Anal. Chem. 1988, 60,86 R-90 R (W23) Ravindhranath, K.; Janardhan, P. B. Indian J. Chem.. Sect. A 1986, 25A 405-407. (W24) Ravindranath, K.; Janardhan, P. B. Curr. s c i . 1986, 55, 232-234. (W25) Sharma, S.D.; Misra, S. J. Liq. Chromatogr. 1085, 8, 2991-2998. (W26) Upadhyay, R. K.; Rani, A. Chromatographia 1987, 23, 86-88. J' (w27) Sheikh* s' "'; Naqvi' N' Pak' 1g8" 8 , 507-513' Chem. Abstr. 1987, 106, 167911s. (W26) Schuster, M.; Koenig. K. H. Fresenius' Z.Anal. Chem. 1987, 327, 102-104. Chem. Absh. 1987, 107, 108279q. (W29) Schuster, M. Fresenius' Z. Anal. Chem. 1986, 324, 127-129. Chem. Abstr. 1086, 105, 162912r. (W30) Rao, A. L. J.; Chopra, S.J . Inst. Chem. (India) 1985, 57, 197-199. I
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(W31) Koenig, K. H.; Kessier, I.; Schuster, M.; Steinbrech, B. Fresenius' Z. Anal. Chem. 1985, 322, 33-35. Chem. Abstr. 1985, 103, 226600~. (W32) Beiikov, V. A,: Shatz, V. D.; Lukevics, E. J. Chromatogr. 1987, 388, 161-177. ("33) Bottura, G.; Pavesi, M. A. Microchem. J. 1987, 35, 112-119. (w34) Janjic, T. J,; Tesic, Z, L.; k i i n a r , M, J,; Radivojsa, p, N,; Gelap, M, B, J. Chromatogr. 1985, 331, 273-283. (W35) Sharma, S. D.;Misra, S. J. Liq. Chromatogr. 1985, 8, 1731-1738. (W36) Timerbaev, A. R.; Petrukhin, 0. M.; Zolotov, Yu. A. Fresenius' Z. Anal. Chem. 1987, 327, 87-101. (W37) Steinbrech, 6. J. Liq. Chromatogr. 1987, 10, 1-48.
The New Electron Microscopy: Imaging the Chemistry of Nature C. E. Fiori
National Institutes of Health, Bethesda, Maryland 20892
The electron microscope permits the direct observation of the microstructure of matter far beyond the resolving capabilities of the conventional light microscope. Consequently, the instrument has had a major, and often revolutionary, impact on many disciplines. During the last several years improvements in electron optics, vacuum systems, X-ray and electron spectrometers, specimen preparation techniques, and the development of powerful and inexpensive laboratory computers have extended the capability of the electron microscope to directly image and quantify the chemical elements which comprise a specimen. This added ability to provide compositional maps has substantially enhanced the importance of the electron microscope. This review will only cover compositional mapping utilizing electron microscopes (>80 kV) operating in the scanning transmission mode (STEM) since this is currently the only mode to permit simultaneous detection of all the various signals. References will include both biological and materials science applications since the subject matter is germane to both. The period covered by this review is essentially the last 10 years.
INTRODUCTION Electron microscopy was mostly developed in Germany between 1930 and 1940. Over the years, the electron microscope and it's variants, the scanning transmission electron microscope, the electron beam X-ray microanalyzer, and the scanning electron microscope, have contributed profoundly to a number of disciplines such as geology, metallurgy, and biology, to name just a few. Reduced to the simplest description, the illuminating electrons of the microscope can interact with individual specimen atoms either elastically or inelastically. In the f i t case the illuminating electrons change direction as a result of the interaction but maintain essentially the same energy they had before the interaction. In the second case the reverse happens and the electron will lose energy due to the interaction but will not change direction to an appreciable degree. The beam electrons can also interact with periodic or quasi-periodic groupings of atoms and by considering the wave properties of these interactions the resulting diffraction phenomena can be understood and utilized to provide structural information. The majority of current applications in electron microscopy utilize the elastic scattering process and diffraction effects. Up until quite recently the only medium onto which this information could practically be recorded was photographic film. By appropriately adjusting the electron optics of the microscope one would select a particular contrast mechanism and cause the selected electrons to expose photographic film. This technique can only capture one small amount of the information coming from the specimen via the scattered electrons. 86 R
Recently, several developments in technology have radically altered this state of affairs. New detectors have become available which are sensitive to a wide range of the possible signals. In particular, the signals which result from the inelastic interactions can now be recorded. Examples include the energy dispersive (EDS) X-ray detector (1-5) and the electron energy loss spectroscopy (EELS) detector (6-14). Sufficiently powerful digital computers of acceptable cost have become available which can directly record and immediately process the immense data flow coming from the detectors. Several powers of 10 improvement in attainable vacuum has recently become available in a number of commericial microscopes. This level of pressure (lov9Torr) is required to prevent the effects of Contamination which can limit the sensitivity and accuracy of analytical results. If mass loss is a consideration the condition of the vacuum system is even more important. If small traces of water or oxygen are present, severe etching can occur so that mass loss may be enhanced (15-17). Lastly, new specimen preparation procedures have been developed which utilize cryogenic techniques (18-23). This last point is critical in biological and polymer applications. By application of technological advances which have occurred in the last several years, a new electron microscopy is possible. This new microscopy is capable of providing more than just the morphology and structure of a specimen at high magnification but also the distributions of the constituent chemical elements. We will begin by first describing image formation followed by a description of the analytical signals and the detectors for these signals, we will then discuss the required hardware, and, finally, we will discuss the construction of an imaging analytical electron microscope.
IMAGE FORMATION AND DIGITIZATION Until quite recently the majority of computer applications in electron microscopy have involved the digitization of a micrograph recorded on a photographic emulsion. Such digitization (24) was carried out by a rotating drum or flat bed microdensitometer or a flying spot scanner. Subsequent processing of the digitized image was carried out in a large central computer. It was obvious from the beginning that the electron detector and analytical spectrometer outputs from a scanning mode microscope could be directly digitized. There have recently been several reports of such work now that computers with sufficient memory, mass storage, and speeds have become available at an acceptable cost (see, for example, ref 25-35). In the conventional (without computer) scanning electron column instruments, we move the electron beam over the face of a recording oscilloscope in synchronism with the beam over
This article not subject to U S . Copyright. Published 1988 by the American Chemical Society
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is obtained hy scanning the primary electron beam over a usually rectangular area of the specimen and changing the intensity of the electron beam of the display oscilloscope (which is scanning in synchronism with the primary heam) by a signal derived from some component of the beamspecimen interaction process. Confusion can result when more than one type of signal (e.g. bright or darkfield, characteristic or continuous X-ray, etc.) are recorded simultaneously. We can now, obviously, have different “images” of the same area all derived from one and the same raster scan. Consequently, it becomes necessary to specify which signal (or combination of signals) was used and the area scanned for each “image”.
the specimen surface. When a detector receives an increase in its signal from the specimen (e.g. hrightfield, X-rays, etc.), the beam on the recording oscilloscope is increased in brightness. Consequently, an “image” is “painted” onto the face of the display tube. The synchronous beams are displaced by imparting horizontal and vertical velocity components V , and Vy. V , is typically between 500 and 2000 times Vy: The direction in which the velocity component is greater IS historically called the line direction and the other direction is called the frame direction. When the beam reaches the end of a line on the oscilloscope, it is inhibited from producing light (blanked) and moved to the beginning of the next line, and the scan is repeated. The ‘conventional” display system has been used since the earliest days of scanning electron column devices since only simple analog electronic devices are required. A method alternative to continuous beam rastering is discrete rastering accomplished with what is usually called a ‘digital scan generator”. In this technique the x and y velocity components of the synchronous electron beams are not constant hut remain zero for a finite period of time and then the beam is rapidly stepped to the next point. The displacements along the line direction are equal. When the end of the line is reached, the beam is moved hack to the beginning of the line and displaced one step along the frame axis with the step size equal to the line step. Each point in the image at which the beam dwells is called a ‘pixel”. Digital scan generators as used in scanning electron column devices typically use between 100 and 4000 pixels along the frame and line directions. The digital method of scan generation is very convenient for applications with the digital computer. Images contain an enormous amount of information, and so mathematical manipulations involving images require the use of a computer. It can he expected that the digital technique will replace the ‘conventional” method in most future microscopes. The application of an on-line and interactive computer results in a substantial improvement in several key capabilities of a scanning electron column. These capabilities derive mainly from the ability of the computer to record all of the information generated and detected when the primary electron beam interacts with a location on the specimen. Consequently, beam-induced radiation damage is minimized (an important consideration in biological or polymer applications). The recorded information may be modified, and most importantly, combined, in a great variety of ways before presentation to the operatoranalyst, permitting structure and/or analytical information to he seen in a micrograph where none could be seen before. When the computer is actually controlling the microscope and data acquisition, it can be considered to be “on-line”. Once the analytical requirements have been specified by the operator, the computer controls, for example, the position of the electron beam, the acquisition parameters for the analytical spectrometers (EDS or EELS), and gain and integration times of the brightfield or darkfield electron detectors. The computer can be considered ‘interactive” if it returns an answer quickly enough to be useful in the analytical strategy of the operator. For example, if the operator requires an X-ray analysis, the computer can process the acquired spectra to obtain atomic concentrations. The operator can utilize the results of such rapid calculations on data to decide whether a particular feature of a specimen should be further examined. We need to clarify a possible confusion in terminology. An ’image” (‘micrograph”, ‘picture”, “area scan“ etc.) of an “area”
INELASTIC ANALYTICAL S I G N A L S Characteristic a n d Continuum X-ray Signals. X-rays ohserved in the electron microscope arise from two types of inelastic or energy-loss interactions between the fast beam electrons and specimen atoms. In one case a beam electron interacts strongly with a core electron and imparts sufficient energy to remove it from the atom. The ejected core electron can have any energy up to the beam energy less the characteristic shell energy. The beam electron is depreciated in energy by whatever kinetic energy the ejected electron has acquired plus the characteristic energy required to remove it. A characteristic X-ray is occasionally emitted when the ionized atom relaxes to a lower energy state by a transition of an electron from a given outer shell to the vacancy in the core shell. The X-ray is called characteristic because its energy equals the energy difference between the two levels involved in the transition, and this difference is characteristic of the element. The second type of inelastic interaction we must consider occurs between a fast beam electron and the nucleus of a target atom. A beam electron can decelerate in the Coulomb field of an atom, that consists of the net field due to the trucleus and core electrons. Depending on the deceleration, a photon is emitted that can have an energy ranging from near zero up to the energy of the beam electron. X-rays that emanate because of this interaction process are referred to as continuum X-rays. They are also commonly called “background”, ‘white”, or “bremsstrahlung” X-rays. By counting characteristic X-rays, we obtain a measure of the number of analyte atoms present in the volume of target excited hy the focused electron beam. By counting continuum X-rays, we obtain a measure approximately proportional to the product of the average atomic number and the mass thickness of the excited volume. If the average atomic number of the target is known, the continuum signal gives a measure of target mass thickness. Electron Energy-Loss Signal. A beam electron can interact strongly enough with a core electron to remove it from the atom. The beam electron is depreciated in energy by whatever kinetic energy the ejected electron has acquired plus the characteristic energy required to remove it. It is possible to determine the identity and quantity of many of the constituent atoms present in that volume of the specimen which the electron beam has illuminated by recording the energy of a large number of electrons which have passed through the specimen at each pixel. The beam electrons passing through the specimen can each interact with more than one atom and with different core levels. The beam electrons may also interact with loosely hound or free electrons in the specimen. Consequently, the electron energy loss signal (EELS) is relatively complicated. However, the required knowledge to interpret these spectra is in place and a large body of literature is developing on the subject. The author gratefully acknowledges Chemical Abstracts Service for providing CA Selects to aid in the literature search used in the preparation of this work. DETECTORS Energy Dispersive X-ray Detector. This detector is a solid-state device with no moving parts and is relatively inexpensive and easy to operate. By far the most common form of the energy dispersive detector is the so-called lithium-diffused-silicon (or Si(Li)) detector (4, 37). X-rays originating from the analytical region of the specimen enter into a cooled, reverse-biased p-i-n (p-type,intrinsic, n-type) lithium-drifted silicon detector. Absorption of each ANALYTICAL CHEMISTRY. VOL. 60, NO. 12. JUNE 15. 1988
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individual X-ray photon leads to the ejection of photoelectrons and Auger electrons that give u most of their energy to the creation of electron-hole pairs. he components of the pairs are swept in opposite directions by the applied bias to form a charge pulse that is then converted to a voltage pulse by a charge sensitive amplifier. The magnitudes of the pulses produced by the detector are on the average proportional to the photon energy of each incoming X-ray. For example, if the detector captures one photon having an energy of 5 keV, the total number of electrons swept from the detector is apC. proximately 1300, which represents a charge of 2 X This is an extraordinarily small charge. The subsequent circuitry must be capable of amplifying this signal by about 1O’O. Cooling of the detector crystal and first stage of the preamplifier to near liquid nitrogen temperature is essential. Since the actual transducer, the Si(Li) diode, is physically small and is contained in a housing that occupies very little space, it is possible to place the detector close to the specimen. This, together with the almost unity response of the detector for most of the X-ray energies of interest, results in high quantum efficiency. This is an important property since it makes possible the use of low current electron probes. The present cost of an EDS detector is $10 000-20 000. Electron Energy Loss Spectrometer (7-12). An energy loss spectrum is obtained by passing the electrons which leave the specimen through a magnetic field. The field will curve the electron path by an amount proportional to the energy of the electron. Consequently, there is a spatial dispersion proportional to the energy the beam electrons lose upon passing through the specimen. There are currently two types of spectrometers used for scanning transmission microscopes: the serial EELS spectrometer and the parallel EELS spectrometer. The serial device has a slit between the magnetic field and a scintillator-photomultiplier detector. Only electrons of a given energy will pass through the slit and be detected. Electrons of any other energy are discarded, resulting in an inefficient collection process. The parallel device does not use a slit but rather allows most of the spatially dispersed electrons to fall on a scintillator which is backed by a photodiode array. A histogram of energy loss results by periodically reading out the charge in each element of the may. This collection method is well suited for imaging purposes. The only disadvantage is cost, since a parallel spectrometer is almost twice that of a serial device. The cost of a parallel EELS spectrometer is currently about $70 000.
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DATA PROCESSING For our purposes data processing can be divided into two parts: spectrum and image processing. Both are required for compositional mapping. Spectrum signal processing is required because EDS and EELS spectra are convolutions of a number of effects. As in many forms of spectroscopy these spectra contain high to medium frequency structures holding the analytical information and medium to lower frequency structures holding other information which is sometimes of no w e and needs to be removed and discarded (background). Statistical effects (noise) are present mostly as high frequencies and the mean values of the analytical structures can be close enough to one another to cause the structures to overlap (interference). Since we are concerned with analytical imaging, spectra are generated at each pixel in the image. Any spectrum processing methods used must contend with short data acquisition times and spectra with poor signal to noise ratios. Image processing follows spectrum processing and considers a pixel in association with other pixels in the same image plane or in other related image planes. For example, if we have scanned an area of a specimen for magnesium, aluminum, and oxygen, we could smooth out statistical variation at each pixel in a plane by averaging each pixel with its nearest neighbors in the same plane. We can also cross correlate information from plane to plane or take advantage of any known stoiin this case. chiometric relationships, such as MgO and A1203 The intensity of the analytical signal at each pixel requires further mathematical transformation to extract chemical information. A s we point out below, high-capacity computer memory and mass storage are now so inexpensive that it is quite reasonable to consider the acquisition and storage of entire spectra at each pixel in an image. This is not a new idea, Legge and Hammond (38) used continuous scanning and stored on 88R
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magnetic tape the x and y coordinate, along with the energy of each detected X-ray photon, in a scanning proton-induced X-ray apparatus. The data from selected coordinates or clusters of coordinates (that do not have to be contiguous) can then be sorted into an X-ray spectrum. This method is equivalent to acquiring an entire X-ray spectrum at each pixel and using a compression algorithm to remove channels which contain zero counts, before storage. This is certainly the most general procedure for storage since virtually no information is discarded. We can come back to this data as often as we like and apply different mathematical or physical procedures to extract further information required. Limited space precludes including any details of data processing. We refer the reader to the following representative literature as a starting point: X-ray spectrum processing (4, 37,39), EDS imaging (33,36,40,41), EELS imaging (15,29-33, 40),conversion of analytical signals to chemical information (42-47), general image processing (41, 48, 49).
HARDWARE Computer. The recent major advances in computer
technology will have a profound effect on wide spread use of compositional mapping and on-line data processing and instrument control for the general community of analytical electron microscopy. Unfortunately, the rate at which advances have been coming has meant that many of the new capabilities have yet to be implemented on commercially available microscopes. Since images are memory intensive and operations on images require large numbers of calculations, the most relevant advances have been in memory size and processor speed. Memory cost at the time this review was written (January 1988) is under $250/megabyte. Basic processor speed, when coupled with a numerical coprocessor, is more than adequate for a wide range of interactive image manipulations. The total cost of a computer system with 8-10 me abytes of main memory and including storage, acquisition, and display hardware is now under $10 000. This is just the cost of an objective lens in some electron microscopes. However, just as the objective lens by itself would be useless, so also the computer. Up until and including the present, no manufacturers of electron microscopes offer their own integrated computer system which will do good compositional mapping. This capability is available from several alternative sources such as the manufacturers of energy dispersive X-ray systems. The cost of these systems is in the range $30 0001OOOOO. It should be noted that there will be a growing trend to take advantage of the inexpensive but extreme1 powerful mass market microcomputers such as the Apple acintosh I1 or the IBM, IBM clone, 80286/386 class computers. These machines have avaiflable a wealth of inexpensive software and hardware accessories. Starting in 1987 at the national meeting of the Microbeam Analysis Society (MAS) and in 1988 at the joint meeting of MAS and the Electron Microscopy Society of America (EMSA),there is now an entire symposium on the use and application of these computers. Software and hardware standards are being discussed and user-written software is exchanged. This trend will clearly grow. Accessories. Image processing requires large amounts of main memory and disk storage. Even a simple operation such as subtractingone 512 X 512 X 8 bit image from another would require 512 kilobytes of main memory to hold the data. Analytical scanning in an STEM can produce even a larger quantitiy of data. For example, a 512 by 512 pixel scan of a specimen while collecting data from EDS, brightfield, and darkfield electron detectors would require several megabytes of disk storage for raw data. Depending on the nature of the various signals, different amounts and type of storage are required for the data. An absolute minimum amount of storage, at each pixel, in STEM applications would be 12 bits which corresponds to a resolution of one part in 4096. Some signals might require 32-bit “words” to store the data at each pixel in the image. Usually this data will be stored on large-capacity disk units with either removable or fixed media packs. The latter units are often called “Winchester” disks and are presently available, for example, with 160-megabyte capacity for a total cost under $3000. These disks have an average access time of about 15 ms and can transfer or accept data at a rate of about a megabyte per second (i.e. about an image a second). These units are also used to store the “operatingsystem” of the computer, user programs, and small
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data fiies. After the acquisition of only a few es,however, storage it becomes apparent that some form of main%val is required. Reel-to-reel nine-track tape drives (capable of storing in the order of 100 images) are excellent for this purpose but are archaic and expensive ($10 000). Recently, “streaming” tape units and tape cartridges have become available at greatly reduced cost which are equally as effective, and more convenient, for archiving purposes. A 60-megabyte “streamer” costa just over $1000. The tape cartridges cost $15 each. These units can back up hard disks at over 4 megabyteslmin. Optical disk technology is currently the most promising for image mass storage. Optical disk are related to the compact disk used in home entertainment systems. Optical disks, however, can be written on once to store the data and then read any number of times. The acronym is WORM, for write once read many. WORMS are now available for the IBM IBM/clone computers for as little $3000 for 400 megabytes of storage. The disks are removable and cost $125. If one does the arithmetic, this form of storage is now cheaper than Polaroid film. WORM drives are about 5 times slower to write to than a regular magnetic disk but can be read at approximatelythe same speed. Models for the Macintosh and other micro computers should be available within months. An EDS or EELS spectrum can be mathematically described by a vector (one-dimensional array). Similarly, an image may be described by a matrix (an M by N dimensional array). There is a computer accessory, generally called an array processor, which can speed up operations on vectors and matrices by several powers of 10. For example, a typical array processor can perform a complex fast Fourier transform of a 1024 point X-ray spectrum in several milliseconds. The present cost of an array processor is approximately $5000-15000. However, an array processor can be tricky to program. Displaying digital images involves much more than exhibiting processed data on a screen. One should be able to alter contrast or brightness, perform pseudo-coloring, annotate with mows, text, and other graphical notation, superimpose graphs (line scans), outline areas of interest, zoom, pan, or combine several images. To have the central processor (CPU) of the main computer do these functions is slow and burdensome and difficult to program. The computer is unavailable for anything else while any of the above is being performed. There are commercialproducts available which are usually called image display systems. These systems have enough memory to store multiple 512 X 512 or 1024 X 1024 by 8-bit images and a number of similarly sized several bit overlays for alphanumeric and graphical information. Eight bits (i.e. 256 “levels”) is totally sufficient for display purposes even though the data displayed, for example, was acquired with 16 bits of resolution (65000 “levels”). It is a trivial matter to “map” the “acquired” resolution into the “displayed” resolution to display all of the data or only certain parts of it. Output can be produced on a standard red-green-blue (RGB) color monitor, refreshed at 30 frames per second directly from the display system memory. The main computer is free to perform other tasks, such as data acquisition, while all of the above-mentioned display functions are being performed on previously acquired images by the display system. The display system is also considerably faster at performing the above since there is usually special hardware included to make the display truly interactive with the operator. One such piece of hardware is an extremely fast integer array processor which operates directly on the 8-bit image planes. This processor can, for example, add one image plane to another in less than one frame time (i.e. l/%th of a second). The cost of this “built-in” array processor is several thousand dollars. At present, the cost of an image display system is betweeen $5000 and $5OOOO. The large range in price is due to the great variety of options available, not all of which are required for basic display of compositional maps. Models are available which plug directly into an IBM, IBM/clone, 80286/386 class microcomputer and will soon be available for the Macintosh I1 and other microcomputers. C O N S T R U C T I O N OF A N IMAGING ANALYTICAL E L E C T R O N M I C R O S C O P E Unfortunately, there is not a single source for an imaging analytical electron microscope. A number of manufacturers provide a basic transmission machine which can be configured
both in the regular transmission and scanning transmission modes. One manufacturer provides a high-resolution dedicated STEM with excellent vacuum capability. One advantage of the dedicated STEM is the relatively large area for the specimen stage and the analytical spectrometers. Several microscope manufacturers provide a serial detection EEL spectrometer. Only one manufacturer of electron microscope accessories provides a parallel detection EEL spectrometer. As of today this manufacturer is the only source although the situation will probably change within the year. There are several sources for the EDS detectors. The manufacturers of the EDS detectors also provide computer systems which can display spectra and images. A number of laboratories have configured their own computer systems. This trend will grow. As one might suspect from this situation, the primary burden of “constructing” an imaging analytical electron microscope falls primarily on the user. Defining and enforcing specifications can be frustrating or impossible, especially when components are purchased at different times. It is the rare exception and not the rule for the system to go together without problems. Fortunately, there are now a great number of successful installations and a growing body of literature on the subject. Most importantly, two professional societies exist (MAS and EMSA) where this new technology is given a prominent position. The MAS national secretary is John Armstrong, Caltech, 170-25, Pasadena, CA 91125, and the EMSA national secretary is Linda Horton, Oak Ridge National Laboratory, PO Box X, Bldg 5500, Oak Ridge, T N 37831. LITERATURE CITED (1) Fiori, C. E.; Newbury, D. E. “Artifacts Observed in Energy Dispersive X-Ray Spectrometry in the Scanning Electron Microscope”. Scanning Electron Mlcrosc. 1978, Voi I , 401-422. (2) Fiori, C. E.; Newbury, D. E. “Artifacts in Energy Dispersive X-ray Spectrometry in the Scannlng Electron Microscope”. Scanning Nectron MlCrOSC. 1980, Voi. 2. 250-258. (3) Fbri, C. E.; Newbury, D. E. “Operation of EnergyDispersive X-Ray Spectrometers in the Analytical Electron Microscope”. Analytlcai Electron MIcroscopy; Geiss, R. H., Ed.; San Francisco Press: San Francisco, CA, 1981; pp 17-24. (4) Helnrich, K. F. J., Newbury, D. E., Mykiebust, R. L., Fiori, C. E., Eds. Energy Disperslve X-Ray Spectrometry; National Bureau of Standards Special Publication 604; U.S. Government Printing Office: Washington DC: 1981; 445 pages. (5) Williams, D. B.; Fiori, C. E.; Goldstein, J. I. “Principles of Energy Dispersive Spectrometry in the Analytical Electron Microscope”. I n frlndpbs of AnalyticaiElectron Microscopy; Joy, D. C., Romig, A. D., Goidstein, J. I., Eds.; Plenum: New York, 1986; Chapter 4. (6) Shuman. H.; Somiyo, A. P. ”Electron energy loss analysis of near-traceelement concentrations of calcium”. Utramicroscopy 1987, 21, 23. (7) Egerton, R. F.; Crozier, P. A. ”A compact parallel-recording detector for EELS”. J . Microsc. (Oxford) 1987, 748, 157-166. (8) Isaacson, M.; Johnson, D. Ulhamicroscopy 1975, 7 , 33. (9) Egerton. R. F. J . Electron Mlcros. Tech. 1984, 7 , 37. (10) Krivanek, 0. L.; Ahn. C. C.; Keeney, R. B. Parallel detection spectrometer using quadrupole lenses. Ultramlcroscopy lS87, 22, 103. (11) Shuman, H.; Kruit, P. Rev. Scl. Instrum. 1985, 56, 231. (12) Jeangulllaume, C.; Tence, M.; Trebbia, P.; Coiiiex, C. Scannlng Electron Mlcrosc. 1983, Voi. 11, 745. (13) Leapman, R. D.;Fiori, C. E.; Gorien, K. E.; Gibson, C. C.; Swyt, C. R. “Combined Elemental and Structural Imaging in a Computer Controlled Analytical Electron Microscope”. Ulhamlcroscopy 1984, 12. 281-292. (14) Coiiiex, C.; Jeanguiiiaume, C.; Mory, J. J. Unrastruct. Res. 1984, 88, 177. (15) Leapman, R. D.; Ornberg, R. L. “Quantitative Electron Energy Loss Spectroscopy in Biology”. Ultramlcroscopy 1988, 24, 25 1-268. 16) Glasser, R. M. “Radiation Damage with Biological Specimens and Organic Materials”. Intrductlon to Anaiytlcal Electron Microscopy; Hren, J. J., Goidstein, J. I..Joy, D. C., Eds.; Plenum: New York, 1979; pp 423-436. 17) Lamvik, M. K.; Kopf, D. A.; Daviiia, S. D. “Mass loss rate in coiiodian is greatly reduced at liquid helium temperature”. J . Mlcrosc. 1987, 148, 211-217. 18) ,Dorge, A.; Rick, R.; Qehring, K.; Thureau, K. “Preparatlon of freezedried cryosections for quantitative X-ray microanalysis of electrolytes in biological soft tissue”. pnuserS Arch. Eur. J . Physlol. 1978, 373, 85-97. 19) Barnard, T.; Seveus, L. “Preparation of Biological Material for X-ray Microanalysis of Diffusible Elements”. J . Mlcrosc. 1977, 112, 281. !O) Andrews, S. B.; Leapman, R. D.; Landis, D. M. D.;Fiori, C. E.; Reese, T. S. ”Elemental distribution in rapid-frozen cerebellar cortex”. J , Cell Bbl. 1984, 99, 424a. (21) Somiyo, A. P.; Urbanics, R.; Vadasz, 0.; Kovach, A. G. 9.;Somiyo, A. V. ”Mitochondrial calcium and cellular electrolytes in brain cortex frozen in situ: electron probe analysis”. Biochem. Blophys . Res. Commun. 1985, 132, 1071-1078. (22) Andrews, S. B.; Leapman, R. D.;Landis, D.M. D.; Fiori, C. E.; Reese, T. S. “Rapid freezing and quantitatlve elemental imaging in neurobiology”. R o c . Mlcrosc. SOC.Canada 1985, 12 (Societe de Mlcroscopie du Canada, 1985), 24-25. ANALYTICAL CHEMISTRY, VOL. 60, NO. 12, JUNE 15, 1988
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Anal. Chem. 1988. 60. 90R-106R (23) Andrews, S. B.; Mazurklewicz, J. E.; Kirk, R. 0. "The distribution of Intracellular ions in the avian salt gland". J. Cell Biol. 1983, 9 6 , 1389-1 399. (24) Sexton. W. Computer Techniques for Image Processing in Nectron Microscopy; Academic: New York, 1978. (25) Fiori, C. E.; Gotien, K. E.; Gibson, C. C. "Comments on the computerization of an analytical electron microscope". Proc, 39th Annual EMSA Meetlng (Chitors Publishing, Baton Rouge, LA, 1981) pp 246-249. (26) Zubin, J.; Wiggens, J. Rev. Sci. Instrum. 1980, 51, 123. (27) Strahm, M.; Butler, J. 37th Ann. R o c . Elect. Microsc. SOC.Am. 1979. 598. (28) Strahm, M.; Butler, J. H. "Fast Digital Data Acquisition and on-line Processing System for an HB5 Scanning Transmission Electron Microscope". Rev. Sci. Instrum. 1081, 52(6); 840-848. (29) Leapman, R. D.; Fiori, C. E.; Gorien. K. E. "Elemental Imaging by EELS an EDXS in the Analytical Electron Microscope: It's Relevance to Trace Element Research". 6bi. Trace Element Res. 1986. (30) Leapman, R. D. Electron Microscopy in Medical Research and Diagnosis-Present and Future Directions: Electron Energy Loss Spectroscopy". J. Electron Micros. Tech. 1988, 4 , 95-101. (31) Leapman, I?.D. "Quantitative Electron Energy Loss Spectroscopy and Elemental Mapping in Biology". Microbeam Analysis ; San Francisco Press: San Francisco, CA, 1986; pp 187-192. (32) Leapman, R. D. "STEM Elemental Mapping by Electron Energy Loss Spectroscopy". Ann. N.Y . Acad. Scl. 1986, 483, 326-338. (33) Leapman, R. D.; Fiori, C. E.; Gorien, K. E.; Gibson, C. C.; Swyt, C. R. "Combined Elemental and Structural Imaging in a Computer Controlled Analytical Electron Microscope". Ultramicroscopy 1884, 12, 281-292. (34) Kowarski, D. "Intelligent interface for a microprocessor controlled scanning transmission electron microscope with X-ray imaging". J. Nec tron Microsc. Tech. 1084, I , 175-184. (35) Gorien, K. E., Barden, L. K., Dei Priore, J. S., Fiori, C. E., Gibson, C. C., and Leapman, R. D. "A Computerlzed Analyticai Electron Microscope for Elemental Imaging". Rev. Sci. Instrum. 1984, 55, 912-921. (36) Fiori, C. E.; Leapman, R. D.; Swyt, C. R.; Andrews, S. B. "Quantitative X-Ray Mapping of Biological Cryosections". Ultramicroscopy 1988, 24, 237-250.
(37) Goidstein, J. I.; Newbury, D. E.; Echiin, P.; Joy, D. C.; Fiori, C. E.: Lifshin, E. Scanning Electron Microscopy and X-Ray Mlcroanalysls: A Text for Siologists, Materials Sclentlsts, and oeologists; Plenum: New York, 1981; 873 pp. (38) Legge G. J. F.; Hammond, I."Total quantltative recording of elemental maps and spectra with a scanning microprobe". J. Microsc. 1979, 117, 20 1-2 10. (39) Statham, P. J. "A Comparative Study of Techniques for Quantitative Analysis of the x-ray Spectra Obtained with a Si(Li) Detector". X-Ray Spectrosc. 1976, 5 , 16-28. (40) Somiyo, A. P. "Compositional mapping in biology: x-rays and electrons". J. Ultrastruct. Res. 1084, 88, 135-142. (41) Newbury, D. E.; Joy, D. C.; Echiin, P.; Fiori, C. E.; Goldstein, J. I.Advanced Scanning Electron Mlcroscopy and Microanalysis, Plenum: New York, 1986; 454 pp. (42) Leapman, R. D.; Fiori, C. E.; Swyt, C. R. "Mass thickness determination by electron energy loss for quantitathre x-ray microanalysis in biology", J. Microsc. 1984, 133, 239-253. (43) Leapman, R. D.; Fiori, C. E.; Swyt, C. R. "Mass Thickness by Inelastic Scattering in Microanalysis of Organic Samples". Analytical Nectron Microscopy, Williams, D. B., Joy, D. C., Eds.; San Francisco Press: San Francisco, CA, 1984; pp 83-88. (44) Kitazawa, T.; Shuman, H.; Somlyo, A. P. "Quantitative electron probe analysis: problems and solutions". Ultramicroscopy 1083, 11, 251-262. (45) Shuman, H.; Somlyo, A. V.; Somiyo, A. P. "Quantitatlve electron probe microanalysis of biological thin sections: methods and validity". Ulhamlcroscopy 1976, 1 , 317-339. (46) Goldstein, J. I.; Williams, D. B.; Cliff, G. "Quantitative x-ray analysis". I n Principles of Analyfical Nectron Microscopy; Joy, D. C. Romig, A. D., Goidstein. J. I., Eds.; Plenum: New York, 1986; Chapter 5. (47) Hail, T. A.; Gupta, 8. L. "EDS quantitation and application to biology". I n Principles of Analytlcal Electron Microscopy; Joy, D.C., Romig, A. D.. Goidstein, J. I., Eds., Plenum: New York. 1986; Chapter 6. (48) Pratt, W. K. Digltal Image Processing; Why-Interscience; New York, 1978. (49) Gonzaiez. R. C.; Wintz, P. Digltal Image Processing; Addison-Wesley: Reading, MA, 1977.
Mossbauer Spectroscopy John G. Stevens* Department of Chemistry, University of North Carolina at Asheville, Asheville, North Carolina 28804-3299
Lawrence H. Bowen Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204
Katherine M. Whatley Department of Physics, University of North Carolina at Asheville, Asheville, North Carolina 28804-3299
The first review of Mossbauer spectroscopy that appeared as part of the Analytical Chemistry fundamental review was published in 1966 making the current review the 12th in the series. The last nine of these have used the facilities of the Mossbauer Effect Data Center. Papers that are included in the current review are those papers that have been received and surveyed by the data center since the last review (1). Papers that are included in the review are mostly from 1985-1987. During the last two years approximately 2500 papers on Mossbauer spectroscopy have been processed by the data center. It is from this compilation of 2500 papers that approximately 300 papers were selected to be in this review. The process for identifying the papers to be used in the review is most difficult. Certainly, the selection process leans toward the individual interests of the three of us. But despite this difficulty, the review represents a good summary of what has been happening in the field of Mossbauer spectroscopy during the past two years. The extensive proceedings of three conferences have been published. The proceedings of the International Conference 90 R
0003-2700/88/0360-90R$06.50/0
on the Applications of the Mossbauer Effect that was held in Alma-Ata, USSR,in September of 1983 have been published as a four-volume wries. Included in it are over 400 short papers and 17 longer invited papers. Topic3 that were covered in the conference and proceedings include the application of the Mossbauer effect in magnetism, chemistry, and material science (2). The proceedings of the International Conference on the Applications of the Mossbauer Effect that was held in Leuven, Belgium, September of 1985, were published as three volumes in the journal Hyperfine Interactions. The proceedings include over 20 invited talks in addition to several hundred shorter papers. Topics of the conference included noncrystalline materials, magnetism electronic structures, metals and alloys, mineralogy, radiation damage, dilute systems, lattice dynamics, biological systems, applications in industry, and developments in theory and experiment (3). The third major proceedings publication was that of the Symposium on the Industrial Applications of the Mossbauer Effect that was held in Honolulu, HI, in December 1984. This volume contains 42 chapters on a variety of topics related to 0 1988 American Chemical Society
