Electron Microscopy James
T
H. McAleor,
Department of Biology, Cafholic University of America, Washington, D.C. 2001 7
HE FOLLOWING may more resemble a news bulletin than a classical comprehensive review. Electron microscopy is developing rapidly. An effort has been made therefore to inform the reader of the latest trends, relying on recent meetings (such as the Electron Microscope Society of America, the IITRI Scanning E M Symposium, and the International E M Congress in Grenoble). The author is primarily a biologist with little knowledge of materials science. The significant advances in this area are left to those competent to report them. During the past two years, freeze etching and scanning electron microscopy, the pioneering fields in 1968, became a major part of the literature. High voltage E M is still being evaluated for its promise of applicability to biology. The high resolution scanning transmission E M (STEM) is the research weapon of the future. Image analysis and enhancement as spin off s from the computer and space industries have vastly broadened the horizons of all electron beam instruments. Standard biological E M techniques have become a routine part of college training and a common supplement to routine pathology. On the industrial front, there was a great deal of E M instrumentation development during the period before the scientific depression in 1969 and 1970. Japan has continued to emerge as a strong contender for the market. The Transmission E M field has all but vanished as a U.S. industry following the withdrawal of RCA and its successor, Forgflo. The Scanning E M field, however, has emerged as a major industry. Transmission EM (TEM). The introduction of the Philips E.M. 300 in 1966 a t the international congress of electron microscopy in Kyoto inspired much of the T E M design which has emerged during the past two years. The Hitachi HU 12 [Kubozoe et al., (%S)], the Joelco 100, and the Akashi [Akashi et al., (I)] from Japan, all show a trend to large instruments with complex vacuum systems, solid state circuits, and super columns. The Siemens 101, appeared as the first major change in 16 years with an improved column and power supply. AEI introduced a medium range instrument, the “Corinth,” with the column upside down, or as some insist, right side up a t last. Philips, introduced the 201, a general purpose, me-
the limiting factor in sections below dium priced instrument. Siemens also 0.5 micron is chromatic aberration introduced a simple instrument the 51 [Krisch and Weichan (%$)I. rather than illumination. It seems less expensive, therefore, to use lower voltr With the advent of the Scanning age with a shorter focal length objective TEM and its short clean ultra high lens to study thick sections in stereo. vacuum column, this trend to large complicated optical systems may soon The use of a high voltage T E M to look a t living cytoplasm does not seem a be reversed in favor of complicated image manipulators, detectors, energy reasonable application since a t the present state of the art it seems unanalyzers, and computers. likely that a rapid sequence of microThe application of dark field and phase contrast methods to the T E M graphs of a mmple in a moist chamber [Matricardi et al. (YO), Moritz et al. [Erickson and Klug (16), Thomson (49), Thon and Willasch (49A)],as well as ( S S ) ] could be made without having so much specimen interaction with the the improvement of the coherence of beam as to render the results useless illumination have increased detail resolueven if a beam stop and an image intion a t high contrast and therefore the tensifier were used. Several microapplicability of TEMs for the study of scopes with voltages of 3 MEV are molecular structure. A significant improvement in TEM constructed or are in development images has come from Fourier trans[Dupuoy and Perrier (IS), Sugato et al. forms generated by optical or computer (4111. Scanning Electron Microscopy. means with subsequent manipulation. This is treated in a later section. There are now about 400 scanning One other improvement which has electron microscopes in the United been incorporated into several of the States and perhaps over twice that more sophisticated TEMs is the capacmany in the world. Cambridge Instruments of England was the first ity to perform as SEMs or STEMS by the appropriate positioning of detectors into the market. The J S M series and the addition of the necessary imagalso had an early lead from 1966 with a converted microprobe. “Ultrascan” ing electronics [Koike et al., (%I)]. which was formed from “K square,” Some of these attachments compete in resolution with SEMs, even if the recently, features a turbomolecular pumping system. Hatachi is one of specimen facilities are generally limited. the larger firms which have recently High Voltage EM. Although there entered the market while others such are still few high voltage microscopes as Siemens, Zeiss, AEI and Philips seem available, efforts to apply and evaluate these tools have progressed to have held back. ETEC [Yew (66)] is a California firm which features a [Cosslett (6), Thomas ( C Y ) ] . Hans highly automated machine in the meRis (38) has tried to unravel the strucdium price range with an eye to the ture of chromosome in stereo thick growing biological market. Welter and sections. A. Szirmae et al. (46) have studied the structure of striated muscle Coates (65) have introduced an ion pumped instrument with a field emisas a function of section thickness and sion gun called the “Kwickscan.” accelerating voltage. High voltage EMS are difficult to Field emission sources produce small focus because the image quality on the intense beam spots. Such guns require a vacuum in the 1Olo Torr range [Crewe screen is poor due to low contrast and electron scatter in the phosphor. The (9) 1; however, this is not compatible with large specimen chambers negatives usually appear much better. Specimen damage is lower a t high voltand porous samples. Lanthanum hexaboride guns which significantly image, but thick sections still show thermal drift in the beam. The real problem, prove intensity can be adapted to most however, is that in the stereo image of a contemporary SEMs. It is probable thick object, the spatial resolution in that differentially pumped field emission the Z axis is about 1/20 that of the X sources will be more important in the and Y axis. The resolution of points future, in improving the resolution of in real space, therefore, is quite poor. the SEM. Sections of a micron thickness or more A much greater immediate improveusually, have too much superimposed ment in the effective resolution of the detail to be of much use. If specimen SEM on biological and some materials damage is not a factor of first imporsamples, however, is offered by the comtance (in sections which have been put bination of SEM with a simultaneous through chemical pergatory already), evaporation of freeze etch replicas [McANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972
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Alear et al. (SI), Fucci et al. (IS)] which can be studied and compared in the TEM. The merger of these two techniques is indicated because they complement each other so well. The SEM has great depth of field and sample capacity as well as a sample which when frozen is dissectable. Freeze etch replicas have greater resolution and detail contrast when viewed in the TEM [Grieve and Sprys ( I S ) , McAlear et al. (SI)]. A variety of experimental cold stages have been designed for SEMs. McAlear et al. (SI) developed a microdissection probe for dissecting frozen samples. A piezo-electric driven probe has also been tested [Pawley and Hayes (37)]. The former uses the microdrive of the specimen stage to move against a fixed needle, while the latter is capable of complex movement and oscillations. It seems clear that lower voltage gives better surface images [Koseige et al., (21)] from poorly scattering surfaces such as frozen samples while high voltage is often used to reveal deeper detail in solid state and materials samples. For these reasons, it is likely that nonbiological SEMs will become larger higher voltage machines with conventional vacuum systems and electron sources while biological machines will become lower voltage high resolution machines with field emission guns, a developed STEM capability, and complex specimen treating features in the stage area. Specimen preparation is still in the highly developmental stages in the SEM [Russ and Kabayo (40), Germinario and McAlear (IGA)] although the rapid freezing techniques of freeze etching may prove the most generally convenient means of treating tissues. Certainly, the veteran critical point method and the more recent freon modification [Cohen & Garner (S)] are becoming more popular as a means of avoiding drying and freeze-drying artifacts. Some remarkable preparations have been made this way, particularly of epithelial surfaces and single cells. So far, however, all of these drying techniques have been rather disappointing in displaying intracellular detail because the gel matrix seems to disintegrate, leaving deep cavities around membrane structures [Masayuki (29)1. The need for uniform metal coating on these dried samples has inspired the development of several devices (67) for twirling and tilting samples a t the same time in order to fill around corners with a thin heavy metal film and thus prevent charging in the electron beam. An alternative method has been offered in the form of a simple sputtering device (68). The use of a beam of inert gas ions to etch surfaces has been applied to materials science and more recently to bio98R
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logical objects [Thompson (48), Echlin etal. ( I C ) ] . SEM images extend human experience. An object appears to have an obliquely lighted surface viewed a t an angle with a great depth of field. The results appear a t once in the form of a positive/negative Polaroid pair. The application of the SEM to routine pathology seems promising since a frozen fractured specimen could be examined within minutes of removal from the patient. The quality of the image and the detail discernible with the SEM should greatly exceed that of the contemporary pathological section , although it is predictable that pathologists will be reluctant to accept these new criteria a t first. Two ETEC instruments have recently been installed a t the National Institutes of Health in Bethesda, Md., and these may provide the means of exploring the potential of SEM pathology. The use of nondispersive detectors for X-ray microanalysis has made it possible to use SEMs as microprobes and to improve the effective resolution of microanalysis in materials science [Russ ( S S ) ] . It is possible to display several images showing different elements in contrasting color, superimposed on the relatively high resolution secondary electron image [Lublin et al.,
W-4)I.
There is some interest in the application of X-ray microanalysis to biology [Tousimis (60)]but elements tend to be dispersed in structure in biological systems and, particularly, the heavier ones involved in enzyme structure and function. Microanalysis in the reflection mode is still limited in resolution by the size of the x-ray emission source. The analysis of thin sections in the transmission mode can produce much higher resolution. The electron beam may also cause fluorescence. This cathode luminescence commonly associated with phosphors is also exhibited by some organic materials. Muir et al. (36) have explored the application to stained and unstained biological materials and suggest that the increased resolution over light optics as well as the analysis prospects are promising. It may be possible to use the Auger (pronounced oh jay) electrons [MacMacDonald and WalDonald (H), drop (28)l produced by the interaction of the beam and the specimen for determining the distribution of elements a t high resolution in the reflection mode. These electrons are of low energy, although of somewhat higher energy than secondary electrons, and are characteristic of the elements from which they are emitted, following excitation of atoms by electron bombardment. Wet stages have been constructed for the SEM in the hope of examining liv-
ANALYTICAL CHEMISTRY, VOL. 44, NO. 5. APRIL 1972
ing cells. An obvious problem arises from the cooling of the sample from the rapid evaporation of water in the vacuum. The slow recording speed of the SEM is a problem in recording dynamic events. For these reasons alone, it seems probable that frozen samples will prove more useful than wet ones. Scanning Transmission Electron Microscopy. A. V. Crewe (9) and his associates have brought about what may well be the greatest single advance in electron microscopy in the past several decades in the development of the high resolution S T E M . The instrument now produces a beam spot as small as 0.5 nm and potentially as small as 0.1 nm and has demonstrated the ability to resolve molecules such as DNA spread on a support film [Crewe et al. ( S ) ] . By combining the signals from the elastically scattered electrons with those which have interacted with atoms inelastically so that their energies have been reduced by increments characteristic of those particular atoms, it is possible to detect the distribution of atomic species to within about 10 Z numbers. It is also possible, therefore, to label different bases along the DNA chain so that a color-coded composite image would demonstrate the base sequence and read out the genetic code directly [Crewe and Beck ( I O ) ] . This instrument demonstrates the characteristics of very high contrast and low specimen damage. It can also be used to study thick samples because chromatic aberration arising within the sample does not limit resolution as it does in the TEM. There is no reason why X-ray analysis, energy loss analysis, and Auger electron analysis couldn’t be carried out simultaneously in the STEM. It is likely that the STEM will supersede the TEM for many applications. J. Cowley (7) has been developing a high voltage STEM with which he hopes to be able to study living cells in the atmosphere. Specimen Preparation. The best specimen preparation is the least preparation. Cryogenic methods in freeze etch replication, the study of frozen fractured surfaces in the SEM, and the study of frozen sections [Christensen ( 6 ) ] in the TEM are indicative of a departure from traditionally based preparations and show a trend toward the application of physical concepts of materials analysis to biological specimens. Freeze etch replication has increased in popularity but still resembles a strange world to most microscopists. Freezing damage and contamination of the surface are the worst problems. Heat radiation damage, platinum and carbon thickness control, and damage to replicas by handling or too vigorous cleaning are frequent difficulties as well. All of the devices available require cy-
cling to atmosphere between runs for removal of the sample, replacement of electrodes, and introduction of a new specimen. There is little to choose in terms of a temporal advantage in operation of the various devices although some allow the processing of a number of samples a t the same time. The cold block devices are by far the least expensive, although until recently they have been more difficult to use because of the necessity of aligning the electrode accurately with tunnels in a protective cover and problems in making reliable heater thermocouple contacts. Most of these problems seem to be resolved in a new simple device (59) which can be used for freeze etch replication or freeze-drying samples for the SEhl. I t can process several samples a t the same time or a single specimen up to l/a-inch in diameter. I t has a built-in handle which also contains a standard thermocouple plug. The sample is transferred under liquid nitrogen which also super cools during the pumpdown cycle and purges the system with N2 gas a t the same time. The sample is fractured outside of the vacuum, thus avoiding mechanical devices or feedthroughs. Complementary replicas are easily made from both sides of the fracture face. The production of complementary replicas in stereo has finally resolved the question of the orientation of the fracture plane in membranes [Veltri and McAlear (51,52)l. It, passes somewhere near the middle of the membrane. This method permitted the identification of several other fracture planes in the cell wall complex of bacteria, but in no case did the fracture plane pass along the surface of the cell membrane. The bumps on the outer facing surface of the inner half of the plasma membrane are seen to come from pits on the inner facing surface of the outer half of the membrane. The need for stereo pairs [Staehelin ( 4 2 ) ] in freeze etch replicas relates to elevation recognition and the identification of artifacts from contamination of the carbon replica. A means for the exploration of structure below the fracture plane has been afforded by the development of a cold stage for an ion milling machine (60). I t may also be possible to use this attachment for the thinning of frozen sections for study in the transmission mode. There is a persistent dream that it may be possible to meaningfully study living cytoplasm a t electron optical resolution levels. Wet stages for the SEM and T E M have been developed and, as previously mentioned, the high voltage STEM in Arizona [J. H. Cowley ( 7 ) ] is being designed for such an attempt. This relates to the superstition among ancient cytologists that life can be equated to its dynamic properties and that to perceive the basis for this
movement is to understand the basis of life. For what it is worth, a medium voltage STEM with field emission cathode for rapid recording and a cold stage for observation of ultrathin frozen sections a t fairly high pressures would seem the only reasonable approach a t this time. After the initial thrill, however, of seeing a few filaments jerk, it probably would not tell us nearly as much as would high resolution materials analysis of the same samples. Image Frocessing. Fourier transforms and computer programs have been used in a number of ways t o improve electron microscope images and to aid in their interpretation. Improvement in contrast and detail can be brought about by noise filtering and edge enhancement [Nathan (SS)]. Conversion of gray levels to hues of color as a means of detail enhancement has been employed using the approximately one thousandfold increase in sensitivity of the human eye to hues of color over sensitivity to shades of gray. The actual improvement in effective resolution by the compensation for spherical aberration has been achieved [Erickson and Klug ( 1 5 ) , Stroke et al. (43), Crewe and Saxon (IOA), Welton (54, 6 6 ) ] . Computer identification of structural detail in micrographs has been successfully employed in the analysis of thin sections of central nervous tissue [Bloom (%')I. Man-assisted computer analysis of micrographs has also shown some promise in making stereologic analysis of micrographs [Gibbard et al., (17)1. The increased use of freeze etch replicas, SEMs [Kimoto and Suganuma ( I 9 ) , Dinnis ( I % ' ) ] and high voltage EMS of thick sections has revived interest in stereography and a reevaluation of its limitation [DeRosier ( I I ) , Lake ( 2 4 ) ] . The availability of a +30° X/Y tilt stage in the AEI 802 [Lucas (26),Swann and Swann (45)]has made it possible to explore the use of multiple stereo in sections. Cine stereo has also been attempted [McAlear and Veltri (W)] but it is too difficult to be popular. Micro Recording and. Electronics. The theoretical limit to the miniaturization of information or the fabrication of electron circuits is now the dimension of atoms since the electron beam which can be used for such purposes has been reduced to this diameter. Practically the need exists to develop new resist materials which would permit highest resolution with minimal beam energy. Using beams about 10 nm in diameter, patterns with 100-nm lines [Chang ( 4 ) ] have been produced [Mueller and Rindfleisch (34)1. Acrylate films have been used as photoresist materials which can be depolymerized by the beam and rendered more soluble.
It is interesting to compare the capacity for integration of information of ultramicrocircuits with the human central nervous system. Electrical conduction is about lo8 times faster than neuronal conduction. The delay a t a transistor is about lo6 times that of a synapse. Circuits with 10 nm by 100 nm components would occupy a volume as small as 1/106 that of a neuron, and the distance between switching points would be reduced by a factor of about l / l O * . We do not have enough information to be able to predict how far it is practically possible to go in ultramicrocircuitry fabrication, the power dissipation problems which may arise, the possible application of thin magnetic films, or the applicability of ovonic circuits and superconduction. It does seem probable, however, that artificial intelligence with a capacity somewhere between lo1*and 1020 times that of the central nervous system, volume per volume, can be produced by man. Furthermore, electron beams near atomic dimensions could possibly record and retrieve information from a volume, bit for bit, less than that required for chemical coding of genetic information. These data do no less than confront man with the real and ominous prospect that he may have the technology to devise self-replicating, inorganic intelligent systems. Conclusion. Two decades of Biological Electron Microscopy have passed from the first thin sections t o the teaching of these results in the 8th grade. We found the cell had membranes within it like those which bounded it and redundantly confirmed this. These membranes had enzymes upon and within them and this fit well with what biochemists said. During this time perhaps several hundred thousand papers with perhaps a million micrographs were published, All of these together constituted a volume of a few mma a t most. The effective resolution was the smallest entity we could identify, a ribosome or a membrane around 6 to 15 nm. The resolution in real space was poorer since seldom was the thickness of the section (30-100 nm) taken into account. The burden of artifacts in these tortured specimens is enough to challenge the credibility of all but the most obvious interpretations. It is little wonder that the wave of discovery in cell ultrastructure seems to have passed by. We still know very little about how cells are actually constructed. It seems clear, however, that the raster microscopes, the SEM and TEM now in rapid development, offer the hope that we may have another far better view of living systems. Suspended in time by rapid freezing, cells will soon be seen with high resolution and understanding of chemi-
ANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972
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cal constitution. We may perceive the froren nerve impulse or the act of synaptic transmission. Further, we may develop understanding of life based on ita molecular organization in 8itU.
We will probably not be able to unravel the central nervous system but should be able to construct far better thinking machines. We may indeed discover that to really understand the mechanisms of life is to better appreciate our own machines. LITERATURE CITED
(1) Akashi, K., et al., Proc. Int. Cmgr. Electron Mictoscopy, Grenoble, 1, 143 (1970). (2) Bloom, F., presented at the Image Anal sis Symposium, Washington Electron bicrosco y SOC.,May 1971 (3) Cohen, A. Garner, G. E:, 29th EMSA, Claitors, Baton Rouge, La., 1971, p 450. (4) Chang T. H. P., SEM Symposium, IITRI, bhicago, Ill., 1971, p 417. (.5) Christensen, A. K., 28th EMSA, Claitors, Baton Rouge, La., 1970, p 294. (6) Cosslett, V. E., ibid., p 4. (7) Cowley, J. H., ibid., p 6. ( 8 ) Crewe, A. V etal., ibid., 250. (9) Crewe, A. q., 29th EMgA, Claitors, Baton Rouge, La., 1971, p 22. (10) Crewe, A. V., Beck, V., ibid., p 40. (10A) Crewe, A. V., Saxon, J., 28th EMSA, Claitors, Baton Rouge, La., 1970, 534. (11) Defiosier, D. J. 28th EMSA, Claitors Baton Rouge, La. 1970 p 246, (12) binnis A. R., SBM dymposium, IITRI, Chic 0,Ill., !971, 43. (13) Dupuoy, Perrier, Proc. Int. Congr. Electron Microscopy, Grenoble, 1, 129 (1970).
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(14) Echlin, P., et ol. 1970 28th EMSA, Claitors, Baton huge, La., 1970, p 286. (15) Erickson, H. P.,Klug, A,, W., p 248. (16) Fucci, R., McAlear, J. H., Stain Technol.,46,249 (1971). (MA) Germinario, L., McAlear, J. H., 1971, Stuiwtechnol., 46, 249. (17) Gibbard, D. W., et al., 29th EMSA, Claitors, Baton Ro e, La., 1971, ~ p (18) Grieve, G. M., S p r y s , J. W., ., p 132. (19) Kimoto, S., Suganuma, T., 1971 J. Eleetronmirroec.,20,73 (1971). (20) Koike, H., et al., 28th EMSA, Claitors, Baton Rouge, La., 1970 384. (21) Kosuge, T., et al. 28th gMSA, Claitors, Baton Rouge, La., 1970, p 390. (22) Krisch, B., Weichan, C., ibid., p 50. (23) Kubozoe, M., et al., ibid., p 47. (24) Lake, J. A., ibid., p90. (25) Lublin, P., 28th EMSA, Claitors, Baton Rouge, La., 1970, p 388. (26) Lucas, J., ibid., p 374. (27) MacDonald, N. C., SEM Symp. TITRI, Chicago, Ill., 1971, p 91. (28) MacDonald, N. C., Waldrop, J. R., 29th EMSA, Claitors, Baton Rouge, La., 1971, p 86. (29) Masayuki, M., ibid., p 496. (30) Matricardi, V. R., et al., ibid., p 468. (31) McAlear, J. H., et al., ibid., p 446. (32) McAlear, J. H., Veltri, B. J., Amer. Cell Biol. SOC.,Boston, Abstracts, 1970. (33) Moritz, R. C., et al., 29th EMSA, Claitors, Baton Rouge, La., 1971, p 44. (34) Mueller, K. H., Rindfleisch, V., ibid., p 48. (35) Muir, M. D., et al., SEM Symp., IITRI, Chicago, Ill., 1971, p 401. (36) Nathan, R., 28th EMSA, Claitors, Baton Rouge, La., 1971, p 28. (37) Pawle J. B., Hayes, T. L., SEM Symp., fiTR1, Chicago, Ill., 1971, p 105. (38) Ris, H., 28th EMSA, Claitorn. Baton Rouge, La., 1970, p 12.
(39) Rues, J. L., SEM Symp., IITRI, Chicsgo, Ill,, 1971. D 65. (40)Rues, J., Kaba o A. 28th EMSA, Chtors, Baton &&e,’ La., 1970, p .win
( 4 5 i u g a t o E., et al., 1970 Proc. Z n t . Conqt. E h r o n MictOecqpy, U r d & , 1,121 (1970). (42)Staehelin, L. A., 28th EMSA, Claitors, Baton Rouge, La., 1970, p
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(4ij%troke G. W., et al. 29th EMSA, Chitors, baton Rouge, La., 1971, p 92. (44)Not mentioned in text. (45) Swann P. R., Swann, G. R., 28th EMSA, klaitors, Baton Rouge, La., 1970,.p 372. (46) Szirmae A., et al., 29th EMSA, Claitors, haton Rouge, La., 1971, p 463. (47) Thomas, L. E. et al., 28th EMSA, Claitors, Baton douge, La., 1970, p 8. (48) Thompson G., ibid., p 500. (49) Thomson, k.G. R., ibid., p 382. (49A) Thon, F., Willasch, A., 29th EMSA, Claitorst Baton Rouge, La., 1971, p 38. (50) Tousimis, A. J., 29th EMSA, Clrutors, Baton Rouee. La.. 1971. D 64. (51) Veltri, B.‘ J.,‘ McAear, J. H., J. Microsc., 93, 191 (1971). (52) Veltri, B. J., McAlear, J. H., Microbwl., 69,in ress. (53) Welter L. Coates; V. J. ’ 1971 29th EdSA, Claitors, Baton kouge, La. 1971, p 32. (54) kelton, T. A., 28th EMSA, Claitors, Baton Rouge, La., 1970 32. (55) Welton, T. A., 29th hbSA, Claitors, Baton Rouge, La., 1971, 94. (56) Yew, N. C., SEM iymp., IITRI, Chicago, Ill., 1971, p 33. (57) Polysciences Inc., Warrington, Pa. (58) E. F. Fullam Inc., Box 444, Schenectady, N.Y. (59) EMventions Inc. Suite 618, 1028 Conn. Ave. NW, Wmhington, D.C., 20036. (60) Commonwealth Scientific Inc., 500 Pendleton St., Arlington, Va.
E.,
Electron Spectroscopy I, Ultraviolet Photoexcitation D. Betteridge, University College Swansea, SA2 8 PP, U.K.
T
HE ECONOMIC DEPRESSION has probably slowed up the rate of development of photoelectron spectroscopy, but the great increase in the rate of publication during the last few months suggests that technique has “taken off .” Since the Report to Analytical Chemists (I), the only published work specifically orientated toward analysis is that of the author’s group (9, S), but significant developments in instrumentation, in the range of samples and in the understanding of the experimentla
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results have taken place. Much of potential interest and importance to the analytical chemist has been published, and this review will attempt to sieve this from the literature on the subject, which is, in the main, written with a bias toward theoretical chemistry. Most of the references are to work published since the completion of the report (I) but in a few instances some earlier work is cited. General, The early results of Turner’s group have been published
ANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972
as a book (4), which is of immense value. Many of the basic inorganic, aliphatic, aromatic, and heterocyclic compounds are presented and discussed. Approximately 300 spectra or spectral details are shown with sufficient clarity to ensure its use as a primary source. A separately available issue of Philosophical Transactions of the .Royal Society of London, Series A , has been devoted to papers on photo electron spectroscopy presented at a meeting of the Society in 1969 (6).
