INSTRUMENTATION
Advisory Panel Jonathan W. Amy Richard A. Durst G. Phillip Hicks
Donald R. Johnson Charles E. Klopfenstein Marvin Margoshes
Harry L. Pardue Howard J. Sloane Ralph E. Thiers
Prospects for Molecular Microscopy Recent improvements in specimen support films and image contrast will help achieve the goal of visualizing single atoms or small molecules by electron microscopy. Other possible applications in chemical identification and structure determination, including the detection of X-rays, fluorescence emission, and secondary electron emission, are likely to be exploited J. WENDELL WIGGINS and MICHAEL BEER Thomas C. Jenkins Department of Biophysics Johns Hopkins University, Baltimore, Md. 21218 t,he electron microscope has emerged as a major device for the determination of the st,ructure of matter. Many recent developments in biology and metallurgy would have been inconceivable without this powerful tool. For all its success, electron microscopy has been beset by a number of fundamental limitations. Particularly, in reaching for the goal of visualizing single atoms or small molecules, t,here have been four substantial problems: insufficient image contrast, specimen damage, insnfficient resolution, and irregularities in the specimen support film. The problem of resolution has been attacked with a vehemence not afforded the others, perhaps because it was the first recognized. This attack and it,s successes have been well reported elsewhere ( 1 ) , Conventional techniques now allow 2-3 A point-to-point resolut,ion, sufficient for many molecular structure investigations. The problem of specimen damage owing to the electron beam is to a great extent unsolved. Indeed, the extent and nature of t,he damage are just beginning to be assessed. Primarily, we will deal with progress in the other two problems, specimen support films and image contrast. VER THE LAST 25 YEARS,
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Advantages of Graphite Crystallite
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Specimen Support Films Specimens are carried into the vacuum system of the elect,ron microscope after mounting on thin support films. These must be as transparent to eleetrans as possible, st,able in the electron
beam, and sufficiently good conductors of heat and electricity t o avoid local accumulation of charges or excessive temperature during irradiation. Finally, the film should be free of irregular structure t o assure that details of the specimen are not obscured by t,he superimposed structure of the support. Until now the most successful supports were thin films of carbon prodnced by evaporation in vacuo onto a smooth surface ( 2 ) . Such a procedore is bound to lead t o variable thickness. This is indeed found; these supports have an irregular structure which leads to a granular image. This granularity sets the limit on the minimum mass which can he detected. Recently, it has been found that evaporated carbon films can be converted to films consisting of crystallites of graphite by subjecting them to a brief heat treatment at 2 7 W C in an atmosphere of argon ( 3 ) . These crystallites are easily recognizable, as shown in Figure 1. The high temperatures require that the films he mount,ed on carbon support grids instead of the more usual copper ones. On examinat,ion in the electron microscope, the graphite crystallites are conspicuously free of the disturbing granularity found in evaporated carbon films. Then the limiting granularity of support films seems largely eliminated in the graphitization, The decrease in granularity is indicated in Figure 2 . Here, t,he int,ensity of the elastically scattered electrons is sampled repeatedly, showing a scan of a graphite film and a carbon film. Since this intensitv
igure 1. Dark field electron microraph of thin graphite film produced by igh temperature heat treatment of carbon film Crystallites 0.1 to 0.2 pm in size are clearly visible. 50,OOOx
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1. JANUARY 1972
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Instrumentation
of scattered electrons is a measure of t,he local thickness, the distribution is related to the distribution of thickness -that is, to the granularity. The distribution is clearly narrower for the graphitized film, even though it was, on t,he average, thicker. This improvement cannot be realized if carbon contamination is introduced during observation. Indeed, if absolutely no cont,amination had been present in the instrument when the micrographs were taken, the difference between the distribut,ions in Figure 2 would have been even greater. However, one important difficulty remains to be solved. The graphite films have extremely different adsorption properties from the carbon ones; therefore, new specimen mounting procedures must be found. Improvements in Image Contrast Figure 2.
Distribution of local scattering intensities from carbon and graphite films
Distribution is a measure Of the thickness Variation. Though overall thickness Of graphite film is considerably greater. thickness variation is considerably less than for carbon film
Figure 3. Row of single thorium atoms a s seen in scanning electron microscope Specimen produced by Spraying on carbon films an equimolar mixture of Th(NO3h and 1, 2. 4. 5benienetetracarboxylic acid. Small S P O ~ E along chains are single atoms Of thorium. Micrograph is roughly 670 A to each side
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ANALYTICAL CHEMISTRY, VOL. 44. NO. 1. JANUARY 1972
We shall now turn t o the important recent development, by Crewe (4) and his collnborators, of the scanning transmission electron microscope (STEM) which has improved the prospects for obtaining better image contrast and some specific chemical information about the components of the specimen. One illustration of the advantage of the STEM is provided by the clear visibility of single heavy atoms (6),as shown in Figure 3. Using similar (though not rigorously identical) specimens, Highton and Beer ( 6 ) estimated that the threshold of visibility for the convent,ional electron microscope is approximately three heavy atoms. The scanning electron microscope of Crewe differs from the conventional scanning type in two basic respects. The high resolution is made possible by the introduction of a unique and ingenious electron gun combining a field emission source, as developed by Muller et al. (7), and a low aberration accelerating lens (8). At the same time they introduced detection of the electrons after transmission through the specimen, which allows great versatility in assessing the interaction with the specimen. To understand the advantages of the STEM, let us state the mode of image formation in both STEM and the transmission electron microscope (TEM). See Figure 4 for a comparison of the schemat,ic diagrams of the T E M and the STEM. In the T E M the entire field of view is illuminated continuously. A magnified image of the field of view is produced on a fluorescent screen or, for permanent recording, a phot,ographic plate. If the optical system were ideal, all electrons incident on the field of view would cont,ribute to the final imq e . The only image cont,rast would he owing to t,he fact that when an elec-
Instrumentation
H a i r p i n filament Grid cap
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Field emission t i p
Anode
Acceleratinq Condenser
lens
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D e f l e c t i o n coils Lens
Specimen Objective
lens
Objective aperture
,-!I
electrons
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P r o j e c t o r lens
Annular detector
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4 1I -
Unscattered electrons ond inelasticallv scattered ‘ electrons
Figure 4. Comparison of schematic diagrams of transmission electron microscope and scanning transmission electron microscope
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Speedrometer
I Transmission
Electron
Microscope
tron is elastically scattered by the specimen, a phase shift I> introduced into its wave function This causes destructil e interference a t the corresponding image point However, the optical svstem is not perfect Since the lensehave an unavoidable spherical aberration, optimum spatial resollition IC obtained by placing an aperture stop ‘it the back focal plane of one of the lenses, usually the objectir e This aperture stop provides additional contrast b> completely removing from the image those electrons scattered through ‘in angle greater than that of tlic aperture. The lense. nlho ha\ e an una\ oidable chromatic aherrdtion Therefore, when a n electron lose5 energy in the scattering process-so-called “inclastic scattering”-it is not focused to the proper image point Then the three processes-int erference, aperture removal, and chromatic defocuslng-all remove electrons from the image of a scattering object iZ calculation including all these effect- is difficult Howei er, several calculations have been made ( 9 ) I n general, when the calculated contract of ‘in object of atomic dimensions 1‘ compared ~51thsubqtrate noise, it. observation seems to bc marginallv powble The TEN can he uqed in the dark field mode qo that the unwattered benni i i exchided from the image by an apprturc, and onll- the qcnttered clwtron‘ form the image I n this mode intcrference contrast effects are con.idernbli
Scannt ng Transmission Electron
Microscope
reduced There are both advantages m d disadvantages in going from bright field to dark field operation The main di*advantages are too little illumination for proper focusing, and each of the methods so far proposed for eliminating the unscattered beam introduces some substantial difficult\ One of the prominent advantages is that miich of the noise from the supporting film originates from interference effects largely eliminated in dark field In the bright field mode the photographic plate is darkened before the optimum number of electrons has been collected I n dark field the unscattered electrons which carry no information are absent, and more scattered electrons may be collected. There are, of course, other problem. t o be overcome in the use of the T E M , but these are also t o be dealt w t h in the STEM The considerations abo\e are mentioned because the7 are preciseli those which make it advantageoilto work n i t h a S T E M The STEM diffcrh from the T E M in its method of illuminating the field of view and forming tlie image I n the STEM the 11liimination is focused on the specimen to a fine spot, the diameter of which determines the spatial resolution limit of the dmice Crewe showed that br ii\iiig the eweedingly bright field emiicion source, spot sizes donn to 5 A diameter mere practicable I n the near future this spot size may be further reduced t o perhaps 1 A This illumina-
tion spot is scanned across the field of view b!* deflection coils in synchronism with the raster of a cathode ray tube. The intensity of illumination of thc CRT is determined by some aspect of the electron transmission through tlic specimen. This method of image formation is formally identical to that of the TEN (IO,I f ) . I t follows that the resolution limit of the TEN and the STEM depends in tlie same way on the performance of the objective lens. If comparable lenses are used in thc two devices, the resolution limits will be compnrable. With instrumclnts using an accelerating voltage of 100 kV, the best resolution attainable today is approsimntely 2.5 A. Howel-er, in prnctice there are significant differences. The priinary advnnt:igc accrues from the focusing of the beam before internctioii n i t h the specimen. Thus, onc doe. not hnve to focus electrons which have nlrcndy lost energy in scattering. The chromatic aberration of the objectivc Ims is then no problem. If one v - i d i e ~ n, selective field of view may be achieved by special use of the deflection coils. Having tlie spatial information in hnnti, one is free to process the rlcctron; tranmitted by the qm3men in an\- inanner which vi11 cnhancc the contrnst (4). If the electrons were sorted according to both eiicrq. and xattcring angle, all the information ~voiildbe estrncted. Complete sortinn
ANALYTICAL CHEMISTRY, VOL. 44, NO. I , JANUARY 1972
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
Instrumentation
is difficult. -4helpful natural circumstance is that elastic scattering involves a considerably larger angle than the inelastic. The incident beam has a divergence angle of about 10--2radians. The typical inelastic scattering angle is less t,han 10-8 radians. Therefore, if the electrons with less than 10-2 radians emergence angle are separated from those with a greater scattering angle, one achieves a reasonable, but not complete, separation of the elast.ically scattered electrons from the remainder. This can be done in practice by placing an annular semiconductor junction detcctor below the specimen to intercept the large angle electrons. The small angle electrons may then be separated according to energy loss in an clwtron spectrometer. Depending on the particular system used to sort the electrons, one ends up with several electrical signal currents which may be conibined in various ways by operational amplifier t’echniques to produce a variety of types of contrast. For rsample, division of t,he total elastic aignal by the total inelastic signal produces an image intensity proportional to the atomic number of the scattcring object ( E ) . Possibilities for Chemical Identification
The electron energy loss spectrum contains a wealth of chemical information about the specimen. There is a valuable relationship between the optical absorption spectrum and the electron energy loss spectrum of a specimen. Both spectra are determined by the dielectric constant. The exact, relation is complicated and is discussed thorough1~-in Ref. 23. In general, any optical absorption of a certain energy is reproduced by a corresponding loss in the electron spectrum. I n addition there nrc electron energy losses owing to collective excitations of the valence electrons. Therefore, the prospect merges that chemical identification ma:’ be accomplished on the molecular scalc just as it is now done for larger samples with a spectrophotometer. Some insight into the specificity of the electron energy loss spectrum may be gained from the data of Crewe et al. ( 1 4 , 1 5 ) , who have examined adenine and thymine. The spectra are detectably different and so encourage optimism in chemical identification by this approach. The limit to what can be accomplished by the new techniques for moleciilar microscopy is probably imposrd by the damage to the specimen from the electron beam. The unscattcred and elastically scattered electrons do nothing to the specimen. The inelns-
tically scattered electrons cause excitations of electrons, or ionization. Either case may lead to dissociation of the molecules involved. Whether or not sufficient informat,ion for identification can be obtained before the character of the specimen is changed is not yet known. There have been both theoretical and experimental studies (25, 1 6 ) . Opinion is divided. The benefits to be gained by successful utilization of the energy loss information are so great that the question must be settled finally by experiment. It is almost certain that energy loss identification can be applied to microscopic st’ructures larger than a few atoms, for example, the organelles of a cell. Depending on what, is indeed the severity of radiation damage, one may conceive of determining the three dimensional structure of molecules along x i t h the subunit identit,y by this technique. Finally, it is worth mentioning that the high resolution scanning microscope can be extended so that other manifestations of the interaction of the elect’ron beam with the specimen are detected. Thus, X-rays, fluorescence emission, and secondary electron emission could all be detected.
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Acknowledgment
The authors gratefully acknowledge the generosity of A. V. Crewe who provided Figure 3. References (1) E. Ruska, A d v a n . O p t . Electron Nicrosc., 1, 116 (1966). (2) D. E. Bradley, B n t . J . Appl. Phys., 5 , 65 (1964). (3) J. R. White, M. Beer, and J. W. Wiggins, Micron., in press. (4) A. V. Creme, Quart R e v . Biophys., 3, 137 (19701. (5) A. V. Crewe, J. Wall, and J. Langmore, Science, 168, 1338 (1970). (6) P. J. Highton and M. Beer, J . R o y . A1!fllicrosc.Soc., 88, 23 (1968). (7) E. W .Muller and T. T. Tsong, “Field Ion Microscouv.” Elsevier (1969) : R.
Gomer,~ “Fieid ’ Emission and ’Field Ionization,’’ Harvard University Press
(1961). (8) A . V. Crewe, D. N. Eggenberger, J. Wall, and L. M. Welter, Rev. Sci. Instrum., 39, 576 (1968). (9) C. B. Eisenhandler and B. M. Siegel, J . A p p l . Phys., 37, 1613 (1966). (10) A . V. C r e w and J. Wall, Optik, 30, 461 (1970). (11) E. Zeitler and M. C. R. Thomson, ibid., 31, 269, 359 (1971). (12) A . V. Crewe, Ber. Bunsenges. P h y s . Chem., 74, 1181 (1970). (13) P. Nozieres and D. Pines, Phys. Rev., 113, 1254 (1959). (14) A . V. Crewe, M. Isaacson, and D. Johnson, Proc. EMSA 28th Ann. Mtg., 262 (19701. (15)-A.-l’. ’Crewe, M. Isaacson, and D. Johnson, ibid., 264 (1970). (16) J. R..Breedlove and G. T . Trammell, Sczence, 170, 1310 (1970).
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