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Electron Microscopy Michael Beer Department of Biophysics, The Johns Hopkins University, Baltimore,
During the past two years, high resolution scanning microscopes have appeared both through manufacturers and assembly in research laboratories. So far, few actual applications have been reported. .Important progress has been made in specimen preservation and the development of electron microscopy designed to avoid radiation damage. These and other results will be briefly reviewed here.
INSTRUMENTS Conventional transmission electron microscopes (CEM) continue to be the principal instruments for obtaining high resolution structural information on biological and even nonbiological materials. During recent years, manufacturers have incorporated into them beam tilting arrangements to allow dark field microscopy, and coils to allow operation in the scanning mode utilizing either transmitted br secondary electrons. Often field emission guns are available as attachments. These are differentially pumped to the necessary vacuum of about Torr with the column at the more usual pressure of perhaps Torr. With such an addition, high resolution Scanning Transmission Electron Microscope (STEM) operation should be possible. Several manufacturers now offer STEM instruments. In ones marketed by AEI and Vacuum Generators, only the gun is a t very high vacuum while the remainder of the column has rubber gaskets and is pumped by diffusion pumps. Siemens offers an instrument capable of perhaps
Md. 2 72 78
2-3 A iesolution, ultrahigh vacuum throughout, computer coupling, and energy analyzer for the transmitted electrons. At present, it is not clear if ultrahigh vacuum will be necessary in the column for the determination of the final detail. For microscopes used as microprobes, the energy of the x-ray fluorescence can be determined by energy dispersive spectrometers. The theoretical and experimental aspects of this field were the subject of a recent conference and its proceedings, which are now published (32),are an excellent report on the present status of the field. Some electron microscopes built for research purposes merit mention. A high resolution STEM at Johns Hopkins University (28) has produced clear images of single atoms. At Cornel1 University, a conventional optics transmission electron microscope with 100-keV beam, superconducting lenses, and ultrahigh vacuum throughout is being tested (18). An electron microscope built a t the Oak Ridge National Laboratories also uses conventional optics and superconducting objective lens (30).
SPECIMEN DAMAGE Electron microscopes now exist which have resolution capabilities near the length of a ckiemical bond. What then prevents electron microscopy from deducing the complete chemical structure of a molecule or an assembly of molecules? For biological materials, there are two difficulties ANALYTICAL CHEMISTRY, VOL. 48, NO. 5, APRIL 1976
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Table I. Damage Resulting from Various Levels of Irradiation Dosage In Electrons/A*
0.5 50
500
Negligible damage to biological structure Poly A spectrum 50% preserved Negatively stained TMV preserved t o at least 25 A
(26)
Hg, Os atoms retained;
(20,27) (29)
Protamine structure apparently retained Negatively stained TMV
(14) (6,29)
(29)
not preserved t o 25 8, In the last line of this table, the dosage of 500 electrons per A2 was not measured but estimated. In the work of Ottensmeyer,this irradiation may be an underestimate. It is puzzling that in one experiment the individual atoms appear to retain their positions while in another, at comparable irradiation, there are gross rearrangements of structure.
which stand in the way of such an accomplishment. The first is damage suffered by the specimen; the second is contrast. First, we will discuss the former. Specimen damage itself is of two types. The first arises from distortion in the structure as a hydrated specimen is desiccated on introduction into the vacuum system. In recent years, a number of important attacks have been made on this problem. Two years ago in the same series, we described the environmental cells developed by Parsons and his collaborators (21). Through these devices, desiccation artifacts can apparently be avoided. Indeed Hui and Parsons have presented electron micrographs of domains in wet lipid bilayers (9).
Recently Taylor and Glaeser ( 2 4 ) showed that a hydrated catalase crystal can be rapidly frozen and then, while still frozen, introduced into the vacuum system of a specially adapted (25) electron microscope. When so examined, the crystal yielded an electron diffraction pattern with reflections out to 3.4 A. Thus, freezing and subsequent examination in the microscope permit the preservation of order to a high degree, and this approach also promises to be an excellent way to avoid the desiccation artifact. In a different approach, Unwin and Henderson (26) showed that the structure of the purple membranes of Halobacterium halobium could be preserved to approximately 7 A if the aqueous phase is replaced by a nonvolatile sugar such as glucose. This procedure is analogous to the well known “negative staining”. The second type of damage which limits electron microscopy is the inevitable disruption of chemical bonds resulting from the inelastic collision of the beam electrons with the specimen. This type of damage was mentioned in my review two years ago. A rather complete discussion of it by Glaeser (in Ref. 22) has appeared recently. Experiments have been carried out in recent years to determine the sensitivity of various materials to irradiation. The structural damage was followed by observing the disappearance of electron diffraction spots of crystalline materials ( 5 )or the changes in the electron energy loss spectra (14) or infrared spectra (23) or the loss. of mass from the specimen (3). Also, Williams and Fisher (29) showed by direct electron microscopy that if electron irradiation is kept at a minimum in negatively stained TMV, the structure is better preserved. Table I gives some measure of the consequences of various levels of irradiation. The dosages have been adjusted to effective dosages at 100 keV. Where published results were given at another acceleratin volta e, V o keV, the dosage found was multiplied by #to correct for the decreased scattering probability of faster electrons. In view of the damage produced during microscopy, how can electron microscopy lead to high resolution structure determination? In a very important paper, Unwin and Henderson (26) and also Kuo and Glaeser (15) recognized that in crystalline objects consisting of a large array of 94R
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identical unit cells, it is possible to take electron micrographs at such a low electron dosage that the probability is negligible that an electron will traverse a region already damaged by a previous electron. At these low dosages, only very noisy micrographs of each unit cell are obtained. However, if there are many unit cells, the large number of noisy micrographs can be averaged and a high quality micrograph obtained. Henderson and Unwin used these principles to deduce the three-dimensional structure of the purple membrane of Halobacterium halobium (8). This approach points the way to the three-dimensional determination of structure through electron microscopy for molecular systems which can be incorporated into appropriate crystalline arrays. Indeed, similar approaches were used in studies of the structure of certain radiation-resistant inorganic niobium compounds (10-13). It should not be surprising that the above mentioned approach also eliminates the problem of contrast. After all, contrast is inadequate only if the signal is submerged in the noise. Averaging over a large number of unit cells leads to a great improvement of the signal-to-noise ratio. This is excellently illustrated by comparing the raw data of Unwin and Henderson-micrographs almost totally lacking in contrast-with the averaged micrograph which has excellent structural information. It is worth noting that the threshold dosage used by Unwin and Henderson below which damage is negligible is 0.5 electron per Az. MOBILITY OF HEAVY ATOMS The visibility of individual heavy atoms ( I ) opens the important possibility that in a chemical or biological preparation, chemical groups can be identified through appropriate labeling by heavy atoms. In this connection, an important technical question must be answered: Does the heavy atom move during preparation or electron microscopic observation? The sharp images of heavy atoms obtained by a number of workers using test specimens (11) or nucleic acids with marked bases (27) and most recently protamine with its amino terminal labeled (20) would suggest that atoms do not move excessively during minimal exposures. On the other hand, clear evidence of mobility was observed by Langmore, Isaacson, and Crewe (17) in preparations of various atoms. They found that during irradiation, 3-10 A jumps occurred although few atoms were actually lost from the specimen. The movement observed during irradiation with dosages of lo4 to lo6 electrons per A2 was small for uranium atoms but apparently of the order of tens of 8, for silver atoms. Langmore and Crewe (16) examined DNA molecules in which particular bases had Hg atoms incorporated. These preparations which appeared attractive for an electron microscopic study of base sequence gave disappointing results. When a high level of irradiation was used, less than five mercury atoms were observed per 100 A of DNA, whereas the chemical data prior to grid preparation suggested about 30 atoms per 100 A. Surprisingly even under very low levels of irradiation ( 5 X C/cm2 or about 1 electron per A2), the mercury atoms were not found on the DNA strands but appeared in the background. It is not known if these heavy atoms became separated from the DNA during the grid preparation or during the earliest stages of electron microscopic observation. At present, there is no conclusive information on the causes of the variable stability of heavy atoms. It could result from greater dosages used in the STEM which is in principle avoidable, or from greater dosage rates in the STEM which is unavoidable or it could simply reflect a difference in the specimens. The elucidation of these phenomena is among the most important tasks facing electron microscopy today. RESULTS AND APPLICATIONS The electron microscope is so generally used in the analysis of structure that any attempt to review the results obtained with such instruments would come close to reviewing major portions of biology, chemistry, and solid-
Mlchael Beer is professor of biophysics at Johns Hopkins University. He was born in Budapest, Hungary, but moved to Canada in 1938. He received a B.A. in physics and chemistry in 1949 and an M.A. in 1950 from the University of Toronto. Dr. Beer earned his Ph.D. in physical chemistry at the University of Manchester, England, in 1953. He was a research associate in physics at the University of Michigan from 1953 to 1954 when he became an assistant professor in physics. After serving as a Fellow of the National Research Council of Canada, Ottawa, from 1956 to 1958, he joined the faculty at Johns Hopkins University. Dr. Beer is president of the Biophysical Society.
state physics. I shall content myself by citing a few excellent reviews which are concerned with particular applications. LITERATURE CITED (1) A. V. Crewe, J. Wall, and J. Langmore, "Visibility of Single Atoms", Science, 168, 1338 (1970); A. V. Crewe, J. Langmore, J. Wall, and M. Beer, "Single Atom Contrast in a Scanning Microscope", Proceedings of 28th Annual EMSA Meeting, 1970, p 250. (2) R. A. Crowther and A. Klug, "Structural Analysis of Macromolecules Assemblies by Image Reconstruction from Electron Micrographs", Ann. Rev. Biochem., 44, 161 (1975). (3) J. Dubochet. "Carbon Loss During Irradiation of T4 Bacteriophages and E. Coli Bacteria i? Electron Microscopes", J. Ultrastruct. Res., 52, 276 (1975). (4) S.C. R. Eigin and H. Weintraub, "Chromosonal Proteins and Chromatin Structure", Ann. Rev. Biochem., 44, 725 (1975). (5) R. M. Glaeser. V. E. Coslett, and U. Veldre, "Low Temperature Electron Microscopy: Radiation Damage in Crystalline Biological Materials", J. Microsc., 12, 133 (1971). (6) R. C. Hart and J. M. Yoshiyama, "Electron Microscopy with Reduced Beam Damage to the Specimen: A Retractable Image Intensifier", J. Ultrastruct. Res., 51, 40 (1975). (7) R. H. Haschemeyer and E. de Harven, "Electron Microscopy of Enzymes", Ann Rev. Biochem., 43, 279 (1974). (8) R. Henderson and P. N. T. Unwin. "ThreeDimensional Model of Purple Membrane Obtained by Electron Microscopy", Nature (London), 257, 28 (1975). (9) S. W. Hui and D. F. Parsons, "Direct Observation of Domains in Wet Lipid Bilayers", Science, 190, 383 (1975). (10)S. lijima. "Direct Observation of Lattice Defects in KNb205 by High Resolution Electron Microscopy", vv Acta Crystaliogr., Sect. A, 29, 18 (1973). (11) S. lijima, S. Kimura, and M. Goto, "Direct Observation of Point Defects in Nb120P9 by High Resolutioii Electron Microscopy", Acta Crystal-
Many of the instrumental and technical questions related to high resolution electron microscopy are discussed in the excellent book edited by Siegel and Beaman (22). In it, articles appear on resolution and contrast, on a p lications in materials science and metallurgy, on various fiological problems relating to beam damage, on energy analysis, and on aspects of microbeam analysis. Crowther and Klug (2) have written a very readable summary of the procedures used and results obtained in deducing three-dimensional structural information from electron micrographs. The many electron microscopic results on the structures of enzvmes have been reviewed bv Haschemeyer and de Harven (7). The electron microscopic studies of DNA have been summarized bv Younehusband and Inman (31). Recent advances inlour understanding of the structure of chromatin is based in an important way on electron microscopy. Some of these developments have been reviewed by Elgin and Weintraub ( 4 ) .
logr. Sect. A, 29,632 (1973). (12) S. lijima and J. G. Allpress, "Structural Studies by High Resolution Electron Microscopy: Tetragonal Tungsten Bronze-Type Structures in the System Nb205-W03", Acta Crystallogr., Sect. A, 30, 22, 29 (1974). (13) S. lijima, S. Kimura, and M. Groto, "High Resolution Microscopy of Nonstiochiometric Nb2*OS4Crystals: Point Defects and Structural Defects", Acta Crystallogr. Part A, 30, 251 (1974). (14) M. Isaacson, D. Johnson, and A. V. Crewe, "Electron Beam Excitation and Damage of Biological Molecules; its implications for Specimen Damage in Electron Microscopy", Radiat. Res., 55, 205 (1973). (15) I. A. M. Kuo and R . M. Glaeser, "Development of Methodology for Low Exposure, High Resolution Electron Microscopy of Biological Speciments", Ultramicroscopy, I , 53 (1975). (16) J. P. Langmore and A. V. Crewe, Progress toward the Sequencing of DNA by Electron Microscopy", Proceedings of 32nd Annual EMSA Meeting, 1974, p 376. (17) J. P. Langmore, M. S.Isaacson, and A. V. Crewe, "The Study of Single Atom Motion in the STEM", 32nd Annual Meeting of EMSA, 1974, p 378. (18) D. L. Musinski, S.T. Wang, and B. M. Siegel, "Liquid Helium Cryostat for the Imaging System of a High Resolution Electron Microscope", 33rd Annual Meeting of Electron Microscopy Society of America, 1975. (19) F. P. Otiensmeyer, R. F. Whiting, E. E. Schmidt, and R . S. Clemens, "Electron Microtephroscopy of Proteins", J. Ultrastruct. Res., 52, 193 (1975). (20) F.P. Ottensmeyer, R. F. Whiting and A. P. Korn, "Three Dimensional Structure of Herring Sperm Protamine Y-1 with the Aid of Dark Field Electron Microscopy", Proc. Nati. Acad. Sci. U.S.A., 72,4953 (1975). (21) D. F. Parsons, V. R,, Matricardi. R . C. Moretz, and J. N. Tuner, "Electron Microscopy and ~~
Diffraction of Wet Unstained and Unfixed Biological Objects", Adv. Biol. M e d . Phys. 15, 162 (1974). (22) B. M. Siegel and D. R. Beaman, Ed., "Electron Microscopy and Microbeam Analysis", Wiiey, New York, N.Y., 1975. (23) K. Stern and G. F. Bahr, "Specimen Damage Caused by the Beam of the Transmission Electron Microscope. A Correlative Reconsideration", J. Ultrastruct. Res.. 31, 526 (1970). (24) K. A. Taylor and R. M. Glaeser, "Electron Diffraction of Frozen Hydrated Protein Crystals", Science, 166, 1036 (1974). (25) K. A. Taylor and R. M. Glaeser, "A Modified Airlock Door for the introduction of Frozen Specimens into the JEM lOOb Electron Microscope", Rev. Sci. instrum., 46, 58 (1975). (26) P. N. T. Unwin and R. Henderson, "Molecular Structure Determination by Electron Microscopy of Unstained Crystalline Specimens", J. Mol. Biol., 94, 425 (1975). (27) R. F. Whiting and F. P. Ottensmeyer, "Heavy Atoms in Model Compounds and Nucleic Acids Imaged by Dark Field Transmission Electron Microscopy", J. Mol. Biol., 67, 173 (1972). (28) J. W. Wiggins, M. Beer, D. C. Woodruff, and J. A. Zubin, "Unique Features of a High Resolution Scanning Transmission Electron Microscope", Proceedings of 32nd Annual EMSA Meeting, 1974. (29) R. C. Williams and H. W. Fisher, "Electron Microscopy of Tobacco Mosaic Virus Under Conditions of Minimal Beam Exposuree", J. Mol. Biol., 52, 121 (1970). (30) R. E. Worsham, W. W. Harris, J. E. Mann, E. G. Richardson, and N. F. Ziegler, "A 150-kV High Coherence Microscope", Proceedings of 32nd Annual EMSA Meeting, 1974, p 412. (31) B. Younghusband and R. B. Inman, "The Electron Microscopy of DNA", Ann. Rev. Biochern., 43, 605 (1974). (32) "Techniques et Applications de la Microanalyse en Biologiee", J. Microsc. Biol. Ceilulaire, 22,No. 2 and 3 (1975).
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