 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 Ef and eV in the sample, and 2) the 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
[4]

 

. One can sum the probability over energies between Ef 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
[4] level . The LDOS near some energy E in an interval is

given by

 

, and the tunnel current at a small bias V is proportional to the LDOS near the Fermi level, which gives important information about the sample . It is desirable to use LDOS to express the current because this value does not change as the volume changes, while probability density does . Thus the tunneling current is given by
[4] [4]

  where
s(0,Ef)

is the LDOS near the Fermi level

of the sample at the sample surface[4]. This current can also be expressed in terms of the

LDOS near the Fermi level of the sample at the tip surface,   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 , the current increases by an order of magnitude, and vice versa.[6]  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 metalinsulator-metal junction.[16] He found that if he solved Schrdingers equation for each side of the junction separately to obtain the wave functions and for each electrode, he could obtain the tunnel matrix, M, from the overlap of these two wave functions[4]. This can be applied to STM by making the electrodes the tip and sample, assigning and 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 z=W at the tip surface[4].  Now, Fermis Golden Rule gives the rate for electron transfer across the barrier, and is written

,  where (E E ) restricts tunneling to occur only between electron levels with the same energy[4]. The tunnel matrix element, given by

,  is a description of the lower energy associated with the interaction of wave functions at the overlap, also called the
[4] resonance energy .

Summing over all the states gives the tunneling current as

 ,  where f is the Fermi function,

s

and

are the density of

states in the sample and tip,

[4] respectively . The Fermi

distribution function describes the filling of electron levels at a given temperature T.   [edit]Early

invention

An earlier, similar invention, the Topografiner of R. Young, J. Ward, and F. Scire from the NIST
[17]

, relied on field

emission. However, Young is credited by the Nobel Committee as the person who

realized that it should be possible to achieve better resolution by using the tunnel effect.         Microscopy Scanning probe microscopy Scanning tunneling spectroscopy Electrochemical scanning tunneling microscope Atomic force microscope Electron microscope Spin polarized scanning tunneling microscopy   [edi
[18]

[edit]See

also

A charge-coupled device (CCD) is a device for the movement of electrical charge, usually from within the device to an area where the charge can be manipulated, for example conversion into a digital value. This is achieved by "shifting" the signals between stages within the device one at a time. CCDs move charge between capacitive bins in the device, with the shift allowing for the transfer of charge between bins.

Often the device is integrated with an image sensor, such as a photoelectric device to produce the charge that is being read, thus making the CCD a major technology fordigital imaging. Although CCDs are not the only technology to allow for light detection, CCDs are widely used in professional, medical, and scientific applications where high-quality image data are required.  
Contents
[hide]

     

1 History 2 Basics of operation 3 Detailed physics of operation 4 Architecture 5 Use in astronomy 6 Color cameras

  

6.1 Sensor sizes

7 Electron-multiplying CCD 8 Frame transfer CCD

     

9 Intensified charge-coupled device 10 See also References 12 External links

[edit]History The charge-coupled device was invented in 1969 at AT&T Bell Labs by Willard Boyle and George E. Smith. The lab was working on semiconductor bubble memory when Boyle and Smith conceived of the design of what they termed, in their notebook, "Charge 'Bubble' Devices".[1] A description of how the device could be used as a shift register and as a linear and area imaging devices was described in this first entry. The essence of the design was the ability to transfer charge along the surface of a semiconductor from one storage capacitor to the next. The concept was similar in principle to thebucket-brigade device (BBD), which was developed at Philips Research Labs during the late 1960's.

The initial paper describing the concept[2] listed possible uses as a memory, a delay line, and an imaging device. The first experimental device[3] demonstrating the principle was a row of closely spaced metal squares on an oxidized silicon surface electrically accessed by wire bonds.

The first working CCD made with integrated circuit technology was a simple 8-bit shift register.[4] This device had input and output circuits and was used to demonstrate use as a shift register and as a crude eight pixel linear imaging device. Development of the device progressed at a rapid rate. By 1971, Bell researchers Michael F. Tompsett et al. were able to capture images with simple linear devices.[5]

Several companies, including Fairchild Semiconductor, RCA and Texas Instruments, picked up on the invention and began development programs. Fairchild's effort, led by ex-Bell researcher Gil Amelio, was the first with commercial devices, and by 1974 had a linear 500element device and a 2-D 100 x 100 pixel device. Under the leadership of Kazuo Iwama, Sony also started a big development effort on CCDs involving a significant investment. Eventually, Sony managed to mass produce CCDs for their camcorders. Before this happened, Iwama died in August 1982. Subsequently, a CCD chip was placed on his tombstone to acknowledge his contribution.
[6]

In January 2006, Boyle and Smith were awarded the National Academy of Engineering Charles Stark Draper Prize,[7] and in 2009 they were awarded the Nobel Prize for Physics,[8] for their work on the CCD.

[edit]Basics

of operation

  
The charge packets (electrons, blue) are collected in potential wells (yellow) created by applying positive voltage at the gate electrodes (G). Applying positive voltage to the gate electrode in the correct sequence transfers the charge packets.

In a CCD for capturing images, there is a photoactive region (an epitaxial layer of silicon), and a transmission region made out of a shift register (the CCD, properly speaking).

An image is projected through a lens onto the capacitor array (the photoactive region), causing each capacitor to accumulate an electric charge proportional to the light intensity at that location. A one-dimensional array, used in line-scan cameras, captures a single slice of the image, while a two-dimensional array, used in video and still cameras, captures a two-dimensional picture corresponding to the scene projected onto the focal plane of the sensor. Once the array has been exposed to the image, a control circuit causes each capacitor to transfer its contents to its neighbor (operating as a shift register). The last capacitor in the array dumps its charge into a charge amplifier, which converts the charge into a voltage. By repeating this process, the controlling circuit converts the entire contents of the array in the semiconductor to a sequence of voltages. In a digital device, these voltages are then sampled, digitized, and usually stored in memory; in an analog device (such as an analog video camera), they are processed into a continuous analog signal (e.g. by feeding the output of the charge amplifier into a low-pass filter) which is then processed and fed out to other circuits for transmission, recording, or other processing.

   
"One-dimensional" CCD image sensorfrom a fax machine.

[edit]Detailed

physics of operation

The photoactive region of the CCD is, generally, an epitaxiallayer of silicon. It has a doping of p+ (Boron) and is grown upon a substrate material, often p++. In buried channel devices, the type of design utilized in most modern CCDs, certain areas of the surface of the silicon are ion implantedwith phosphorus, giving them an n-doped designation. This region defines the channel in which the photogenerated charge packets will travel. The gate oxide, i.e. the capacitor dielectric, is grown on top of the epitaxial layer and substrate.

Later on in the process polysilicon gates are deposited by chemical vapor deposition, patterned withphotolithography, and etched in such a way that the separately phased gates lie perpendicular to the channels. The channels are further defined by utilization of the LOCOS process to produce thechannel stop region.

Channel stops are thermally grown oxides that serve to isolate the charge packets in one column from those in another. These channel stops are produced before the polysilicon gates are, as the LOCOS process utilizes a high temperature step that would destroy the gate material. The channels stops are parallel to, and exclusive of, the channel, or "charge carrying", regions.

Channel stops often have a p+ doped region underlying them, providing a further barrier to the electrons in the charge packets (this discussion of the physics of CCD devices assumes an electrontransfer device, though hole transfer, is possible).

The clocking of the gates, alternately high and low, will forward and reverse bias to the diode that is provided by the buried channel (n-doped) and the epitaxial layer (p-doped). This will cause the CCD to deplete, near the p-n junction and will collect and move the charge packets beneath the gatesand within the channelsof the device.

CCD manufacturing and operation can be optimized for different uses. The above process describes a frame transfer CCD. While CCDs may be manufactured on a heavily doped p++ wafer it is also possible to manufacture a device inside p-wells that have been placed on an nwafer. This second method, reportedly, reduces smear, dark current, and infrared and red response. This method of manufacture is used in the construction of interline transfer devices.

Another version of CCD is called a peristaltic CCD. In a peristaltic charge-coupled device, the charge packet transfer operation is analogous to the peristaltic contraction and dilation of the digestive system. The peristaltic CCD has an additional implant that keeps the charge away from the silicon/silicon dioxide interface and generates a large lateral electric field from one gate to the next. This provides an additional driving force to aid in transfer of the charge packets. [edit]Architecture

The CCD image sensors can be implemented in several different architectures. The most common are full-frame, frame-transfer, and interline. The distinguishing characteristic of each of these architectures is their approach to the problem of shuttering.

In a full-frame device, all of the image area is active, and there is no electronic shutter. A mechanical shutter must be added to this type of sensor or the image smears as the device is clocked or read out.

With a frame-transfer CCD, half of the silicon area is covered by an opaque mask (typically aluminum). The image can be quickly transferred from the image area to the opaque area or storage region with acceptable smear of a few percent. That image can then be read out slowly from the storage region while a new image is integrating or exposing in the active area. Frametransfer devices typically do not require a mechanical shutter and were a common architecture for early solid-state broadcast cameras. The downside to the frame-transfer architecture is that it requires twice the silicon real estate of an equivalent full-frame device; hence, it costs roughly twice as much.

The interline architecture extends this concept one step further and masks every other column of the image sensor for storage. In this device, only one pixel shift has to occur to transfer from image area to storage area; thus, shutter times can be less than a microsecond and smear is essentially eliminated. The advantage is not free, however, as the imaging area is now covered by opaque strips dropping the fill factor to approximately 50 percent and the effective quantum efficiency by an equivalent amount. Modern designs have addressed this deleterious characteristic by adding microlenses on the surface of the device to direct light away from the opaque regions and on the active area. Microlenses can bring the fill factor back up to 90 percent or more depending on pixel size and the overall system's optical design.

   
CCD from a 2.1 megapixel Argus digital camera.

The choice of architecture comes down to one of utility. If the application cannot tolerate an expensive, failure-prone, power-intensive mechanical shutter, an interline device is the right

choice. Consumer snap-shot cameras have used interline devices. On the other hand, for those applications that require the best possible light collection and issues of money, power and time are less important, the full-frame device is the right choice. Astronomers tend to prefer full-frame devices. The frame-transfer falls in between and was a common choice before the fill-factor issue of interline devices was addressed. Today, frame-transfer is usually chosen when an interline architecture is not available, such as in a back-illuminated device.  CCDs containing grids of pixels are used in digital cameras, optical scanners, and video cameras as light-sensing devices. They commonly respond to 70 percent of the incident light (meaning a quantum efficiency of about 70 percent) making them far more efficient than photographic film, which captures only about 2 percent of the incident light.

   
CCD from a 2.1 megapixel Hewlett-Packard digital camera.

Most common types of CCDs are sensitive to near-infrared light, which allows infrared photography, night-visiondevices, and zero lux (or near zero lux) video-recording/photography. For normal silicon-based detectors, the sensitivity is limited to 1.1 m. One other consequence of their sensitivity to infrared is that infrared from remote controls often appears on CCD-based digital cameras or camcorders if they do not have infrared blockers.

Cooling reduces the array's dark current, improving the sensitivity of the CCD to low light intensities, even for ultraviolet and visible wavelengths. Professional observatories often cool their detectors with liquid nitrogento reduce the dark current, and therefore the thermal noise, to negligible levels. [edit]Use

in astronomy

Due to the high quantum efficiencies of CCDs, linearity of their outputs (one count for one photon of light), ease of use compared to photographic plates, and a variety of other reasons, CCDs were very rapidly adopted by astronomers for nearly all UV-to-infrared applications.

Thermal noise and cosmic rays may alter the pixels in the CCD array. To counter such effects, astronomers take several exposures with the CCD shutter closed and opened. The average of images taken with the shutter closed is necessary to lower the random noise. Once developed, the dark frameaverage image is then subtracted from the open-shutter image to remove the dark current and other systematic defects (dead pixels, hot pixels, etc.) in the CCD.

The Hubble Space Telescope, in particular, has a highly developed series of steps (data reduction pipeline) to convert the raw CCD data to useful images. See the references for a more in-depth description of the steps in astronomical CCD image-data correction and processing.
[9]

CCD cameras used in astrophotography often require sturdy mounts to cope with vibrations from wind and other sources, along with the tremendous weight of most imaging platforms. To take long exposures of galaxies and nebulae, many astronomers use a technique known as autoguiding. Most autoguiders use a second CCD chip to monitor deviations during imaging. This chip can rapidly detect errors in tracking and command the mount motors to correct for them.

  
Array of 30 CCDs used on Sloan Digital Sky Survey telescope imaging camera, an example of "drift-scanning."

An interesting unusual astronomical application of CCDs, called drift-scanning, uses a CCD to make a fixed telescope behave like a tracking telescope and follow the motion of the sky. The charges in the CCD are transferred and read in a direction parallel to the motion of the sky, and at the same speed. In this way, the telescope can image a larger region of the sky than its normal field of view. TheSloan Digital Sky Survey is the most famous example of this, using the technique to produce the largest uniform survey of the sky yet accomplished.

In addition to astronomy, CCDs are also used in laboratory analytical instrumentation such as monochromators,spectrometers, and N-slit laser interferometers. [edit]Color
[10]

cameras

  
A Bayer filter on a CCD

   
CCD-Colorsensor

Digital color cameras generally use a Bayer mask over the CCD. Each square of four pixels has one filtered red, one blue, and two green (the human eye is more sensitive to green than either red or blue). The result of this is thatluminance information is collected at every pixel, but the color resolution is lower than the luminance resolution.

Better color separation can be reached by three-CCD devices (3CCD) and a dichroic beam splitter prism, that splits the image into red, green and blue components. Each of the three CCDs is arranged to respond to a particular color. Most professional video camcorders, and some semiprofessional camcorders, use this technique. Another advantage of 3CCD over a Bayer mask device is higher quantum efficiency (and therefore higher light sensitivity for a given aperture size). This is because in a 3CCD device most of the light entering the aperture is captured by a sensor, while a Bayer mask absorbs a high proportion (about 2/3) of the light falling on each CCD pixel.

For still scenes, for instance in microscopy, the resolution of a Bayer mask device can be enhanced by Microscanningtechnology. During the process of color co-site sampling, several frames of the scene are produced. Between acquisitions, the sensor is moved in pixel dimensions, so that each point in the visual field is acquired consecutively by elements of the mask that are sensitive to the red, green and blue components of its color. Eventually every pixel in the image has been scanned at least once in each color and the resolution of the three channels become equivalent (the resolutions of red and blue channels are quadrupled while the green channel is doubled).

 

[edit]Sensor

sizes

Sensors (CCD / CMOS) are often referred to with an inch fraction designation such as 1/1.8" or 2/3" called the optical format. This measurement actually originates back in the 1950s and the time ofVidicon tubes. Compact digital cameras and Digicams typically have much smaller sensors than a digital SLR and are thus less sensitive to light and inherently more prone to noise. Some examples of the CCDs found in modern cameras can be found in this table in a Digital Photography Review article 
Aspec t Ratio

Type

Widt h mm 2.300

Heigh t mm 1.730

Diagon al mm 2.878

Area 2 mm

Relativ e Area

  

1/6"

  

4:3

  

  

  

  

3.979

  

1.000

1/4" 1/3.6 " 1/3.2 " 1/3"

4:3

3.200

2.400

4.000

7.680

1.930

4:3

4.000

3.000

5.000

12.000

3.016

 

4:3

 

4.536

 

3.416

 

5.678

 

15.495

 

3.894

4:3

4.800

3.600

6.000

17.280

4.343

1/2.7 " 1/2" 1/1.8 " 2/3"

    

4:3

    

5.270

     

3.960

    

6.592

    

20.869

    

5.245

 

4:3

6.400

4.800

8.000

30.720

7.721

4:3

7.176

5.319

8.932

38.169

9.593

 

4:3

8.800 12.80 0 18.00 0

6.600

11.000

58.080 122.88 0 243.00 0

14.597

1"

4:3

9.600 13.50 0

16.000

30.882

4/3"

4:3

22.500

61.070

Other image sizes as a comparison

APSC

3:2

25.10 0

16.70 0

30.148

419.17 0

105.34 6

35m m

3:2

36.00 0

24.00 0

43.267

864.00 0

217.14 0

 

645

4:3

56.00 0

41.50 0

69.701

2324.0 00

584.06 6

[edit]Electron-multiplying

CCD

  
Electrons are transferred serially through the gain stages making up the multiplication register of an EMCCD. The high voltages used in these serial transfers induce the creation of additional charge carriers through impact ionisation.

  
There is a dispersion (variation) in the number of electrons output by the multiplication register for a given (fixed) number of input electrons (shown in the legend on the right). The probability distribution for the number of output electrons is plotted logarithmically on the vertical axis for a simulation of a multiplication register. Also shown are results from the empirical fit equation shown on this page.

An electron-multiplying CCD (EMCCD, also known as an L3Vision CCD, L3CCD or Impactron CCD) is a charge-coupled device in which a gain register is placed between the shift register and the output amplifier. The gain register is split up into a large number of stages. In each stage the electrons are multiplied by impact ionization in a similar way to an avalanche diode. The gain probability at every stage of the register is small (P < 2%) but as the number of elements is large (N > 500), the overall gain can be very high (g = (1 + P) ), with single input electrons giving many thousands of output electrons. Reading a signal from a CCD gives a noise background, typically a few electrons. In an EMCCD this noise is superimposed on many thousands of electrons rather than a single electron; the devices thus have negligible readout noise.
N

EMCCDs show a similar sensitivity to Intensified CCDs(ICCDs). However, as with ICCDs, the gain that is applied in the gain register is stochastic and the exact gain that has been applied to a pixel's charge is impossible to know. At high gains (> 30), this uncertainty has the same effect on the signal-to-noise ratio (SNR) as halving the quantum efficiency with respect to operation with a gain of unity. However, at very low light levels (where the quantum efficiency is most important) it can be assumed that a pixel either contains an electron - or not. This removes the noise associated with the stochastic multiplication at the cost of counting multiple electrons in the same pixel as a single electron. The dispersion in the gain is shown in the graph on the right. For multiplication registers with many elements and large gains it is well modelled by the equation:

 

if where P is the probability of getting n output electrons given m input electrons and a total mean multiplication register gain of g.

Because of the lower costs and the somewhat better resolution EMCCDs are capable of replacing ICCDs in many applications. ICCDs still have the advantage that they can be gated very fast and thus are useful in applications like range-gated imaging. EMCCD cameras indispensably need a cooling system to cool the chip down to temperatures around 170 K. This cooling system unfortunately adds additional costs to the EMCCD imaging system and often yields heavy condensation problems in the application.

The low-light capabilities of L3CCDs are starting to find use in astronomy. In particular their low noise at high readout speeds makes them very useful for lucky imaging of faint stars, and high speed photon counting photometry.

Commercial EMCCD cameras typically have clock-induced charge and darkcurrent (dependent on the extent of cooling) that leads to an effective readout noise ranging from 0.01 to 1 electrons per pixel read. Custom-built deep-cooled non-inverting mode EMCCD cameras have provided effective readout noise lower than 0.1 electrons per pixel read [1] for lucky imaging observations. [edit]Frame

transfer CCD

  
A frame transfer CCD for startracker applications.

   
Vertical smear.

A frame transfer CCD is a specialized CCD, often used in astronomy and someprofessional video cameras, designed for high exposure efficiency and correctness.

The normal functioning of a CCD, astronomical or otherwise, can be divided into two phases: exposure and readout. During the first phase, the CCD passively collects incoming photons, storing electrons in its cells. After the exposure time is passed, the cells are read out one line at a time.

During the readout phase, cells are shifted down the entire area of the CCD. While they are shifted, they continue to collect light. Thus, if the shifting is not fast enough, errors can result from light that falls on a cell holding charge during the transfer. These errors are referred to as "vertical smear" and cause a strong light source to create a vertical line above and below its exact location. In addition, the CCD cannot be used to collect light while it is being read out. Unfortunately, a faster shifting requires a faster readout, and a faster readout can introduce errors in the cell charge measurement, leading to a higher noise level.

A frame transfer CCD solves both problems: it has a hidden, not normally used, area containing as many cells as the area exposed to light. Typically, this area is covered by a reflective material such as aluminium. When the exposure time is up, the cells are transferred very rapidly to the hidden area. Here, safe from any incoming light, cells can be read out at any speed one deems necessary to correctly measure the cells' charge. At the same time, the exposed part of the CCD is collecting light again, so no delay occurs between successive exposures.

The disadvantage of such a CCD is the higher cost: the cell area is basically doubled, and more complex control electronics are needed. [edit]Intensified

charge-coupled device

An intensified charge-coupled device (ICCD) is a CCD that is optically connected to an image intensifier that is mounted in front of the CCD.

An image intensifier includes three functional elements: a photocathode, a micro-channel plate (MCP) and a phosphor screen. These three elements are mounted one close behind the other in the mentioned sequence. The photons which are coming from the light source fall onto the photocathode, thereby generating photoelectrons. The photoelectrons are accelerated towards the MCP by an electrical control voltage, applied between photocathode and MCP. The electrons are multiplied inside of the MCP and thereafter accelerated towards the phosphor screen. The phosphor screen finally converts the multiplied electrons back to photons which are guided to the CCD by a fiber optic or a lens.

An image intensifier inherently includes a shutter functionality: If the control voltage between the photocathode and the MCP is reversed, the emitted photoelectrons are not accelerated towards the MCP but return to the photocathode. Thus, no electrons are multiplied and emitted by the MCP, no electrons are going to the phosphor screen and no light is emitted from the image intensifier. In this case no light falls onto the CCD, which means that the shutter is closed. The process of reversing the control voltage at the photocathode is called gating and therefore ICCDs are also called gateable CCD cameras.

Beside of the extremely high sensitivity of ICCD cameras, which enable single photon detection, the gateability is one of the major advantages of the ICCD over the EMCCD cameras. The highest performing ICCD cameras enable shutter times as short as 200 picoseconds.

ICCD cameras are in general somewhat higher in price than EMCCD cameras because they need the expensive image intensifier. On the other hand EMCCD cameras need a cooling system to cool the EMCCD chip down to temperatures around 170 K. This cooling system adds additional costs to the EMCCD camera and often yields heavy condensation problems in the application.

 

ICCDs are used in night vision devices and in a large variety of scientific applications.

X-ray fluorescence (XRF) is the emission of characteristic "secondary" (or fluorescent) X-raysfrom a material that has been excited by bombarding with high-energy X-rays or gamma rays. The phenomenon is widely used forelemental analysis and chemical analysis, particularly in the investigation of metals, glass,ceramics and building materials, and for research in geochemistry,forensic science and archaeology.

  
1 Underlying physics

Contents
[hide]

     

Characteristic radiation Primary radiation Dispersion Detection X-ray intensity

Chemical analysis

Energy dispersive spectrometry

     

Si(Li) detectors Wafer detectors Amplifiers Processing Usage

Wavelength dispersive spectrometry

            

Sample presentation Monochromators Analysis Lines Crystals Detectors Extracting analytical results

Other spectroscopic methods using the same principle Instrument qualification See also Notes References 8 External links

[edit]Underlying

physics

  

Physics of X-ray fluorescence, in a schematic representation.

When materials are exposed to short-wavelength X-rays or to gamma rays, ionisation of their componentatoms may take place. Ionisation consists of the ejection of one or more electrons from the atom, and may take place if the atom is exposed to radiation with an energy greater than its ionisation potential. X-rays and gamma rays can be energetic enough to expel tightly held electrons from the inner orbitals of the atom. The removal of an electron in this way renders the electronic structure of the atom unstable, and electrons in higher orbitals "fall" into the lower orbital to fill the hole left behind. In falling, energy is released in the form of a photon, the energy of which is equal to the energy difference of the two orbitals involved. Thus, the material emits radiation, which has energy characteristic of the atoms present. The term fluorescence is applied to phenomena in which the absorption of radiation of a specific energy results in the re-emission of radiation of a different energy (generally lower).

  
Figure 1: Electronic transitions in a calcium atom. Remember, when electrons are jumping down, one of the electrons in the lower orbital is missing.

  
Figure 2: Typical energy dispersive XRF spectrum

    
Figure 3: Spectrum of a rhodium target tube operated at 60 kV, showing continuous spectrum and K lines

[edit]Characteristic

radiation

Each element has electronic orbitals of characteristic energy. Following removal of an inner electron by an energetic photon provided by a primary radiation source, an electron from an outer shell drops into its place. There are a limited number of ways in which this can happen, as shown in figure 1. The main transitions are given names: an L K transition is traditionally called K , an M K transition is called K , an M L transition is called L , and so on. Each of these transitions yields a fluorescent photon with a characteristic energy equal to the difference in energy of the initial and final orbital. The wavelength of this fluorescent radiation can be calculated from Planck's Law:

The fluorescent radiation can be analysed either by sorting the energies of the photons (energy-dispersive analysis) or by separating the wavelengths of the radiation (wavelength-dispersive analysis). Once sorted, the intensity of each characteristic radiation is directly related to the amount of each element in the material. This is the basis of a powerful technique in analytical chemistry. Figure 2 shows the typical form of the sharp fluorescent spectral lines obtained in the wavelength-dispersive method (see Moseley's law).
[edit]Primary

 

radiation

In order to excite the atoms, a source of radiation is required, with sufficient energy to expel tightly held inner electrons. Conventional X-ray generators are most commonly used, because their output can readily be "tuned" for the application, and because higher power can be deployed relative to other techniques. However, gamma ray sources can be used without the need for an elaborate power supply, allowing an easier use in small portable instruments. When the energy source is a synchrotron or the Xrays are focused by an optic like a polycapillary, the X-ray beam can be very small and very intense. As a result, atomic information on the sub-micrometer scale can be obtained. X-ray generators in the range 2060 kV in order to the K line, which allows excitation of a broad range of atoms. The continuous spectrum consists of "bremsstrahlung" radiation: radiation produced when high energy electrons passing through the tube are progressively decelerated by the material of the tube anode (the "target"). A typical tube output spectrum is shown in figure 3.
[edit]Dispersion

 

In energy dispersive analysis, the fluorescent X-rays emitted by the material sample are directed into a solid-state detector which produces a "continuous" distribution of pulses, the voltages of which are proportional to the incoming photon energies. This signal is processed by a multichannel analyser (MCA) which produces an accumulating digital spectrum that can be processed to obtain analytical data. In wavelength dispersive analysis, the fluorescent

X-rays emitted by the material sample are directed into a diffraction grating monochromator. The diffraction grating used is usually a single crystal. By varying the angle of incidence and take-off on the crystal, a single X-ray wavelength can be selected. The wavelength obtained is given by the Bragg Equation:
 

where d is the spacing of atomic layers parallel to the crystal surface.

[edit]Detection

 

In energy dispersive analysis, dispersion and detection are a single operation, as already mentioned above. Proportional counters or various types of solid state detectors (PIN diode, Si(Li), Ge(Li), Silicon Drift Detector SDD) are used. They all share the same detection principle: An incoming X-ray photonionises a large number of detector atoms with the amount of charge produced being proportional to the energy of the incoming photon. The charge is then collected and the process repeats itself for the next photon. Detector speed is obviously critical, as all charge carriers measured have to come from the same photon to measure the photon energy correctly (peak length discrimination is used to eliminate events that seem to have been produced by two X-ray photons arriving almost simultaneously). The spectrum is then built up by dividing the energy spectrum into discrete bins and counting the number of pulses registered within each energy bin. EDXRF detector types vary in resolution, speed and the means of cooling (a low number of free charge carriers is critical in the solid state detectors): proportional counters with resolutions of several hundred eV cover the low end of the performance spectrum, followed by PIN diode detectors, while the Si(Li), Ge(Li) and Silicon Drift Detectors (SDD) occupy the high end of the performance scale. In wavelength dispersive analysis, the single-wavelength radiation produced by the monochromator is passed into a photomultiplier, a detector similar to a Geiger counter, which counts individual

photons as they pass through. The counter is a chamber containing a gas that is ionised by X-ray photons. A central electrode is charged at (typically) +1700 V with respect to the conducting chamber walls, and each photon triggers a pulse-like cascade of current across this field. The signal is amplified and transformed into an accumulating digital count. These counts are then processed to obtain analytical data.
  [edit]X-ray

intensity

The fluorescence process is inefficient, and the secondary radiation is much weaker than the primary beam. Furthermore, the secondary radiation from lighter elements is of relatively low energy (long wavelength) and has low penetrating power, and is severely attenuated if the beam passes through air for any distance. Because of this, for high-performance analysis, the path from tube to sample to detector is maintained under high vacuum (around 10 Pa residual pressure). This means in practice that most of the working parts of the instrument have to be located in a large vacuum chamber. The problems of maintaining moving parts in vacuo, and of rapidly introducing and withdrawing the sample without losing vacuum, pose major challenges for the design of the instrument. For less demanding applications, or when the sample is damaged by a vacuum (e.g. a volatile sample), a helium-swept X-ray chamber can be substituted, with some loss of low-Z (Z = atomic number) intensities.
[edit]Chemical

 

analysis

The use of a primary X-ray beam to excite fluorescent radiation from the sample was first proposed byGlocker and Schreiber in 1928[1]. Today, the method is used as a non-destructive analytical technique, and as a process control tool in many extractive and processing industries. In principle, the lightest element that can be analysed is beryllium (Z = 4), but due to instrumental limitations and low X-ray yields for the light elements, it is often difficult to quantify elements lighter than sodium (Z = 11), unless background

corrections and very comprehensive interelement corrections are made.

    
Figure 4: Schematic arrangement of EDX spectrometer

[edit]Energy

dispersive spectrometry

In energy dispersive spectrometers (EDX or EDS), the detector allows the determination of the energy of the photon when it is detected. Detectors historically have been based on silicon semiconductors, in the form of lithium-drifted silicon crystals, or high-purity silicon wafers.

    
Figure 5: Schematic form of a Si(Li) detector

[edit]Si(Li) detectors

These consist essentially of a 35 mm thick silicon junction type pi-n diode (same as PIN diode) with a bias of -1000 V across it. The lithium-drifted centre part forms the non-conducting i-layer, where

Li compensates the residual acceptors which would otherwise make the layer p-type. When an X-ray photon passes through, it causes a swarm of electron-hole pairs to form, and this causes a voltage pulse. To obtain sufficiently low conductivity, the detector must be maintained at low temperature, and liquid-nitrogen must be used for the best resolution. With some loss of resolution, the much more convenient Peltier cooling can be employed.[2]
  [edit]Wafer detectors

More recently, high-purity silicon wafers with low conductivity have become routinely available. Cooled by the Peltier effect, this provides a cheap and convenient detector, although the liquid nitrogen cooled Si(Li) detector still has the best resolution (i.e. ability to distinguish different photon energies).
[edit]Amplifiers

 

The pulses generated by the detector are processed by pulseshaping amplifiers. It takes time for the amplifier to shape the pulse for optimum resolution, and there is therefore a trade-off between resolution and count-rate: long processing time for good resolution results in "pulse pile-up" in which the pulses from successive photons overlap. Multi-photon events are, however, typically more drawn out in time (photons did not arrive exactly at the same time) than single photon events and pulse-length discrimination can thus be used to filter most of these out. Even so, a small number of pileup peaks will remain and pile-up correction should be built into the software in applications that require trace analysis. To make the most efficient use of the detector, the tube current should be reduced to keep multi-photon events (before discrimination) at a reasonable level, e.g. 520%.
[edit]Processing

 

Considerable computer power is dedicated to correcting for pulsepile up and for extraction of data from poorly resolved spectra. These elaborate correction processes tend to be based on empirical relationships that may change with time, so that

continuous vigilance is required in order to obtain chemical data of adequate precision.

  [edit]Usage

EDX spectrometers are superior to WDX spectrometers in that they are smaller, simpler in design and have fewer engineered parts. They can also use miniature X-ray tubes or gamma sources. This makes them cheaper and allows miniaturization and portability. This type of instrument is commonly used for portable quality control screening applications, such as testing toys for Lead (Pb) content, sorting scrap metals, and measuring the lead content of residential paint. On the other hand, the low resolution and problems with low count rate and long dead-time makes them inferior for high-precision analysis. They are, however, very effective for high-speed, multi-elemental analysis. Field Portable XRF analysers currently on the market weigh less than 2 kg, and have limits of detection on the order of 2 parts per million of Lead (Pb) in pure sand.

  
Figure 6: Schematic arrangement of wavelength dispersive spectrometer

 

Chemist operates a goniometer used for X-ray fluorescence analysis of individual grains of mineral specimens, U.S. Geological Survey, 1958.

 

[edit]Wavelength

dispersive spectrometry

In wavelength dispersive spectrometers (WDX or WDS), the photons are separated by diffraction on a single crystal before being detected. Although wavelength dispersive spectrometers are occasionally used to scan a wide range of wavelengths, producing a spectrum plot as in EDS, they are usually set up to make measurements only at the wavelength of the emission lines of the elements of interest. This is achieved in two different ways: "Simultaneous" spectrometers have a number of "channels" dedicated to analysis of a single element, each consisting of a fixed-geometry crystal monochromator, a detector, and processing electronics. This allows a number of elements to be measured simultaneously, and in the case of high-powered instruments, complete high-precision analyses can be obtained in under 30 s. Another advantage of this arrangement is that the fixed-geometry monochromators have no continuously moving parts, and so are very reliable. Reliability is important in production environments where instruments are expected to work without interruption for months at a time. Disadvantages of simultaneous spectrometers include relatively high cost for complex analyses, since each channel used is expensive. The number of elements that can be measured is limited to 1520, because of space limitations on the number of monochromators that can be crowded around the fluorescing sample. The need to accommodate multiple monochromators means that a rather open arrangement around the sample is required, leading to relatively long tube-samplecrystal distances, which leads to lower detected intensities and more scattering. The instrument is inflexible, because if a new element is to be measured, a new measurement channel has to be bought and installed. "Sequential" spectrometers have a single variable-geometry monochromator (but usually with an arrangement for selecting from a choice of crystals), a single detector assembly (but usually with

more than one detector arranged in tandem), and a single electronic pack. The instrument is programmed to move through a sequence of wavelengths, in each case selecting the appropriate X-ray tube power, the appropriate crystal, and the appropriate detector arrangement. The length of the measurement program is essentially unlimited, so this arrangement is very flexible. Because there is only one monochromator, the tube-sample-crystal distances can be kept very short, resulting in minimal loss of detected intensity. The obvious disadvantage is relatively long analysis time, particularly when many elements are being analysed, not only because the elements are measured in sequence, but also because a certain amount of time is taken in readjusting the monochromator geometry between measurements. Furthermore, the frenzied activity of the monochromator during an analysis program is a challenge for mechanical reliability. However, modern sequential instruments can achieve reliability almost as good as that of simultaneous instruments, even in continuoususage applications.
  [edit]Sample presentation

In order to keep the geometry of the tube-sample-detector assembly constant, the sample is normally prepared as a flat disc, typically of diameter 2050 mm. This is located at a standardized, small distance from the tube window. Because the X-ray intensity follows an inverse-square law, the tolerances for this placement and for the flatness of the surface must be very tight in order to maintain a repeatable X-ray flux. Ways of obtaining sample discs vary: metals may be machined to shape, minerals may be finely ground and pressed into a tablet, and glasses may be cast to the required shape. A further reason for obtaining a flat and representative sample surface is that the secondary X-rays from lighter elements often only emit from the top few micrometers of the sample. In order to further reduce the effect of surface irregularities, the sample is usually spun at 520 rpm. It is necessary to ensure that the sample is sufficiently thick to absorb the entire primary beam. For higher-Z materials, a few millimetres

thickness is adequate, but for a light-element matrix such as coal, a thickness of 3040 mm is needed.

    
Figure 7: Bragg diffraction condition

[edit]Monochromators

The common feature of monochromators is the maintenance of a symmetrical geometry between the sample, the crystal and the detector. In this geometry the Bragg diffraction condition is obtained. The X-ray emission lines are very narrow (see figure 2), so the angles must be defined with considerable precision. This is achieved in two ways: Flat crystal with Soller collimators The Soller collimator is a stack of parallel metal plates, spaced a few tenths of a millimetre apart. To improve angle resolution, one must lengthen the collimator, and/or reduce the plate spacing. This arrangement has the advantage of simplicity and relatively low cost, but the collimators reduce intensity and increase scattering, and reduce the area of sample and crystal that can be "seen". The simplicity of the geometry is especially useful for variable-geometry monochromators.

 

 
Figure 8: Flat crystal with Soller collimators

  
Figure 9: Curved crystal with slits

 

Curved crystal with slits The Rowland circle geometry ensures that the slits are both in focus, but in order for the Bragg condition to be met at all points, the crystal must first be bent to a radius of 2R (where R is the radius of the Rowland circle), then ground to a radius of R. This arrangement allows higher intensities (typically 8-fold) with higher resolution (typically 4-fold) and lower background. However, the mechanics of keeping Rowland circle geometry in a variable-angle monochromator is extremely difficult. In the case of fixed-angle monochromators (for use in simultaneous spectrometers), crystals bent to a logarithmic spiral shape give the best focusing performance. The manufacture of curved crystals to acceptable tolerances increases their price considerably.
[edit]Analysis Lines

 

The spectral lines used for chemical analysis are selected on the basis of intensity, accessibility by the instrument, and lack of line overlaps. Typical lines used, and their wavelengths, are as follows:
e l   w a  e l   w a  e l   w a  e l   w a

e m e n t

v e l e n g t h ( n m )

e m e n t

v e l e n g t h ( n m )

e m e n t

v e l e n g t h ( n m )

e m e n t

v e l e n g t h ( n m )  0 . 1 3 1 3 0 . 1 2 7 6 0 . 1 2 4 1 0 . 1 2 0 7 0 . 1

   L i  2 2 . 8  N i 

0 . 1 6 5 8 0 . 1 5 4 1 0 . 1 4 3 5 0 . 1 3 4 0 0 . 1

   I

0 . 3 1 4 9 0 . 3 0 1 6 0 . 2 8 9 2 0 . 2 7 7 6 0 . 2

P t

   B e  1 1 . 4  C u 

  X e 

  A u 

   B  6 . 7 6  Z n 

  C s 

  H g 

   C  4 . 4 7  G a 

  B a 

  T l 

3 . 1

G e

L a

P b

2 5 4

6 6 6   B i 

1 7 5 0 . 1 1 4 4 0 . 1 1 1 4 0 . 1 0 8 5 0 . 1 0 5 7

  O 

2 . 3 6 2

  A s 

0 . 1 1 7 6 0 . 1 1 0 5 0 . 1 0 4 0 0 . 0 9 8 0 1 0 . 0 9 2 5 6 0 . 0 8 7

  C e 

0 . 2 5 6 2 0 . 2 4 6 3 0 . 2 3 7 0 0 . 2 2 8 2

  F

1 . 8 3 2

  S e 

  P r 

  P o 

  N e

1 . 4 6 1

  B r 

  N d 

  A t 

   N a  1 . 1 9 1 K r 

  P m 

  R n 

   M g  0 . 9 8 9 R b 

  S m 

0 . 2 2 0 0 0 . 2 1 2

  F r 

0 . 1 0 3 1 0 . 1 0 0

  A l

0 . 8 3 4

  S r 

  E u 

  R a 

5 3  0 . 7 1 2 6    Y 0 . 0 8 2 8 8 0 . 0 7 8 5 9 0 . 0 7 4 6 2 0 . 0 7 0 9 4 0 . 0 6 7 5 1 0 . 0 6 4 3   G d 

  S i

0 . 2 0 4 7

  A c 

0 . 0 9 8 0 0 . 0 9 5 6

  P

0 . 6 1 5 8

 Z r 

  T b 

0 . 1 9 7 7

  T h 

  S

0 . 5 3 7 3

 N b 

  D y 

0 . 1 9 0 9

  P a 

0 . 0 9 3 3

  C l

0 . 4 7 2 9

 M o 

  H o 

0 . 1 8 4 5

   U

0 . 0 9 1 1 0 . 0 8 8 8

  A r

0 . 4 1 9 3 0 . 3 7 4 2

 T c 

  E r 

0 . 1 7 8 4 0 . 1 7 2 7

  N p 

  K

  R u 

  T m 

  P u 

0 . 0 8 6

3  R h  0 . 0 6 1 3 6 0 . 0 5 8 5 9 0 . 0 5 5 9 9 0 . 0 5 3 5 7 0 . 3 7 7 2 0 . 3 6 0 0

8 0 . 0 8 4 7

  C a

0 . 3 3 5 9

  Y b 

0 . 1 6 7 2

  A m 

  S c

0 . 3 0 3 2

 P d 

  L u 

0 . 1 6 2 0 0 . 1 5 7 0 0 . 1 5 2 2 0 . 1 4 7 6 0 . 1 4 3 3

  C m 

0 . 0 8 2 8

  T i

0 . 2 7 4 9

 A g 

  H f 

  B k 

0 . 0 8 0 9

   V

0 . 2 5 0 4 0 . 2 2 9 0 0 . 2 1 0 2

 C d 

  T a 

  C f 

0 . 0 7 9 1 0 . 0 7 7 3 0 . 0 7 5 6

  C r 

  I n 

   W

  E s 

  M n 

  S n 

  R e 

  F m 

  F e 

0 . 1 9 3 6 0 . 1 7 8 9

  S b 

0 . 3 4 3 9 0 . 3 2 8 9

  O s 

0 . 1 3 9 1 0 . 1 3 5 1

  M d 

0 . 0 7 4 0 0 . 0 7 2 4

  C o 

  T e 

  I r 

  N o 

Other lines are often used, depending on the type of sample and equipment available.
[edit]Crystals

           

The desirable characteristics of a diffraction crystal are: High diffraction intensity High dispersion Narrow diffracted peak width High peak-to-background Absence of interfering elements Low thermal coefficient of expansion Stability in air and on exposure to X-rays Ready availability Low cost Crystals with simple structure tend to give the best diffraction performance. Crystals containing heavy atoms can diffract well, but also fluoresce themselves, causing interference. Crystals that are water-soluble, volatile or organic tend to give poor stability. Commonly used crystal materials include LiF (lithium fluoride), ADP (ammonium dihydrogen phosphate), Ge (germanium), graphite, InSb (indium antimonide), PE (tetrakis-(hydroxymethyl)-

methane: penta-erythritol), KAP (potassium hydrogen phthalate), RbAP (rubidium hydrogen phthalate) and TlAP (thallium(I) hydrogen phthalate). In addition, there is an increasing use of "layered synthetic microstructures", which are "sandwich" structured materials comprising successive thick layers of low atomic number matrix, and monoatomic layers of a heavy element. These can in principle be custom-manufactured to diffract any desired long wavelength, and are used extensively for elements in the range Li to Mg.


Properties of commonly used crystals

 mat erial  pl an e  d (n m) mi n (n m)  0. 05 3 0. 03 7 0. 02 4 0. 13 9 0. 08 5 0. 08 8 0. 09 8 0. 11   ma x (n m) 0.3 79   inte nsity ther mal exp ansi on  dura bility

LiF

20 0

0.2 014 0.1 424

++++ +

+++

+++

LiF

22 0

0.2 68

+++

++

+++

LiF

42 0 10 1 11 1 00 1 11 1 00 2

0.0 901

0.1 69

++

++

+++

ADP

0.5 320

1.0 00 0.6 14 0.6 30

++

++

Ge

0.3 266

+++

+++

grap hite

0.3 354

++++

+++

InSb

0.3 740 0.4 371

0.7 03 0.8 21

++++

+++

PE

+++

+++ ++

4  10 10 10 10 11 1 10 10 40 0  1.3 25  0. 34 6 0. 34 1 0. 08 2 0. 33 8  2.4 90  ++

KAP

++

++

RbA P

1.3 05 0.3 135 1.2 95 0.5 86 6.0 0

2.4 53

++

++

++

Si

0.5 89

++

+++

TlAP

2.4 34

+++

++

++

 

YB66 6n m LSM

  1. 56 6 

 11. 276

+++

++

 

[edit]Detectors

Detectors used for wavelength dispersive spectrometry need to have high pulse processing speeds in order to cope with the very high photon count rates that can be obtained. In addition, they need sufficient energy resolution to allow filtering-out of background noise and spurious photons from the primary beam or from crystal fluorescence. There are four common types of detector: gas flow proportional counters sealed gas detectors scintillation counters semiconductor detectors

   

   
Figure 10: Arrangement of gas flow proportional counter

Gas flow proportional counters are used mainly for detection of longer wavelengths. Gas flows through it continuously. Where there are multiple detectors, the gas is passed through them in series, then led to waste. The gas is usually 90% argon, 10% methane ("P10"), although the argon may be replaced with neon or helium where very long wavelengths (over 5 nm) are to be detected. The argon is ionised by incoming X-ray photons, and the electric field multiplies this charge into a measurable pulse. The methane suppresses the formation of fluorescent photons caused by recombination of the argon ions with stray electrons. The anode wire is typically tungsten or nichrome of 2060 m diameter. Since the pulse strength obtained is essentially proportional to the ratio of the detector chamber diameter to the wire diameter, a fine wire is needed, but it must also be strong enough to be maintained under tension so that it remains precisely straight and concentric with the detector. The window needs to be conductive, thin enough to transmit the X-rays effectively, but thick and strong enough to minimize diffusion of the detector gas into the high vacuum of the monochromator chamber. Materials often used are beryllium metal, aluminised PET film and aluminised polypropylene. Ultrathin windows (down to 1 m) for use with low-penetration long

wavelengths are very expensive. The pulses are sorted electronically by "pulse height selection" in order to isolate those pulses deriving from the secondary X-ray photons being counted.


Sealed gas detectors are similar to the gas flow proportional counter, except that the gas does not flow though it. The gas is usually krypton or xenon at a few atmospheres pressure. They are applied usually to wavelengths in the 0.150.6 nm range. They are applicable in principle to longer wavelengths, but are limited by the problem of manufacturing a thin window capable of withstanding the high pressure difference. Scintillation counters consist of a scintillating crystal (typically of sodium iodide doped with thallium) attached to a photomultiplier. The crystal produces a group of scintillations for each photon absorbed, the number being proportional to the photon energy. This translates into a pulse from the photomultiplier of voltage proportional to the photon energy. The crystal must be protected with a relatively thick aluminium/beryllium foil window, which limits the use of the detector to wavelengths below 0.25 nm. Scintillation counters are often connected in series with a gas flow proportional counter: the latter is provided with an outlet window opposite the inlet, to which the scintillation counter is attached. This arrangement is particularly used in sequential spectrometers. Semiconductor detectors can be used in theory, and their applications are increasing as their technology improves, but historically their use for WDX has been restricted by their slow response (see EDX).

  
A glass "bead" specimen for XRF analysis being cast at around 1100 C in a Herzog automated fusion machine in a cement plant quality control laboratory. 1 (top): fusing, 2: preheating the mould, 3: pouring the melt, 4: cooling the "bead"

 

[edit]Extracting analytical results

At first sight, the translation of X-ray photon count-rates into elemental concentrations would appear to be straightforward: WDX separates the X-ray lines efficiently, and the rate of generation of

secondary photons is proportional to the element concentration. However, the number of photons leaving the sample is also affected by the physical properties of the sample: so-called "matrix effects". These fall broadly into three categories:
   

X-ray absorption X-ray enhancement sample macroscopic effects All elements absorb X-rays to some extent. Each element has a characteristic absorption spectrum which consists of a "saw-tooth" succession of fringes, each step-change of which has wavelength close to an emission line of the element. Absorption attenuates the secondary X-rays leaving the sample. For example, the mass absorption coefficient of silicon at the wavelength of the aluminium K line is 50 m /kg, whereas that of iron is 377 m /kg. This means that a given concentration of aluminium in a matrix of iron gives only one seventh of the count rate compared with the same concentration of aluminium in a silicon matrix. Fortunately, mass absorption coefficients are well known and can be calculated. However, to calculate the absorption for a multi-element sample, the composition must be known. For analysis of an unknown sample, an iterative procedure is therefore used. It will be noted that, to derive the mass absorption accurately, data for the concentration of elements not measured by XRF may be needed, and various strategies are employed to estimate these. As an example, in cement analysis, the concentration of oxygen (which is not measured) is calculated by assuming that all other elements are present as standard oxides. Enhancement occurs where the secondary X-rays emitted by a heavier element are sufficiently energetic to stimulate additional secondary emission from a lighter element. This phenomenon can also be modelled, and corrections can be made provided that the full matrix composition can be deduced. Sample macroscopic effects consist of effects of inhomogeneities of the sample, and unrepresentative conditions at

its surface. Samples are ideally homogeneous and isotropic, but they often deviate from this ideal. Mixtures of multiple crystalline components in mineral powders can result in absorption effects that deviate from those calculable from theory. When a powder is pressed into a tablet, the finer minerals concentrate at the surface. Spherical grains tend to migrate to the surface more than do angular grains. In machined metals, the softer components of an alloy tend to smear across the surface. Considerable care and ingenuity are required to minimize these effects. Because they are artifacts of the method of sample preparation, these effects can not be compensated by theoretical corrections, and must be "calibrated in". This means that the calibration materials and the unknowns must be compositionally and mechanically similar, and a given calibration is applicable only to a limited range of materials. Glasses most closely approach the ideal of homogeneity and isotropy, and for accurate work, minerals are usually prepared by dissolving them in a borate glass, and casting them into a flat disc or "bead". Prepared in this form, a virtually universal calibration is applicable.


Further corrections that are often employed include background correction and line overlap correction. The background signal in an XRF spectrum derives primarily from scattering of primary beam photons by the sample surface. Scattering varies with the sample mass absorption, being greatest when mean atomic number is low. When measuring trace amounts of an element, or when measuring on a variable light matrix, background correction becomes necessary. This is really only feasible on a sequential spectrometer. Line overlap is a common problem, bearing in mind that the spectrum of a complex mineral can contain several hundred measurable lines. Sometimes it can be overcome by measuring a less-intense, but overlap-free line, but in certain instances a correction is inevitable. For instance, the K is the only usable line for measuring sodium, and it overlaps the zinc L (L2M4) line. Thus zinc, if present, must be analysed in order to properly correct the sodium value.

 

[edit]Other

spectroscopic methods using the same principle

It is also possible to create a characteristic secondary X-ray emission using other incident radiation to excite the sample: electron beam: electron microprobe (or Castaing microprobe); ion beam: particle induced X-ray emission (PIXE). When radiated by an X-ray beam, the sample also emits other radiations that can be used for analysis: electrons ejected by the photoelectric effect: X-ray photoelectron spectroscopy (XPS), also calledelectron spectroscopy for chemical analysis (ESCA) The de-excitation also ejects Auger electrons, but Auger electron spectroscopy (AES) normally uses an electron beam as the probe. Confocal microscopy X-ray fluorescence imaging is a newer technique that allow control over depth, in addition to horizontal and vertical aiming, for example, when analyzing buried layers in a painting.[3]
[edit]Instrument

  

 

qualification

A 2001 review,[4] addresses the application of portable instrumentation from QA/QC perspectives. It provides a guide to the development of a set of SOPs if regulatory compliance guidelines are not available. Further information: Verification and Validation
[edit]

 

 

X-Ray Fluorescence (XRF)

Karl Wirth, Macalester College and Andy Barth, Indiana University~Purdue University, Indianapolis

 

What is X-Ray Fluorescence (XRF)

An X-ray fluorescence (XRF) spectrometer is an x-ray instrument used for routine, relatively non-destructive chemical analyses of rocks, minerals, sediments and fluids. It works on wavelength-dispersive spectroscopic principles that are similar to an electron microprobe (EPMA). However, an XRF cannot generally make analyses at the small spot sizes typical of EPMA work (2-5 microns), so it is typically used for

bulk analyses of larger fractions of geological materials. The relative ease and low cost of sample preparation, and the stability and ease of use of x-ray spectrometers make this one of the most widely used methods for analysis of major and trace elements in rocks, minerals, and sediment.  

Fundamental Principles of X-Ray Fluorescence (XRF)

The XRF method depends on fundamental principles that are common to several other instrumental methods involving interactions between electron beams and xrays with samples, including: X-ray spectroscopy (e.g., SEM -EDS), X-ray diffraction (XRD), and wavelength dispersive spectroscopy (microprobe WDS). The analysis of major and trace elements in geological materials by x-ray fluorescence is made possible by the behavior of atoms when they interact with radiation. When materials are excited with high-energy, short wavelength radiation (e.g., X-rays), they can become ionized. If the energy of the radiation is sufficient to dislodge a tightly-held inner electron, the atom becomes unstable and an outer electron replaces the missing inner electron. When this happens, energy is released due to the decreased binding energy of the inner electron orbital compared with an outer one. The emitted radiation is of lower energy than the primary incident X-rays and is termed fluorescent radiation. Because the energy of the emitted photon is characteristic of a transition between specific electron orbitals in a particular element, the resulting fluorescent X-rays can be used to detect the abundances of elements that are present in the sample.

 

X-Ray Fluorescence (XRF) Instrumentation - How Does It Work?

The analysis of major and trace elements in geological materials by XRF is made possible by the behavior of atoms when they interact with X-radiation. An XRF spectrometer works because if a sample is illuminated by an intense X-ray beam, known as the incident beam, some of the energy is scattered, but some is also absorbed within the sample in a manner that depends on its chemistry. The incident X-ray beam is typically produced from a Rh target, although W, Mo, Cr and others can also be used, depending on the application.

  

Show Caption

When this primary X-ray beam illuminates the sample, it is said to be excited. The excited sample in turn emits X-rays along a spectrum of wavelengths characteristic of the types of atoms present in the sample. How does this happen? The atoms in

the sample absorb X-ray energy by ionizing, ejecting electrons from the lower (usually K and L) energy levels. The ejected electrons are replaced by electrons from an outer, higher energy orbital. When this happens, energy is released due to the decreased binding energy of the inner electron orbital compared with an outer one. This energy release is in the form of emission of characteristic X-rays indicating the type of atom present. If a sample has many elements present, as is typical for most minerals and rocks, the use of a Wavelength Dispersive Spectrometer much like that in an EPMA allows the separation of a complex emitted X-ray spectrum into characteristic wavelengths for each element present. Various types of detectors (gas flow proportional and scintillation) are used to measure the intensity of the emitted beam. The flow counter is commonly utilized for measuring long wavelength (>0.15 nm) X-rays that are typical of K spectra from elements lighter than Zn. The scintillation detector is commonly used to analyze shorter wavelengths in the X-ray spectrum (K spectra of element from Nb to I; L spectra of Th and U). X-rays of intermediate wavelength (K spectra produced from Zn to Zr and L spectra from Ba and the rare earth elements) are generally measured by using both detectors in tandem. The intensity of the energy measured by these detectors is proportional to the abundance of the element in the sample. The exact value of this proportionality for each element is derived by comparison to mineral or rock standards whose composition is known from prior analyses by other techniques.              

Applications
X-Ray fluorescence is used in a wide range of applications, including research in igneous, sedimentary, and metamorphic petrology soil surveys mining (e.g., measuring the grade of ore) cement production ceramic and glass manufacturing metallurgy (e.g., quality control) environmental studies (e.g., analyses of particulate matter on air filters) petroleum industry (e.g., sulfur content of crude oils and petroleum products) field analysis in geological and environmental studies (using portable, hand-held XRF spectrometers) X-Ray fluorescence is particularly well-suited for investigations that involve bulk chemical analyses of major elements (Si, Ti, Al, Fe, Mn, Mg, Ca, Na, K, P) in rock and sediment bulk chemical analyses of trace elements (in abundances >1 ppm; Ba, Ce, Co, Cr, Cu, Ga, La, Nb, Ni, Rb, Sc, Sr, Rh, U, V, Y, Zr, Zn) in rock and sediment - detection limits for trace elements are typically on the order of a few parts per million X-ray fluorescence is limited to analysis of relatively large samples, typically > 1 gram materials that can be prepared in powder form and effectively homogenized materials for which compositionally similar, well-characterized standards are available materials containing high abundances of elements for which absorption and fluorescence effects are reasonably well understood

    

In most cases for rocks, ores, sediments and minerals, the sample is ground to a fine powder. At this point it may be analyzed directly, especially in the case of trace element analyses. However, the very wide range in abundances of different elements, especially iron, and the wide range of sizes of grains in a powdered sample, makes the proportionality comparison to the standards particularly troublesome. For this reason, it is common practice to mix the powdered sample with a chemical flux and use a furnace or gas burner to melt the powdered sample. Melting creates a homogenous glass that can be analyzed and the abundances of the (now somewhat diluted) elements calculated.

       

Strengths and Limitations of X-Ray Fluorescence (XRF)?

Strengths
X-Ray fluorescence is particularly well-suited for investigations that involve: bulk chemical analyses of major elements (Si, Ti, Al, Fe, Mn, Mg, Ca, Na, K, P) in rock and sediment bulk chemical analyses of trace elements (>1 ppm; Ba, Ce, Co, Cr, Cu, Ga, La, Nb, Ni, Rb, Sc, Sr, Rh, U, V, Y, Zr, Zn) in rock and sediment

Limitations
In theory the XRF has the ability to detect X-ray emission from virtually all elements, depending on the wavelength and intensity of incident x-rays. However... In practice, most commercially available instruments are very limited in their ability to precisely and accurately measure the abundances of elements with Z<11 in most natural earth materials. XRF analyses cannot distinguish variations among isotopes of an element, so these analyses are routinely done with other instruments (see TIMS and SIMS). XRF analyses cannot distinguish ions of the same element in different valence states, so these analyses of rocks and minerals are done with techniques such as wet chemical analysis or Mossbauer spectroscopy.

 

 

User's Guide - Sample Collection and Preparation

Virtually any solid or liquid material can be analyzed, if adequate standards are available. For rocks and minerals, typical commercial instruments require a sample constituting at least several grams of material, although the sample collected may be much larger. For XRF chemical analyses of rocks, samples are collected that are several times larger than the largest size grain or particle in the rock. This initial sample then suffers a series of crushing steps to reduce it to an average grain size of a few millimeters to a centimeter, when it can be reduced by splitting to a small representative sample of a few tens to hundreds of grams. This small sample split is then ground into a fine powder by any of a variety of techniques to create the XRF sample. Care must be taken particularly at this step to be aware of the composition of the crushing implements, which will inevitably contaminate the sample to some extent.

Data Collection, Results and Presentation

          

X-Ray spectrum Data table Detection limits Precision Accuracy Database and Plotting Evaluation of Data Quality (flyers, trends, discriminant fields) Geochemical Plots

Basic Theory of X-ray Fluorescence

 X-ray Fluorescence Introduction Although X-ray fluorescence spectroscopy is no longer regarded as a new instrumental technique for elemental analysis, ongoing evolutionary developments continue to redefine the role of this important analytical tool. From the demonstration of the first principles in the 1960s to the development of the first commercial instruments in the 1970s, the increasing availability of affordable computational power has a least been as important to the desirability and acceptance of the technology as innovative hardware design. With the widespread availability and use of a 32-bit microprocessor personal computer as the industry standard platform, X-ray fluorescence spectroscopy has become a useful and complimentary laboratory tool to other techniques.  X-Ray Fluorescence Theory An electron can be ejected from its atomic orbital by the absorption of a light wave (photon) of sufficient energy. The energy of the photon (hv) must be greater than the energy with which the electron is bound to the nucleus of the atom. When an inner orbital electron is ejected from an atom, an electron from a higher energy level orbital will be transferred to the lower energy level orbital. During this transition a photon maybe emitted from the atom. This fluorescent light is called the characteristic X-ray of the element. The energy of the emitted photon will be equal to the difference in energies between the two orbitals occupied by the electron making the transition. Because the energy difference between two specific orbital shells, in a given element, is always the same (i.e. characteristic of a particular element), the photon emitted when an electron moves between these two levels, will always have the same energy. Therefore,

by determining the energy (wavelength) of the X-ray light (photon) emitted by a particular element, it is possible to determine the identity of that element. For a particular energy (wavelength) of fluorescent light emitted by an element, the number of photons per unit time (generally referred to as peak intensity or count rate) is related to the amount of that analyte in the sample. The counting rates for all detectable elements within a sample are usually calculated by counting, for a set amount of time, the number of photons that are detected for the various analytes characteristic X-ray energy lines. It is important to note that these fluorescent lines are actually observed as peaks with a semi-Gaussian distribution because of the imperfect resolution of modern detector technology. Therefore, by determining the energy of the X-ray peaks in a samples spectrum, and by calculating the count rate of the various elemental peaks, it is possible to qualitatively establish the elemental composition of the samples and to quantitatively measure the concentration of these elements.
Nuclear quadrupole resonance spectroscopy or NQR is a chemical analysis technique related to nuclear magnetic resonance (NMR).[1] [edit]Principle In NMR, nuclei with spin 1/2 have a magnetic dipole moment so that their energies are split by a magnetic field, allowing resonance absorption of energy related to the difference between the ground state energy and the excited state. In NQR, on the other hand, nuclei with spin 1 , such as N,
14 35

 

Cland Cu, also have an electric quadrupole moment so that their energies are split by

63

an electric field gradient, created by the electronic bonds in the local environment. Since unlike NMR, NQR is done in an environment without a static (or DC) magnetic field, it is sometimes called "zero field NMR". Many NQR transition frequencies depend strongly upon temperature.  Any nucleus with more than one unpaired nuclear particle (protons or neutrons) will have a charge distribution which results in an electric quadrupole moment. Allowed nuclear energy levels are shifted unequally due to the interaction of the nuclear charge with an electric field gradient supplied by the non-uniform distribution electron density (e.g. from bonding electrons) and/or surrounding ions. The NQR effect results when transitions are induced between these nuclear levels by an externally appliedradio frequency (RF) magnetic field. The technique is very sensitive to the nature and symmetry of the bonding around the nucleus. The energy level shifts are much larger than the chemical shifts measured in NMR. Due to symmetry, the shifts become averaged to zero in the liquid phase, so NQR spectra can only be measured for solids.   [edit]Applications There are several research groups around the world currently working on ways to use NQR to detect explosives. Units designed to detect landmines[2] and explosives concealed in luggage have been tested. A detection system consists of a radio frequency (RF) power source, a coil to

produce the magnetic excitation field and a detector circuit which monitors for a RF NQR response coming from the explosive component of the object.  Another practical use for NQR is measuring the water/gas/oil coming out of an oil well in realtime. This particular technique allows local or remote monitoring of the extraction process, calculation of the well's remaining capacity and the water/detergents ratio the input pump must send to efficiently extract oil.[citation
needed]

 

BULK EXPLOSIVE DETECTION TECHNIQUES Biological and chemical methods for detecting explosive vapors currently are limited by incomplete knowledge of how explosive vapors migrate in the shallow subsurface. An additional category of explosive detection technologies overcomes this limitation by searching for the bulk explosive inside the mine. Methods being explored for this purpose include nuclear quadrupole resonance (NQR) and a variety of methods that use the interaction of neutrons with components of the explosive. These technologies emerged from interest in detecting bulk explosives in passenger baggage for the airline industry and in investigating the potential presence of explosive devices in other settings.

        

Contents
[hide]

1 Description 2 Strengths 3 Limitations 4 Summary Evaluation 5 Links

Description
NQR is a radio frequency (RF) technique that can be used to interrogate and detect specific chemical compounds, including explosives. An NQR device induces an RF pulse of an appropriate frequency in the subsurface via a coil suspended above ground. This RF pulse causes the explosives nuclei to resonate and induces an electric potential in a receiver coil. This phenomenon is similar to that exploited by magnetic resonance imaging (MRI) used in medical testing, but NQR uses the internal electric field gradient of the crystalline material rather than an external static magnetic field to initially align the nuclei.

 

Strengths
NQR has a number of features that make it particularly well suited for landmine detection. The primary attraction of NQR is its specificity to landmines: In principle, it signals only in the presence of bulk quantities of specific explosives. Unlike many other technologies, its false alarm rate is not driven by ground clutter but rather by its signal-to-noise ratio (SNR). The SNR increases with the square root of the interrogation time and also increases linearly with the mass of the explosive. Thus, with sufficient interrogation time, NQR can achieve nearly perfect operating characteristics (probability of detection near one with probability of false alarm near zero). This makes NQR more attractive as a confirmation sensor used to interrogate only those locations identified by other detectors (e.g., GPR,EMI) as likely mine locations. Interrogation times of 0.53.0 minutes may be sufficient for performance that leads to high probability of detection (more than 0.99) and low probability of false alarm (less than 0.05). The NQR signal from cyclotrimethylenenitramine (RDX) is particularly large, implying high performance and small interrogation times (less than three seconds) for detection of mines containing RDX. Another positive feature of NQR is that it is relatively robust to diverse soil conditions; for example, because it requires bulk concentration of explosives to declare, it is not misled by trace explosive residues as can be the case with vapor-sensing techniques.

 

Limitations
The major weakness of NQR is the fact that, because of its nuclear properties, TNT, which comprises the explosive fill of most landmines, provides a substantially weaker signal than either RDX or tetryl, posing a formidable SNR problem. Moreover, TNT inherently requires longer interrogation times because its nuclear properties preclude interrogation more frequently than once per five to ten seconds. Another significant limitation is the susceptibility of NQR to RF interference from the environment. This is especially problematic for TNT detection because the frequencies required to induce a response from TNT (790900 kHz) are in the AM radio band. When present, radio signals overwhelm the response from TNT. An additional weakness is that NQR cannot locate explosives that are encased in metal because the RF waves will not penetrate the case. This is not a major weakness because a large majority of antipersonnel mines have plastic cases, and EMI detection can successfully detect those with metal cases. NQR also cannot detect liquid explosives, but very few antipersonnel mines use liquid explosives. NQR is very sensitive to the distance between the detection coil and the explosive. Therefore, the detection coil must be operated very close to the ground, which can be problematic in rough or

highly vegetated terrain. Moreover, current implementations require stationary detection for optimal results; detection in motion substantially degrades the SNR.  

Summary Evaluation
NQR is an explosive-specific detection technology that offers considerable promise as a technique for reducing false alarms as compared with such conventional detection approaches as EMI. It offers opportunities for improvement not addressed by competing technologiesmost notably that its potency derives from unique explosive signatures of mines. In principle, this specificity affords it the possibility of a zero false alarm rate against nonmetal mines. Currently, the most promising role of NQR is that of a confirmation sensor used in conjunction with a conventional scanning sensor or as part of an integrated multisensor detection system.

CHEMISTRY: ON NUCLEAR QUADRUPOLE RESONANCE The following points are made by J.B. Miller and G.A. Barrall (American Scientist 2005 93:50): 1) Nuclear quadrupole resonance (NQR) has much in common with nuclear magnetic resonance (NMR), the fundamental physical process that makes magnetic resonance imaging possible. Nuclear magnetic resonance, first demonstrated in 1946, takes advantage of the fact that certain atomic nuclei possess magnetic dipole moments -- that is, these nuclei act like tiny bar magnets, each with a north magnetic pole at one end and a south magnetic pole at the other. The laws of quantum mechanics dictate that when such nuclei are subjected to an externally applied magnetic field, they must align themselves along it. But the magnetic moments of these nuclei, usually depicted as arrows, are allowed only two possible orientations: in the same direction as the applied magnetic field or opposite to it. 2) Although alignment with the applied field is favored (this being the lower-energy condition), the energy difference between the two orientations is such that thermal agitation is usually sufficient to ensure that only slightly more than half the nuclei are in the lower-energy state. The key is that the nuclei can occupy two distinct states separated by a well-defined increment in energy. (It will be well defined as long as the applied magnetic field is uniform.) In that sense, the situation is much like that of an electron in an atom, which can be in the "ground" state or in a higher-energy "excited" state.

3) A ground-state electron shifts to an excited state when the atom receives a dollop of electromagnetic radiation of just the right energy to put it there -- that is, when it absorbs a photon of just the right frequency. Conversely, if this excited-state electron falls back to the ground state, the atom will emit a photon of the exact same frequency to carry away the difference in energy. In NMR, the energy difference between states is much less than for the electronic states of an atom, so the relevant frequencies are much lower. Rather than dealing with optical frequencies, NMR typically involves oscillations of just a few tens to hundreds of megahertz, which includes the band where broadcast FM radio stations operate. 4) Nuclear quadrupole resonance is similar in concept, but unlike nuclear magnetic resonance it does not rely on nuclei aligning themselves in an externally applied magnetic field. Instead, NQR exploits the fact that some nuclei possess an electric quadrupole moment, which can be thought of as arising from two back-to-back electric dipoles (positive and negative charges separated by a short distance). Why do some atomic nuclei have an electric quadrupole moment? Physicists would say because they have a spin quantum number greater than 1/2. A more intuitive explanation is because the positive electric charge these nuclei carry is not distributed with perfect spherical symmetry. 5) Consider for a moment a spherical nucleus with its positive charge distributed uniformly throughout. Now squeeze that nucleus in your mind's eye so that what was originally shaped like a basketball is flattened into a pumpkin. A pumpkin of positive charge can be thought of, to a rough approximation, as being the sum of a sphere of positive charge and two oppositely directed electric dipoles, one at the top and one at the bottom. That is, the only requirement for an electric quadrupole moment is that the nucleus be squashed (or stretched) along one axis. 6) When a nucleus possessing such an electric quadrupole moment is subjected to an electric field that varies from place to place, interesting things happen. The intrinsic electric quadrupole moment of the nucleus and the electric-field gradient imposed from outside together create distinct energy states. This result is analogous to the multiple energy states in NMR, where the critical ingredients were the intrinsic magnetic dipole moment of the nucleus and a magnetic field imposed from the outside.

7) The key difference between NMR and NQR is the definition of "outside." In NMR, the outside magnetic field arises because the experimenter has invested considerable effort in setting it up, perhaps using a superconducting electromagnet. In NQR, the required electric field (or, more precisely, the required electric-field gradient) comes free: It reflects the local arrangement of electrons around the nucleus under study. That arrangement, in turn, depends not only on the nature of the atom but also on its chemical environment. This feature accounts for one of the chief benefits of NQR -- the method is exquisitely sensitive to chemistry.[1-4]
 X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam ofX-rays strikes a crystal and diffracts into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a threedimensional 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 theirchemical bonds, their disorder and various other information.  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 electronicor 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 asreflections. 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 ofFourier 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 ofelectron 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.  
Contents
[hide]

1 History

   

Early scientific history of crystals and X-rays X-ray analysis of crystals Development from 1912 to 1920

Contributions to chemistry and material science

   

Mineralogy and metallurgy Early organic and small biological molecules Biological macromolecular crystallography

Relationship to other scattering techniques

   
Methods

Elastic vs. inelastic scattering Other X-ray techniques Electron and neutron diffraction

Overview of single-crystal X-ray diffraction

   

Procedure Limitations

Crystallization Data collection

  

Mounting the crystal X-ray sources Recording the reflections

Data analysis

    

Crystal symmetry, unit cell, and image scaling Initial phasing Model building and phase refinement

Deposition of the structure

Diffraction theory

        
See also

Intuitive understanding by Bragg's law Scattering as a Fourier transform Friedel and Bijvoet mates Ewald's sphere Patterson function Advantages of a crystal

References Further reading

     

International Tables for Crystallography Bound collections of articles Textbooks Applied computational data analysis Historical

External links

     

Tutorials Primary databases Derivative databases 9.4 Structural validation

[edit]History [edit]Early

scientific history of crystals and X-rays

  
Drawing of square (Figure A, above) and hexagonal (Figure B, below) packing fromKepler's work, Strena seu de Nive Sexangula.

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.[1]

  
As shown by X-ray crystallography, the hexagonal symmetry of snowflakes results from the tetrahedral arrangement ofhydrogen bonds about each water molecule. The water molecules are arranged similarly to the silicon atoms in the tridymitepolymorph of SiO2. 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 Ren Just Hay (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 indiceswhich are
[2]

still used today for identifying crystal faces. Hay'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,
[3]

Auguste Bravais,

[4]

Yevgraf Fyodorov,

[5]

Arthur Schnflies[6] 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.
[7]

  
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-rays were discovered by Wilhelm Conrad Rntgen 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 oflight 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 linesakin to those observed in the visible wavelengths. Single-slit experiments in the laboratory of Arnold Sommerfeldsuggested the wavelength of X-rays was about 1 Angstrm. 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,[8] but it was not broadly accepted until 1922,[9][10] when Arthur Compton confirmed it by the scattering of X-rays from electrons.
[11]

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.[12][13][14][15] Nevertheless, Bragg's view was not broadly accepted and the observation of X-ray diffraction in 1912[16] confirmed for most scientists that Xrays were a form of electromagnetic radiation.  [edit]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 equals an integer multiple of

the wavelength . In that case, part of the incoming beam is deflected by an angle 2 , 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 byBragg's law: 

Here d is the spacing between diffracting planes,

is the incident angle, n is any integer, and

is

the wavelength of the beam. These specific directions appear as spots on thediffraction pattern called reflections. Thus, X-ray diffractionresults from an electromagnetic wave (the X-ray) impinging on a regular array of scatterers (the repeating arrangement of atoms within the crystal).  X-rays are used to produce the diffraction pattern because their wavelength is typically the

same order of magnitude (1-100 ngstrms) 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 artificialdiffraction 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 ngstrms) 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.[16][17] 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/m 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.  [edit]Development

from 1912 to 1920

 

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 arrangedtetrahedrally and held together by singlecovalent 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.

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.[19][20][21] 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 1914 was that of table salt.[22][23][24] 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 ionic compounds.[25] The structure ofdiamond was solved in the same year,[26][27] proving the tetrahedral arrangement of its chemical bonds and showing that the length of CC single bond was 1.52 ngstrms. Other early structures includedcopper, and pyrite (FeS2)
[29] [28]

calcium fluoride (CaF2, also known as fluorite), calcite (CaCO3)

in 1914; spinel (MgAl2O4) in 1915;[30][31] the rutile and anatase forms

of titanium dioxide (TiO2) in 1916;[32] pyrochroite Mn(OH)2 and, by extension, brucite Mg(OH)2 in 1919;.[33][34] Also in 1919sodium nitrate (NaNO3) and cesium dichloroiodide (CsICl2) were determined by Ralph Walter Graystone Wyckoff, and the wurtzite (hexagonal ZnS) structure became known in 1920.[35]  The structure of graphite was solved in 1916[36] by the related method of powder diffraction,[37] which was developed by Peter Debye and Paul Scherrer and, independently, by Albert Hull in 1917.[38] The structure of graphite was determined from single-crystal diffraction

in 1924 by two groups independently. [edit]Contributions

[39][40]

Hull also used the powder method to determine the and magnesium.
[42]

structures of various metals, such as iron  

[41]

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,[26]the octahedral bonding of metals observed in ammonium hexachloroplatinate (IV),[43] and the resonance observed in the planar carbonate group[29] and in aromatic molecules.[44] Kathleen Lonsdale's 1928 structure of hexamethylbenzene[45] established the hexagonal symmetry of benzeneand showed a clear difference in bond length between the aliphatic CC bonds and aromatic CC bonds; this finding led to the idea of resonance between chemical bonds, which had profound consequences for the development of chemistry.[46] Her conclusions were anticipated by William Henry Bragg, who published models of naphthalene and anthracene in 1921 based on other molecules, an early form of molecular replacement.[44][47]

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 andanatase 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,[48][49][50] metal-metal quadruple bonds,[51][52][53] and three-center, two-electron bonds.[54] X-ray crystallography or, strictly speaking, an inelastic Compton scattering experiment has also provided evidence for the partly covalent character of hydrogen bonds.[55] In the field of organometallic chemistry, the Xray structure offerrocene initiated scientific studies of sandwich compounds,[56][57] while that of Zeise's saltstimulated research into "back bonding" and metal-pi complexes.[58][59][60][61] 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.   [edit]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.[62][63][64][65][66][67] Most notably, Linus Pauling's structure of the alloy Mg2Sn[68] led to his theory of the stability and structure of complex ionic crystals.[69] [edit]Early

organic and small biological molecules

  
The three-dimensional structure ofpenicillin, for which Dorothy Crowfoot Hodgkin was awarded the Nobel Prize in Chemistry in 1964. The green, white, red, yellow and blue spheres represent atoms ofcarbon, hydrogen, oxygen, sulfur andnitrogen, respectively.

The first structure of an organic compound,hexamethylenetetramine, was solved in 1923. biological membranes.[71][72][73][74][75][76][77][78][79] In the 1930s, the structures of much larger

[70]

This

was followed by several studies of long-chain fatty acids, which are an important component of molecules with two-dimensional complexity began to be solved. A significant advance was the structure of phthalocyanine,[80] 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 withDorothy Crowfoot Hodgkin, who solved the structures ofcholesterol (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 ofinsulin, on which she worked for over thirty years.[81]

  
Ribbon diagram of the structure ofmyoglobin, 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 subatomic 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).

 

[edit]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 ofsperm whale myoglobin by Max Perutz and Sir John Cowdery Kendrew, for which they were awarded the Nobel Prize in Chemistry in 1962.[82] Since that success, over 48970 X-ray crystal structures of proteins, nucleic acids and other biological molecules have been determined.[83]For comparison, the nearest competing method in terms of structures analyzed is nuclear magnetic resonance (NMR) spectroscopy, which has resolved 7806 chemical structures.[84] 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 interacts with its protein target and what changes might improve it.[85] 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, such as ion channels andreceptors.[86][87] [edit]Relationship

  

to other scattering techniques

Further information: X-ray scattering techniques [edit]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 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 (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.

 

[edit]Other

X-ray techniques

Other forms of elastic X-ray scattering include powder diffraction, SAXS and several types of Xray 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. A Greninger chart can be used  
[88]

to interpret the back reflection Laue photograph.

[edit]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 rescattered 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.

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, H2O, and heavy water, D2O. [edit]Methods [edit]Overview

 

of single-crystal X-ray diffraction

   
Workflow for solving the structure of a molecule by X-ray crystallography.

The oldest and most precise method of X-raycrystallography 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.
[89]

Each spot is called

areflection, 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 ngstrm and to within a few tenths of adegree, respectively. The atoms in a crystal are not static, but oscillate about their mean positions, usually by less than a few tenths of an ngstrm. X-ray crystallography allows measuring the size of these oscillations.   [edit]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 ortwinning.  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.    [edit]Limitations See also: Resolution (electron density) 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" crystallographyare 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.   [edit]Crystallization Further information: crystallization, recrystallization, and Protein Crystallization

  
A protein crystal seen under amicroscope. 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 diffractionquality 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.

   
Three methods of preparing crystals, A: Hanging drop. B: Sitting drop. C: Microdialysis

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, ideally to a diffractionquality crystal.[94] 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.
[95]

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.[96] 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 perexperiment when compared to crystallization trials setup by hand (in the order of 1 microliter).[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.   [edit]Data

collection

[edit]Mounting the crystal

   
Animation showing the five motions possible with a four-circle kappa goniometer. The rotations about each of the four angles , , and 2 leave the crystal within the X-ray beam, but change the crystal orientation. The detector

(red box) can be slid closer or further away from the crystal, allowing higher resolution data to be taken (if closer) or better discernment of the Bragg peaks (if further away).

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 (themother 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.[98]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 often crack if flash-frozen; therefore, they are generally pre-soaked in a cryoprotectant solution before freezing.[99] 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 the beam; the loop/capillary axis. When the angle, which rotates about an axis perpendicular to axis; and, finally, the and angle about the rotation allows axes are aligned. The angle, about an axis at ~50 to the angle is zero, the

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 [edit]X-ray sources Further information: Diffractometer and Synchrotron 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. axis only. An older type of goniometer is the four-circle goniometer, and its relatives such as the six-circle goniometer.   

   
A diffractometer

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 innershell electrons of the metal. The most common metal used iscopper, which can be kept cool easily, due to its highthermal conductivity, and which produces strong K and K lines. The K line is sometimes suppressed with a thin (~10 m) 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 )  [edit]Recording the reflections

  
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.

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 withphotographic 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 cases, data sets on multiple crystals must be taken.     [edit]Data
[100]

analysis

[edit]Crystal symmetry, unit cell, and image scaling Further information: Space group 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.[101] 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 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.    [edit]Initial phasing Further information: Phase problem 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 acomplex 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 (140 pm), direct methods can be used to obtain phase information, by exploiting known phase relationships between certain groups of reflections.[102][103]  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.[104]  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. 
[105]

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. [edit]Model building and phase refinement
[104]

  
A protein crystal structure at 2.7 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.

 

Further information: Molecular modeling 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 R-factor defined as

A similar quality criterion is Rfree, 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, Rfree should be approximately the resolution in ngstrms divided by 10; thus, a data-set with 2 resolution should yield a finalRfree ~ 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.[clarification needed]

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.

 

[edit]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. [edit]Diffraction

  

theory

Further information: Dynamical theory of diffraction and Bragg diffraction The main goal of X-ray crystallography is to determine the density of electrons f(r) 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 divided by the wavelength of the oscillation. The corresponding formula for a Fourier transform will be used below

  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 |F(q)| and aphase (q) related by the equation   The intensities of the reflections observed in X-ray diffraction give us the magnitudes |F(q)| but not the phases (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 f(r).  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.  [edit]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, k, l), 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 multiple n of the X-ray wavelength . between the plane and the X-ray results in a path-length difference that is an integer

  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 2 . 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.   [edit]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 kin, and so the strength of the incoming wave at time t=0 is given by

  At position r within the sample, let there be a density of scatterers f(r); 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    where S is the proportionality constant. Let's consider the fraction of scattered waves that leave with an outgoing wave-vector of kout and strike the screen at rscreen. Since no energy is lost (elastic, not inelastic scattering), the wavelengths are the same as are the magnitudes of the wave-vectors |kin|=|kout|. From the time that the photon is scattered at r until it is absorbed at rscreen, the photon undergoes a change in phase   The net radiation arriving at rscreen is the sum of all the scattered waves throughout the crystal

  which may be written as a Fourier transform

  where q = kout - kin. The measured intensity of the reflection will be square of this amplitude  

[edit]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 f(r) at a position r is always a real number. As noted above, f(r) is the inverse transform of its Fourier transform F(q); however, such an inverse transform is a complex number in general. To ensure that f(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)   The equality of their magnitudes ensures that the Friedel mates have the same intensity |F|2. 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.  The Friedel-mate constraint can be derived from the definition of the inverse Fourier transform

  Since Euler's formula states that eix = 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 f(r) is real if and only if the second integral Isin is zero for all values of r. In turn, this is true if and only if the above constraint is satisfied

  since Isin = Isin implies that Isin=0.   [edit]Ewald's

sphere

Further information: 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 kout and kin have the same length, due to the elastic scattering, since the wavelength 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 Xray beam, the values of|F(q)|can 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.   [edit]Patterson

function

Further information: Patterson function A well-known result of Fourier transforms is the autocorrelation theorem, which states that the autocorrelation c(r) of a function f(r)

  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[106]) 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-1) 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.  [edit]Advantages

of a

crystal
 In principle, an atomic structure could be determined from applying X-ray scattering to noncrystalline 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 cellsrepeated indefinitely in three independent directions. Such periodic systems have a Fourier transformthat 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. In other words, using a crystal concentrates the 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.     Bragg diffraction Bravais lattice Crystallographic database [edit]See

also

         

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)

BASIC PHOTOCHEMISTRY

Kendric C. Smith Stanford University School of Medicine 927 Mears Ct., Stanford, CA 94305-1041 kendric@stanford.edu www.stanford.edu/~kendric/

Photochemistry is the underlying mechanism for all of photobiology. When a molecule absorbs a photon of light, its electronic structure changes, and it reacts differently with other molecules. The energy that is absorbed from light can result in photochemical changes in the absorbing molecule, or in an adjacent molecule (e.g.,

photosensitization). The energy can also be given off as heat, or as lower energy light, i.e., fluorescence or phosphorescence, in order to return the molecule to its ground state. Each type of molecule has a different preference for which of these different mechanisms it uses to get rid of absorbed photon energy, e.g., some prefer fluorescence over chemistry.

The Basic Laws of Photochemistry The First Law of Photochemistry states that light must be absorbed for photochemistry to occur. Although this is a very simple law, some people in Low Level Light Therapy believe that light can produce effects without being absorbed. More education is required! The Second Law of Photochemistry states that for each photon of light absorbed by a chemical system, only one molecule is activated for a photochemical reaction. This law is true for ordinary light intensities, however, with high-powered lasers, two-photon reactions can occur, i.e., the molecule is raised to a higher energy state than produced by single-photon absorption. The Bunsen-Roscoe Law of Reciprocity states that a photochemical effect is directly proportional to the total energy dose, irrespective of the time required to deliver the dose. This law is true for chemicals in a test tube, but the response of cells to radiation usually involves a sequence of interacting biological reactions, making a linear "dose x time" relationship highly unlikely. There is no reciprocity when damage is produced, e.g., DNA damage, but there can be reciprocity over a narrow range of doses for photoreceptors that trigger a response, such as phytochrome (see module on Basic Photomorphogenesis).

Electromagnetic Radiation Electromagnetic radiation consists of waves of electric and magnetic fields traveling in space at right angles to one another (Figure 1).

Figure 1. An electromagnetic wave showing the perpendicularlyoriented waves of electric and magnetic fields, and the characteristic wavelength ( ) of the radiation.

The electromagnetic spectrum is composed of different wavelengths of light having different photon energies, and is classified into the regions shown in Figure 2. Note that the regions of interest for photochemistry, i.e., visible and ultraviolet (UV), are only a small part of the full electromagnetic spectrum. Longer wavelengths, e.g., far infrared, tend to cause the vibrational excitation of molecules, which results in heating. Shorter wavelength X-rays cause ionization.

Figure 2. The electromagnetic spectrum highlighting the visible region, which along with the ultraviolet region, is capable of producing photochemical changes in molecules.

The Jablonski Diagram The energy gained by a molecule when it absorbs a photon causes an electron to be promoted to a higher electronic energy level. Figure 3 illustrates the principal photophysical radiative and nonradiative processes displayed by organic molecules in solution. The symbols So, S1, T2, etc., refer to the ground electronic state (So), first excited singlet state (S1), second excited triplet state (T2), and so on. The horizontal lines represent the vibrational levels of each electronic state. Straight arrows indicate radiative transitions, and curly arrows indicate non-radiative transitions. The boxes detail the electronic spins in each orbital, with electrons shown as up and down arrows, to distinguish their spin. Note that all transitions from one electronic state to another originate from the lowest vibrational level of the initial electronic

state. For example, fluorescence occurs only from S1, because the higher singlet states (S2, etc.) decay so rapidly by internal conversion that fluorescence from these states cannot compete.

Figure 3. The basic concepts of this Jablonski diagram are presented in the Basic Photophysics module. This version emphasizes the spins of electrons in each of the singlet states (paired, i.e., opposite orientation, spins) compared to the triplet states (unpaired, i.e., same orientation, spins).

Electronically Excited States The absorption of a UV or visible photon by a molecule produces an electronically excited state. The distribution of the electrons surrounding the nuclei change, as well as the forces between the atomic nuclei of a molecule. As a result, molecules in electronically excited states often have very different chemical and physical properties than their electronic ground states. For example, hydroxy naphthalene becomes a strong acid in its excited state. The

ground state of -hydroxy naphthalene has a pKa of 9.2, but this is reduced to 0.4 in the excited singlet state. Molecules such as this are known as photoacids.

The Beer-Lambert Law The absorption of photons of light is described by the Beer-Lambert Law, a relationship that indicates a decrease in intensity as a beam passes through a medium that can absorb it. Consider a parallel beam of monochromatic light of initial intensity, lo, passing through a homogeneous absorbing medium (Figure 4).

Figure 4. Schematic representation showing that light of initial intensity, lo, passing through an absorbing medium in a cuvette with light path, l, will emerge with a final intensity, lt.

In spectroscopy, absorbance, A, and Optical Density, OD, are used somewhat interchangeably. Optical Density can be expressed as: OD = log10(1/T) where T is the transmittance. It can also be expressed as: A = OD = -log10(lt/lo) [using the symbols in Figure 4]

Note the log scale. An OD of 1 will have a transmittance of 0.1, and a % transmission of 10, but at OD = 2, the transmittance is 0.01, and the % transmission = 1. Another way of expressing this information is to use the BeerLambert Law. It states that the absorbance, A, of a molecular species is linearly related to the path length (centimeter), l, the absorber concentration (moles/liter), c, and the proportionality constant, , called the molar extinction coefficient of the absorbing molecular species (liters/mole-cm) [a measure of how strongly a chemical species absorbs light at a given wavelength]. A= cl

Energy Level Diagram One way to view the properties of molecular excited states is shown by the potential energy diagram in Figure 5. This diagram, known as aFranck-Condon energy level diagram, shows potential energy curves for the ground state (So), and first excited singlet state (S1) of an organic molecule as a function of nuclear configuration. These curves are sometimes referred to as potential energy wells, because of their shape. The horizontal lines within each curve represent the vibrational levels of each electronic state. The lowest vibrational state for each energy level is designated as 0, and the levels above it are successively 1, 2, etc. The band assignments in brackets (e.g., (0, 1)) indicate, respectively, the vibrational level of the initial state, and of the final state involved in a transition.

Figure 5. Franck-Condon Energy Level Diagram.

The horizontal axis is the nuclear configuration, which can be thought of as the distance between nuclei. When considering two atoms bonded to each other, the bottom of the well corresponds to the equilibrium bond length. Because excitation involves the movement of charge density into an antibonding orbital, the equilibrium bond length in S1 is generally longer than in S0. This is illustrated in the diagram by the displacement of the S1 well to the right of the S0 well. The absorption of light takes place on a much faster time scale (~ 1015 s) than molecular vibration (~ 1012 s), and hence the initially formed excited state must have the same nuclear configuration as the ground state. This transition is called the vertical or Franck-Condon transition, and results in the molecule having excess vibrational energy. The excess vibrational energy can be dissipated through the process of vibrational relaxation, i.e., the

process of internal conversion, which returns the molecule to the lowest vibrational level of S1. Fluorescence usually occurs from the lowest vibrational level of S1. Because these transitions occur at lower energies than absorption, fluorescence is observed at longer wavelengths ( ) than absorption (i.e., lower energy), as shown in the lower right corner of Figure 5.

Quantum Yields and Lifetimes The energy that a molecule gains when it absorbs light is subsequently lost by a molecule in one of several ways. As shown graphically in the Jablonski diagram (Figure 3), it can lose the energy as heat as it returns to the ground state (internal conversion). Alternately, it can lose the energy as light (fluorescence), usually on a nanosecond time scale. A third pathway is intersystem crossing to a triplet state, from which energy can also be lost as light (phosphorescence), but over much longer times (microseconds or longer). And finally, the energy can be transferred to another molecule. The quantum yield of a process is the probability that an absorbed photon undergoes one particular process. Thus, one can define a quantum yield for fluorescence, a quantum yield for phosphorescence, or a quantum yield for other pathways. Each quantum yield is typically a number between 0 and 1 (except under unusual circumstances), and the total of all quantum yields for a particular absorption event should sum to one. Note that these processes are competing. If conditions are altered such that the quantum yield for fluorescence is increased, then the quantum yield for some other process(es) must decrease. Consider a molecule, M, that is exposed to light, and absorbs photons at the rate, Iabs. As shown in the following formulas, the

excited singlet state of the molecule, 1M*, can fluoresce emitting a photon, h f. It can lose energy as heat, and move to the triplet excited state, 3M*, by intersystem crossing (isc). Finally, it can lose energy as heat, and move to the ground state by internal conversion (ic). The rate of each of these loss processes will be proportional to the concentration of the excited singlet state, 1M*, and a rate constant, k, for each process, as given in the second column below.

Because the lifetime of the singlet excited state is relatively short, we can assume that all of the excited singlet states that are formed by light absorption will rapidly decay through one of the three means just described. This is the "steady state" approximation. Since the rate of formation of the excited singlet state is Iabs , then the sum of the rates of loss, each given above, must be equal to the rate of formation.

From this relationship, one can determine the quantum yield for each process. It is simply the rate of that process as a fraction of all pathways for the loss of energy.

Intermolecular Processes: Excited State Quenching

When a second molecule interacts with a molecule in an excited state, new ways may be created for the excited state species to lose its energy of excitation. Such interactions (collisions) can induce the loss of energy in the form of heat, which is called physical quenching, or it can cause the energy to be transferred to the second molecule with or without the transfer of an electron. The former is called simply energy transfer, and the latter electron transfer. Formally, one can write:

When this kind of quenching occurs it reduces the concentration of the excited state more rapidly than if the quencher were not present. This means that fluorescence, which is proportional to the concentration of the excited state will also be reduced. By monitoring fluorescence from the excited state molecule, one can determine the concentration of a quencher, if its rate constant of interaction with the excited state is known. Once again, consider the molecule, M, that can decay by fluorescence, intersystem crossing or internal conversion. Now add a fourth possiblility, quenching. The reactions and rates are then given by:

Note that the quenching reaction rate depends on 2 reactants, M and Q, and the rate constant is a bimolecular rate constant. Once again, one can make the steady state approximation, and compare the rate of fluorescence, kf[1M*] in the presence of the quencher to the same quantity in the absence of the quencher. This leads to the following relationship:

Where is the intensity, or rate of fluorescence, without a quencher; is the intensity, or rate of fluorescence, with a quencher; is the quencher rate coefficient; is the fluorescence lifetime of A without a quencher present, and is the concentration of the quencher. This relationship is referred to as the Stern-Volmer equation.

Types of Photochemical Reactions 1. Linear addition to an unsaturated molecule, e.g., the pyrimidine base, thymne, in DNA can combine with the amino acid residue, cysteine, in proteins. This is a model for the photochemical crosslinking of DNA and proteins by UV radiation (see the section on Ultraviolet Radiation Photobiology).

2. Cycloaddition of unsaturated molecules, e.g., two thymines can react to form a ring product, the thymine dimer, an important class of products formed in DNA by UV radiation (see the section on Ultraviolet Radiation Photobiology).

3. Photofragmentation, e.g., the side chain of riboflavin can split off to form lumiflavin.

4. Photooxidation, e.g., the ring structure of cholesterol can add a peroxy group.

5. Photohydration, e.g., uracil can add a molecule of water to it 5-6 double bond when UV irradiated.

6. Cis-Trans Isomerization, e.g., all-trans retinal can be converted to 11-cis retinal.

7. Photorearrangement, e.g., 7-dehydrocholesterol can be converted to vitamin D3.

8. Energy Transfer, e.g., all photosensitized reactions (see section on Photosensitization).

Conclusion Additional information can be found in the Suggested Reading section, and in the Photochemistry module by Yuri V. Il'ichev.

Suggested Reading Coxon, JM and Halton, B, Organic Photochemistry, Cambridge University Press, 1987 Gilbert, A and Baggott, J, Essentials of Molecular Photochemistry, Blackwell Science Ltd, 1990.

Kagan, J, Organic Photochemistry: Principles and Applications, Academic Press, 1993. Klessinger, M and Michl, J, Excited States and Photo-Chemistry of Organic Molecules, Wiley-VCH, 1995. Smith, K.C., The Science of Photobiology, 2nd Ed. Plenum Pub Corp, 1989. Turro, NT, Modern Molecular Photochemistry, University Science Books, 1991. Wayne, CE and Wayne, RP, Photochemistry, Oxford University Press, 1996. Online Articles Franck-Condon Principle, Wikipedia Virtual Textbook of Organic Chemistry, William Reusch [Note especially: "Other Topics: Photochemistry"]

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