Electron Microscopy - Analytical Chemistry (ACS Publications)

THE REACTION BETWEEN DIPHENYLAMINE AND NITRATES IN ULTRAVIOLET LIGHT. B. B. Coldwell , S. R. McLean. Canadian Journal of Chemistry 1959 ...
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ELECTRON MICROSCOPY F. A. HAMM General Aniline & Film Corp., Easton, P a .

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ROGRESS in electron microscopy as an applied science has been very real. Although few, if any, problems have been solved in the real sense of the word through use of the electron microscope, applications of the instrument seem to have overshadowed developments of new or better electron microscopes. This is the natural consequence of the ability to extend in a rather accurate way the range of human vision. Although the recent publication (12) on Muller’s point microscope may have aroused interest in striving for the ultimate in resolution, most of the significant results have been derived from reproducible and reasonably accurate studies of structures on the order of 100 A. or greater. Certainly in the fields of biology and metallography, the electron microscope is extensively considered along with the light microscope in a corroborative way. Extended use of electron microscopy is being enjoyed to the greatest extent in the field of biology, manifest by the many installations of the instrument in universities, medical schools, and medical research institutes. Refinements in specimen preparation have been realized, but the success and confidence displayed by the average worker are probably primarily due to the greater thoroughness with Fhich electron microscopical researches are being conducted. The word “artifact” is used less, and its meaning is often recognized as implying an observation that may be inherent but a t the same time understood. Artifacts often no longer degrade or detach significance from a micrograph. This review is not intended to include a complete bibliography of recent publications. The third addendum to the Electron hficroscope Society of Bmerica bibliography is near completion a t the time of this writing. As before, this addendum is distributed free of charge to all of the society’s members. The original Keysort card bibliography and the two addenda include some 2200 references. Furthermore, a committee a t the Kational Bureau of Standards has recently published a bibliography of electron microscopy (38). Those interested in electron microscopy are indeed fortunate in having ready access to these literature references. INSTRUMENTS

Any dependence by a foreign country on this country for electron microscopes that might have prevailed no longer exists. Many were surprised to learn at the eighth annual meeting of the Electron Microscope Society of America ( 1 4 ) that five different electron microscopes are being produced commercially in Japan. Four are magnetic and one is of the electrostatic type. Resolutions on the order of 50 A. are claimed. Apparently no literature describing these instruments is available. Two distinctly new instruments were recently made available ( 2 % ) , the Philips EM-100 and the RCA-EMT instruments. Deliveries on both have been made. The latter instrument appears to be a good replacement for the RCA-EMC, and is considered by many as a valuable auxiliary instrument, especially useful in shouldering the routine burdens now placed on the larger, more universal instruments. Cosslett (11) and Ellis (16) have compared and described the so-called console models. The relatively new Metropolitan-Vickers EM3 100-kv. instrument has been thoroughly described by Haine et al. (80). This instrument has a vertical column, unlike the Philips instrument, but they are similar in that very high magnifications and 100-kv. accelerating potentials may be employed. It has been reported that the instrument and micrograph exhibit a t the International Congress for Electron Microscopy held

a t Paris, France, September 14 to 22, 1950, was the greatest ever witnessed. The Triib Tauber (Swiss), Siemens-100 (German), Zeiss-AEG (German), CSF (French), Metropolitan-Vickers EM3 and Horizontal (English), Philips EM-100 (Dutch), and RCAE M U were all on display. Based on the papers presented a t the Paris conference, it appears that considerably more instrumentation and “gadgeteering” are being carried out abroad. England and Germany seem to be making the most progress, The Gabor technique (16, 19, 5 2 ) has been somewhat revised and its principles have been more clearly described. Theoretically better resolution and contrast are possible than with conventional microscopy. Haine and Dyson (19) are primarily responsible for the newer approach to diffraction microscopy. An ordinary electron microscope is used to form the diffraction image, subsequently reconstructed with visible light in a phase contrast microscope. Bragg (7) has published a simple explanation of Gabor’s image reconstruction by using light analogies. Haine (Paris conference) described a novel technique for stabilizing high direct current voltages. In principle the change in trajectory of the illuminating electron under the influence of an applied magnetic field is a function of its speed (wave length). This constitutes a velocity spectrometer which replaces the usual feed-back resistance voltage divider. The most fundamental considerations of electron optics appear to be emanating from the German laboratories. Men like W. Glaser, 0. Scherzer, E. IT. Muller, and G. Mollenstedt (40) presented papers a t the Paris conference pertaining to chromatic and spherical aberrations and the compensations therefor. Such information will eventually be available in the literature. Boersch ( 6 ) has described a type of electron filter to remove electrons strongly scattered by the specimen by means of an electrostatic field. Muller (18) gave a demonstration of his point (field) electron microscope, showing the resolution of copper phthalocyanine molecules. Heavy atoms may be “detected.” This type of microscope holds much promise for the resolution of molecules. However, the sample must exhibit very high thermal and vacuum stability, and the vacuum must be much better than normally realized in routine electron microscopy or molecular distillation. Consequently, the application of this technique for general purposes may be self-limiting. The researchers in this country have been somewhat more reluctant to advocate the use of higher accelerating potentials. Ellis (14)has critically evaluated the use of accelerating voltages up to 150 Irv. The problem resolves itself into what orders of contrast and resolution are desired for different specimen thicknesses. The gain in penetrating power with increasing voltage may be offset by poorer contrast and limited resolution because of overlapping structure. Differences in thickness or scattering power within a given specimen may also make image interpretation difficult. Certainly greater electron beam stability and greater specimen stability are derived from the use of higher voltages. Picard et al. ( 1 4 ) described the new RCA 100-kv. electron microscope, which will presumably be available in the near future. Instruments providing a choice of accelerating potentials from 40 to 100 kv. are now commercially available; significant results made possible through the use of higher accelerating voltages are awaited. Columbe ( 1 4 ) described a type of electron shadow microscope using the General Electric electron diffraction instrument. Low magnification (250X) evaluation of the specimen as an aid in choosing the best field for diffraction should be useful.

ANALYTICAL CHEMISTRY

18 The difficulties normally encountered in metal evaporation, such as filament breakagc and metal sputtering, may be eliminated through the use of high frequency (‘70megacycles) induction heating as described by Picard et al. (14). For specific uses this technique should be valuable. Hoaever, due consideration must be given to adequate coupling between frequency and metal to be evaporated, high vacuum requirements to minimize residual gas ionization, and the decrease in evaporation efficiency with time because of the poor coupling at elevated temperatures. Particle size measurements with the electron microscopr have long been discussed, but little quantitative information has been forthcoming. This is really not surprising to those who have attempted it. Froula ( 1 4 ) , however, has devised two useful gadgets that should obviate the tedious, time-consuming photography normally necessary in studies of particle size. The first method involves the magnification of the specimen dimension to a given size by means of a continuously variable magnification control. In the second method, a 0.200-inch circular aperture is moved along a vertical axis below the projector lens until its shadow circumscribes the particle image. Both methods obviously require calibration. Hillier’s ( 2 7 ) removable intermediate lens for extending the range of the RCA-EMU instrument is a worth-while contribution. A magnification range of twenty-five-fold without changing pole pieces is the notable feature. RlacDonald (37) has published a review of instruments available in this country and has made design suggestions for the future. Easier and more precise focus control has gained in popularity because it permits a greater percentage of well focused micrographs. Meryman (39) has described an electromagnetic device placed in the specimen chamber of the RCA-EMU instrument. In effect it is similar to the older electrostatic deflection type of gadget. New possibilities for using standard electron microscopes as diffraction cameras for analyzing liquid vapors have been described by Heston and Cutter (26). A small sample holder with needle valve assembly is fitted into the regular diffraction stage of an RCA-ERIU instrument. Jacob (30) in a somewhat theoretical paper has discussed the geometry of an electron beam a t crossover in an immersion objective a t various voltages, cathode distances, etc. The two-wave-length microscope has been discussed by Buerger (8). In his instrument x-rays are used up to the object diffraction image. Light rays are then brought into phase to produce an electron density map of the crystal, showing spatial arrangement of the atoms. Vseful magnification up to several hundred thousand diameters is realized. A photoelectron autoradiographic technique using an emission microscope has been described by Barker (6). Again the applications are self-limiting because the specimen must be activated to emit electrons and the exposure times are long. Sinipson and Bronkhorst (66) have designed a type of universal stage for rotating the specimen along two axes not parallel to the optic axis. Their interests are primarily the plotting of field strengths around ferromagnetic crystals A distorted image of the supporting mesh is superimposed on the crystal image; magnetic field strengths are determined from the mesh image distortion. Lattice constants for thin films have been determined by Schulz (54)by using a multiple reflection electron beam scattering technique. The effects of vibration and lens asymmetries on the resolution of an RCA-EMU instrument have been described by Hamm (23, 24). Remedial procedures are evaluated. APPLICATIONS

General. It is now apparent that the applications of electron microscopy to specific problems in biology and metallography somewhat overshadow the various other fields.

Backus and JYillianis (4)developed a valuable technique for use in qualitative or quantitative assays suitable, for example, for determining virus counts or particle weights. The method consists of spray-drying of tiny droplets which are representative of the sample. Volatile pH adjusting electrolytes are used. Replica techniques are now well established and are being extensively used. However, Hass and McFarland ( 2 5 )described a new two-step aluminum oxide replica technique which produces stable replicas exhibiting a minimum of inherent structure Electrolytic oxidation of the aluminum in contact with the specimen, follovr-ed by dissolution of the remaining aluminum and x backing layer of magnesium, represents the general procedure Kahler and Lloyd (31 ) have compared various shadowing techniques from the standpoint of inherent errors in measuring particle sizes. They prefer a double shadowing from opposite sides. I t is somewhat surprising that more m ork on dyed textile fibers has not been reported. Sections, both longitudinal and transverse, and replicas of dyed fibers can be prepared. Hock (69) has reported on this subject using degraded cellulose fibrils as hi3 subject. Kaye (32, S?) has reported on replica studies of sjnthetic fibers, using an aluminum-beryllium alloy for substrate and replica preparation. A good correlation between these results and modern theories of dyeing should some day be possible. .4 dearth of information relevant to catalysts and fine metal particles in general may be indicative of the difficulties encountered in critically studying such materials. Electron microscopy data should be correlative with other data determined by adsorption and x-ray investigations. 1IcCartney e t al. (36)have published a good, comprehensive elrctron microscopical study of metal oxide catalysts. Rochow ( 5 1 ) is continuing his resinographic program and recently has been concerned with organosilicone macromolecules Replicas of fractured surfaces constitute the general technique. Richard and Smith ( 4 7 ) have studied gel formation of polymerplasticizer systems. Mottlau (41), Wittebort, and Baker et al. ( 1 4 ) have described specimen preparation techniques for lubricating greases. Identification of the solid components, such as soaps, and evaluation of chemical and physical treatments on these greases are now possible. Recent research on soaps primarily from the standpoint of their cleansing power has also been disclosed (IS). Polystyrene latex spheres (Dow Chemical 580-G) are still the subject of controversy, although their use as size standards has been well established. Gerould ( 1 7 ) has tabulated the results of various types of size determinations from seventeen laboratories. Kern and Kern (34)have offered what appears to be a reasonable explanation for discrepancies in the measured sizes of these latex spheres and other particles. I n brief, they contend that unshadowed portions build up a negative charge which then acts as a negative lens, decreasing the image size. Yudowitch ( 1 4 ) has calculated a relatively high size for the latex spheres from loa-angle x-ray scattering. One of the most significant recent contributions in the field of electron microscopy has been developed by Sennett (22, 55) and his eo-workers a t Toronto. Structural factors of thin evaporated films of metals have been correlated with their optical and electrical characteristics. It has been reported that excellent dispersions of organic pigments were illustrated by Cooper a t the Paris conference. A spray-drying technique is used, somewhat similar to that reported by Backus and Williams (4). Cosslett ( 1 0 ) has reported in a general way on the problem of preparing electron microscope specimens. Reed and Millard ( 4 6 ) , also in England, have described a replica technique for studying photographic emulsions before and after development to gain information about grain size, shape, etc. The details for recording motion pictures of electron microscope images have been described by Preuss and Watson (44,59).

V O L U M E 2 3 , N O . 1, J A N U A R Y 1 9 5 1 Those investigators who firid it possible to utilize this technique can study instantaneous specimen changes, and of course motion pictures sometimes serve Tvell for lecture purposes. Caution must be exercised in avoiding an excess of x-ray dosage from the various ports and final viewing screen xvhen strong illumination is used. JVatson and Preuss (60) have described techniques for measuring this x-ray radiation. Metallurgical. I t was pointed out in last year’s review ( 2 1 ) that m:my of those concerned Irith electron metallography were about ready to present their results. This came to pass a t the Detroit meeting of the Electron Microscope Society of America ( 1 4 ) . The group now known as Subcommittee XI on Electron 3Iicrostructure of Steel of A.S.T.hI. Committee E-4 deservcls niuc,h credit for havine organized and carried out a cooperativr rcwarch program. The program is estensive in scope, as illustrated by the numerous structures already investigated, such as (’oarse and fine pearlite, bainite, tempered (at different temperatures) martensite, carbides, etc. Critical evaluation of replica techniques and etchants based on light and electron micrographs prepared in different laboratories serves to check on the validity o f their observations. Some seventeen members of this committee are cooperating on this program. Although these electron metallographers have been slon- in publishing their resuks, caution and good judgment hare been esercised, so that their work must be respected. A survey of two years’ work was presented by Prllisier ( 1 4 ) , Veil and Reed (61) have oramined directly very thin films of metals (nickel on zinc) electroplated, rather than resorting to the use of replicas. Better resolution is claimed, dt,hough this approach is limited by the nature of the specimen. Haas (18)has discussed graticules as guides in electron metallography. He described an instrumrnt for cutting fine lines on a polished metal surface so that the same area may be replicated repeatedly. I t seems apparent that the electron microscope used in conjunction with the light microscope will serve a useful purpose in correlating microstructure of steels with their preparation and physical properties. Biological. The application of electron microscopy to biological subjects is now universal, in the sense that almost, every conceivable type of living structure has been investigated. Biological systems are tremendously complex, to say the least. For this reason very feyr positive statements of practical significance are made-for example, in the study of healthy and malignant tissue, viruses (plant and animal), bacteria, blood cells, etc., little or no information of unequivocal correlation LTith clinical data has been forthcoming. Positive identification of microorganisms for diagnosis of infectious diseases, identification of cancer-producing substance8 (viruses), correlation between type and number of microorganic structures conirnensurate with cert:iin levels of infectivity, etc., are typical problems that are constantly kept in the minds of this group of electron microscopists. Because Ire can assume that these investigators now have confidence in their techniques, some of these problems may eventually be solved, but only after considerably morr information has been obtained. I t lvould be illogical to predict that progress in this field rrill be rapid. Severtheless the sanguine interest of many and the prospwts of resolving the intricacies of a t least some forms of life will continue to stimulate high grbde research in this field. Because of the wealth of published information, the reader is advised to consult the particular journal most appropriate to the type of biological structure of interest. One of the more significant achievements resulting from the study of bacteria and tissue sections is that of “fixation” by means of electron bombardment ( 1 4 ) . Low levels of illumination cause chemical changes, so that bacteria, tissue sections, embedding media, collodion substrates, etc., exhibit much lower solubilities and greater heat stability. Valence forces are presumably affected. A “ k e d ” substrate may therefore be used in preparing specimens in ways not possible before insolubilization.

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.inother new technique that demands particular attention was developpd by Anderson ( I ) , who has succeeded in minimizing the serious surface tension forces normally encountered in drying biological specimens. Although his technique may be a compromise because of the use of organic solvents and liquid carbon dioside, certainly the drastically deleterious effects of strong surface tension forces are obviated as manifest by the striking threedimensionality of his stereoscopic images. Angulo and Watson (2) have investigated liver cell nuclei from laboratory animals. The same authors (3)note the presence of filamentary bodies in skin tissue fluid from a patient with the pinta disease similar to those of influenza, poliomyelitis, and the pores. A degraded erythrocyte is postulated as the common source. A possible application of the electron microscope for diagnostic purposes is conjectured by Coriell arid his co-workers (9). The elementary bodies, presumably a virus, isolated from lesions of thr herpes simplex disease, appear to exhibit a unique shape. The preservation of nuclear and cytoplasmic material in bacteria does much to introduce contrast; however, electron mirroscopy has revealed little new information concerning these structures. RIudd and Smith (42) in a study of E. coli found that the electron-scattering powers of these structures, when fixed with osmium, are the antitheses of those exhibited when treated with hydrochloric acid. Smith (5Y)has also described an improved technique for staining chromatinic. bodies in bacteria. Robinson and Bishop (60) have described methods for preparing bone and tooth specimens. A good technique for examining red blood cells has been tleveloped in France (Paris conference). A drop of fresh blood is spread on a collodion film, incubatrd, and floated off on water, shadowed, and examined in the electron microscope. Apparently the reaction at the solid interface bet\veen collodion and blood is analogous to the clotting of blood after a cut. Those interested in electron microscopy and other instrumentid approaches to studies of bacteria \vould do \vel1 to consult Racferial Proceedings of 1‘350 ( 6 8 ) . X o w that it has definitely been establiahcd that slow speeds are desirable for ultrat,hin sectioning, the problems of sperinien advance in the microtome, specimen fixation, and embedding are being argued ( 1 4 ) . There have developed t,wo schools of thought in regard to specimen advance in the microtome. Hillier and his eo-workers (28) have modified a commercial Spencer microtome so as to minimize static friction, thermal expansion, external vibration, ctc. Sewman et al. ( 4 3 )originated the idea of advancing the specimen supporting block by means of thermal espansion after cooling. Tissue sections of good quality have been prepared in both nays. The advisability of removing the embedding medium, the distortion introduced through embedding, the evaluation of staining techniques, etc., have all been considered (14). h i a l sect,ioning through an nppreciable portion of the specimm is now possible (28) and is probably a prerequisite to a critical study of certain structures. Richards (48,49), who n-as formerly concerncd with light microscope microtomy, has now given some thought to knife sharpness and static electricity. Transverse and longitudinal sections of nerve fibers ( 6 3 ) permit a much more detailed study of these structures. Bakrr (14) has studied tn-o-componrnt greases through the medium of thin sections of frozen specimens. FOREIGN DEVELOPMENTS

The International Congress for Electron Microscopy held in Paris, briefly described above, has set a notable precedent for a similar gathering which is to be held biennially. Undoubtedly many people contributed to the success of this meeting, but Locquin of the Kational Museum of Natural History in Paris was primarily responsible for the arrangements. It was the first meeting of its kind to which German workers were invited. The

ANALYTICAL CHEMISTRY

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French deserve commendation for extending this invitation. Among the approximately 300 people who attended, many well knoxvn names appeared. Anderson, Boersch, Cosslett, Gabor, Glaser, Hillier, Kellenberger, hIahl, Mollenstedt, Muller, Ruksa, Schemer, and V O I ~Borries are a few of the authorities who were present. The exhibition of electron micrographs from countries such as Australia, Japan, and Sweden also added interest to the meeting. The German workers seem t o be emphasizing instrumentation. The biological applications appear to be concentrated in England. The Department of Biomolecular Structure a t the University of 1,ecds is one of thc largest groups in this field.

Coriell, L. I., Rake, G., Blank, H., and Scott, McS. T.’F., J . Bact., 59, 61-8 (1950).

Cosslett, V. E., Nature, 165, 1009 (1950). Ibid., 166, 305-6 (1950). Discovery, 274-5 (1950). D y e r , CII, 621--2 (1949).

Electron Microscope Society of America, J . Applied Phys. (January 1951); A4bstractsof papers presented Sept. 14 to 16, 1950, at Detroit, Mich. Ellis, S.G., Rev. Sci. Instruments, 21, 255-7 (1950). Gahor, D., Proc. Roy. SOC.,197, 454 (1949). Gerould, C. H., J . Applied Phys., 21, 1 8 3 4 (1950). Haas, E., .Vature, 166, 482 (1950). Haine, hI. E., and Dyson, J., Ibid.. 166, 315 (1950). Haine, M. E., Page, R. S.,and Garfitt, R. G., J . Applied Phys., 21, 173-82 (19501.

FUTURE DEVELOPMENTS

The interests of the members of the Electron 1\Iicroscopcl Society of America are gradually being focused onto decidedly different subjects. The concent,rated efforts on electron metallography and biology are cited as examples. A further breakdown of the subject of‘ biology into the plant and animal kingdoms, or of t,issue cells into liver, nerve, or muscle classifications serves to eniphasizo the degrcr t o which electron microsropy ha3 become spechlized. a n y projection into the future is pure conjecture and must be done advisedly. Nevertheless, it is conceivable that these specialized interests may affect the proceedings of the Electron hlicroscope Socaiety. Those attending the eighth annual meeting in Detroit ( 1 4 ) could sense this, and the atmosphere prevailing a t this mceting might be interpretrd :is a harometrr of things to come. One possible developnient may result in certain groups of electron microscopists preferring to attend other meetings of a type more specifically related to their interests. If we consider those electron microscopists not primarily concerned with biology or metallography, it may be argued that their interests are focused on subjects of a more general “colloidal” nature. Strict definition of terms is t o be avoided. It then also develops that these colloidal electron microscopists are for the most part working in industrial laboratories. Come what may. the development of electron microscopy and the affairs of the American society are awaited with interest. The proton microscope is no longer being discussed as a likely probabilit,y, and its serious limitations are now well known. Kirkpatrick (%), the pioneer in this field, has reviewed the x-ray microscope as an instrument and as a research tool. The limiting aspects, diffraction and geomet,rical effects, have been discussed by Prince (45). It may be a fair statement to predict some useful but restricted applications of x-ray microscopy hecause of the greater penetrating power of x-rays; however. the practical resolution may fall somewhere between the light and electron microscope. Because of the variety of specimens being examined in the various types of commercial electron microscopes, the year 1951 may yield reports demonstrating the advantages of using higher accelerating voltages, depending on the nature of tho specimen.

Hamm, F. .1.,AAx.kr,. CHEY.,22, 26-30 (1950). Ibid., pp. 958-8. Hamm, F. A,, J . Applied Phys., 21, 271-8 (1950). Hamm, F. A, and Snowden, F. C., Ea’. Sci. Instruments, 21, 426-31 (1950).

Hass, G., and McFarland, M.E., J . Applied Phys., 21, 435-6 (1950).

Heston, €3. O., and Cutter, P. R., R ~ PSei. . I n s t n m e n f s . 21, 60812 (1950).

Hillier, J., J . Applied Phys., 21, 785-90 (1950). Hillier, J., and Gettner, hl. E., Ibid., 21, 889 (1950). Hock, C. W., Textile Research J., 20, 141 (1950). Jacob, L., J. d p p l i e d Phys., 21, 966-70 (1950). Kahler, H., and Lloyd, B. J., Jr., Ibid., 21, 699-704 (1950). Kaye, Kilbur, Ibid., 20, 1209-14 (1949). Kaye, Wilbur, PS.4 Journal, 16, 62 (1950). Kern, S. F., and Kern, R. A, J . Applied Phys., 21, 705-7 (1950). Kirkpatrick, P., Xature, 166, 251 (1950). LIcCartney, J. T., et al., J . Phys. & Colloid C h e m , 54, 505 (1950). MacDonald, W. W., Electronics, 23, 66-9 (1950). Marten, C., et al., Satl. Bur. Standards, Circ. 502 (19501. bferynian, H. T., Rev. Sci. Instruments, 20, 955 (1949). RIollenstedt, G., Optik, 5 , 499-517 (1949). hlottlau, 9.P., J . Applied Phys., 20, 1055-9 (1949). hIudd, S., and Smith, A. G., J. Bact., 59, 561-73 (1950). Seaman, S.B.. et al., Science, 110, 66 (1949). Preuss, L. E., and Tatson, J. H. L., J . Applied Phys.. 21, 902-3 (1950).

Prince, E., Ibid., 21, 698 (1950). Reed, R., and Millard, A,, PYOC. Leeds Phil. Lit. SOC..5, 81-8 (1948).

Richard, K. R., and Smith. P. .L, J . Chem. Phys., 18,230 (1950). Richards, 0. IT., Rec. Sci. Instritmenfs, 21, 670 (1950). Richards, 0. W.,and Jenkins, R. L., Science, 111, 624-5 (1950). Robinson, R. 1., and Bishop, 17. IT., Ibid., 111, 655-7 (1950). Rochow, T. G., and Rochow, E. G., I b i d . , 111, 271-5 (1950). Rogers, 0. L., S a t i t r e , 166, 237 (1950). Rozsa, G., Morgan, C., Szent-Gyorgyi, A , , and FYyckoff, R . W.G., Science, 112, 42-3 (1950).

Schulz, L. G., J . Applied Phys.. 21, 942 (1950). Sennett, R. S., and Scott, G . D., J . Optical Soc. A m . , 40, 203-11 (1950).

Simpson, J. A., and Bronkhorst, A. I-.,Rec. Sci. Insfrutnents, 21, 669-70 (1950).

Smith, A. G., J . Bact., 59, 575-87 (1950). Soc. Am. Bacteriologists, Bnctwial Proceedings (May 14 to 18, 1950).

Watson, J. H. L., and Preuss, L. E., J . Applied Phys., 21, 904-7 (1950).

Vatson, J. H. L., and Preuss, L. E., Science,112, 407-9 (1950) Weil, R., and Read, H. J., J . Applied Phys., 21, 1068 (1950). RECEIVED S o r e m b e r 17, 1950.

.ACKNOWLEDGMENT

The author is indebted to James Hillier, RCA Laboratories, Princeton, S . J., for t,he information on the Paris conference. LITERATURE CITED

(1) Anderson, T. F., J . Applied Phys., 21, 724 (1950). (2) Angulo, J. J., and Watson, J. H. L., Science, 111, 670-2 (1950). (3) Angulo, J. J., Watson, J. H. L., and Olarte, J., J. Bact., 60, 12938 (1950). (4) Bsokus, R. C., and Williams, R. C., J . A p p l i e d Phys., 21, 11-15 (1960). ( 6 ) Barker, A. N., Research, 3, 431 (1950). (6) Bocrwh, H., Optik, 5, 436-50 (1949). (7) Bragg, W.L., Nature, 166, 399-400 (1960). ( 8 ) Buerger, M.J., J . AppZiedPhys., 21, 909-17 (1950).

SKINNER L SHERMAN. I N C