ANALYTICAL CHEMISTRY Kinsinger, W.G., Hillier, J., Picard, R. G., and Zieler, H. W., J . Applied Phys., 17,989-96 (1946). Kirchner, F., Naturwissenschaftm, 34, 34 (1947). Klemperer, O., Proc. Phys. Soc. (London). 59, 302-23 (1947). Klemperer, 0.. and Mayo, B. J., J . Inst. Elec. Engrs. (London), III,95, 135 (1948).
Koch, P. A., Freytag, H., and von Ardenne, M., Glastech. Be?.. 21,249-55 (1943).
Konig, H., ‘~~aturwissenschaften, 34, 108 (1947). Kurotchkin, T. J., Libby, R. L., Gagnon, E., and Cox, H. R., J . Immunol., 55, 283 (1947). Kushnir, Yu. M . , Elektrichestro, 5, 3-16 (1947). Laplume, J., Cahiers phys., 1947, 55-66. Liebmann, G., J. Sci. Instruments, 25, 37-43 (February 1948). Liebmann, G., Phil. Mag., 37, 677-85 (1946). Loring, H. S.,Proc. SOC.Exptl. Biol. Med., 64, 101 (January 1947).
Mahl, H., and Recknagel, A., 2. Physik, 122,660-79 (1944). Mandle. R. J., Proc. Soc. Exptl. B i d . illed., 64, 362-6 (1947). Markham. R., Smith, K. M., and Wyckoff, R. W.G., Sature, 159,574 (1947). Ibid., 161, 760 (1948).
Marton, L., and Bol, K., J . Applied Phys., 18,522-9 (1947). Mateosian, E. der, Iron Age, 159, 51-3 (1947). Matolsky, A. G., Nature, 161, 353 (1948). Mollenstedt, G., Reichsber. Physik, 1, 10-11 (1944). Mossman, H. W., and Noer, H. R., Anat. Record, 97, 253 (February 1947). Muller, H.O., Chem. Zentr., 1, 71 (1947). Kutting, G. C., and Borasky, R., J . A m . Leather Chemists Assoc., 43, 96 (1948).
O’Daniel, H., and Kedesp, H., .’lhturwissenschaften, 34, 55 (1947).
Osier, G., and Stanley, W.M., Brit. J . Exptl. Path., 27, 261-5 (1946).
Passey, R. D., Dmochowski, L., Astbury, W. T., and Keed. R., Nature, 159, 635 (1947). Ibid., 161,759 (1948). Pease, D. C., and Baker, It. F., Proc. S O C Erptl. . Biol. . I l e d . , 67, 470 (1948). Picard, R. C., and Reissner, J., Rea. Sci. Instruments, 17, 484-9 (1946). Poole, J. B. le, Philips Tech. Reo., 2, 33-45 (1947). porter,K , R., and Thompson, H , p,, ~ ~ 7, 431 ~ (1947). Quynn, J. T . , Rer. Sci. Instruments, 19, 472 (1948).
cancer
(130) Raether, H., Nature, 161, 311 (1948). (131) Rake, G., Rake, H., Hamre, D., and GroupL., V., Proc. Soc. Erptl. B i d . Med., 63, 489 (1946). (132) Ramberg, E. G., and Hillier, J., J . Applied Phys., 19, 679 (1945). U. 6. Patent 2,424,041 (June 10, 1947). (133) Ramo, S., (134) Rami, S., S a t w e , 160, 712 (1947). (135) Reed, R., and Rudall, K. M., Biochim. et Biophys. Acta, 2, 19-26 (January 1948). (136) Kees, A . L. G., Proc. S O C C‘hem. . I n d . Victoria, 46, No. 2, 794813 (1946). (137) Regenstreif, E., A n n . RadioBlectricitB,2, 348-58 (1947). (138) Kochow, T . G., Coven, G. E., and Davis, E. G., Paper Trade J . , 3-8 (February 26, 1948). (139) Kochow, T. G., and Gilbert, R. L., “Protective and Decorative Coatings,” Vol. V, Chapt. 5 , ed. by J. J. -Mattiello, Sew York, John Wiley B: Sons, 1946. (140) Roginskii, S . Z., Shekhter, A. B., and Sakharova, S. V., Compt. rend. acad. sci. C.R.S.S., 52, 687-9 (1946). (141) Kuess, G. L., Chem. Zentr., 1, 71 (1947). (142) Seman. 0. I.. J . Tech. Phus. (C.S.S.R.1. 16. 291-308 (1946). (143) Shekhter, -4., Roginskii, and Sakharova, S. V.,Acta Physicochim.. U.R.S.S., 21, 849-52 (1946). (144) Sigurgeirsson, T., and Stanley, W. AI., Phytopathology. 37, 26-38 (1947). (145) Uchastkina. Z. V., Bumazh. Prom., 21, 23-33 (1946). (146) Van Dorsten, A. C.. Ooderkamo, IT. J.. Poole, J. B. le, Philips Tech. Ren., 9, 193-201 (1947). 1117) Van Iterson. W.. Biochim. et Biovhus. Acta. 1 . 527 11947). (148) \‘an Thiel, P. H., and Van Iterson, I T , Proc. Koninkl. S e d e r land. Akad. Wetenschap., 50, 88 (1937). (149) Von Ments, M., and Poole, J. B. le, Applied Sci. Res., B1, 1-17 (1947). (150) Walton. IT. H.. Trans. Inst. Chem. Enors. (London), and Soc. Chem. I n d . , Roads and Building RIaterials Group, Advance Copy, Feb. 4, 1947, 44-50. (151) Watson, J. H. L., J . Applied Phys.. 16, 996-1005 (1945). (152) Ibid., 18, 153-61 (1947). (153) Ibid., 19, 110-11 (1948). (154) I W . , 713 (1948). (155) Watjon, J. H. L., J . Phys. & (‘oiloid Chem., 51, 654-61 (1947). (156) Watson, J. H. L.3 Trans. Electrochem. sot., 92, 4 (1947). (157) Weimer. P. K., and Rose, A . , Proc. Inst. Radio Engrs. and Waves and Electrons, 35, 1273-9 (1947). (158) IC. ~ Williamson, ~ ~ I., J . Sci. ~ Instruments, h ,24, 242-3 (1947). (159) Ryckoff, R. W. G., Science, 104, 21-6 (1946).
s.,
RECEIVED October 28, 1948.
LIGHT MICROSCOPY EDWIN E. JELLEY Kodak Research Laboratories, Rochester, N . Y .
T
HE Symposium on Light and Electron Microscopy held in
Chicago (3) served to emphasize that the very rapid expantion and development of electron microscopic techniques have served to stimulate further interest in light microscopy. Many of the papers presented at the symposium stressed the correlation of electron and light microscopy (66). Other publications which compare the results given by light and electron microscopy include the study of pigments (32, 135) and of metals (36). Pijper (110) describes a dark-field study of typhoid bacillus in which sunlight was used as an illuminant; the results, recorded on motion picture film, show that what has been considered for many years t o be active bacterial flagella really are passive niucous twirls. One of the important contributions which electron microscopy has made to light microscopy is the replica technique. Although replicas had been used in light microscopy before the days of the electron microscope, i t needed the recent work with the electron microscope to develop the refinements in technique which have made the replica so important in present-day research. Schaefer proposed making replicas containing dye for use with the optical
microscope (193). The metal shadowing technique worked out by Williams and Wyckoff (146) is proving to be very important for light microscopy. rlccording to this publication, satisfactory replicas are obtained with collodion, Formvar, and Faxfilm, and aluminum is a suitable metal for shadowing these replicas for optical microscopy. This technique gives a very high contrast image, so that the microscope objectives may be used a t their maximum resolving power. The application of the shadowing technique in the preparation of replicas of tooth surfaces is described by Scott and Wyckoff (126). The application of replica technique3 to the study of ceramic surfaces with the the optical microscope is described by Allen and Friedberg (1). Another technique, which was developed for electron microscopy and is also applicable to light microscopy, is the use of the high speed microtome for cutting extra thin sections of biological and other material (39,66,57,106,19~).The paper by Seidel and Winter (166) has deepened the mystery as to esactly what has been achieved by Rife in his universal microscope. The article states that this marvelous instrument, which has a resolving power greatly surpassing theoretical limits, contains 5862 parts,
V O L U M E 21, NO. 1, J A N U A R Y 1 9 4 9 but does not give an adequate description of the parts or a scientific eyplanation of their function. As Rife has not published details of the construction and principle of his microscope, it is t o be hoped that a group of selected scientists will have an opportunity of investigating it, so that the scientific \vorld. in general can be given an accurate appraisal, and optical theory be reinvestigated if necessary. h new microscopic principle has been described by Gabor (Z), according to which a point source of radiation is made t o yield a comples diffraction pattern of the object. This diffraction pattern is photographed and subsequently unscrambled by optical means t o yield an enlarged image of the object. It is believed that this method v J l be of use in electron microscopy, but its possibilities in light microscopy remain t o be determined. TRANSMITTED LIGHT
JIicroscopists have long been troubled with the necessity for changing t,he illuminating system of the microscope when working alternately with low and high powers. If the microscope is set up t o give Kohler illumination with a high-power objective, changing to a low-power objective with the same condenser gives a very small illuminated field, so that some sort of auxiliary illuminating system or condenser is necessary if the microscopist is to leave the Kohler system in proper alignment. Benford (21) has described a substage condenser ryhich can be used for high powers, low powers, and dark-field illumination, in which the illuminated field corresponds to the field of view of the objective. A h o t h e rattempt to achieve a more or less universal condenser is described by Hall ( 6 4 ) ,in which the microscope is equipped with a variable-focus condenser. This condenser, which is a tu-0-lens hhh6 type, has the top lens fixed t o the microscope stage. The bottom lens is racked to its top position with a high-power objective, and to its bottom position for low-power objectives. A condenser of this type is considered adequate for much student and industrial work. In using the Kohler system of illumination, when the system has been properly set up, and the substage diaphragm has been opened to give a 4/5ths cone of light in the Objective, the illumination is usually very much too bright for comfortable work, and its intensity must Be reduced by means of a filter. Benford ( 2 1 ) has described a set of Tnconel neutral filters for this purpose which are said to be very good for color photomicrography. Copeland (40) has described the adaptation of a Polaroid variable-density filter from an American hir Force gun sight for this purpose. By merely rotating one of the cpmponent polarizers the intensity of the light can be adjusted to a comfortable working value. Dempstcr ( 4 7 ) has given a detailed discussion of microscope illumination with special referencc to the problem of glare of both optical and visual origin. REFLECTING 1IICROSCOPES
The ultraviolet reflecting microscope of Burch (36) has been applied to the study of nuclear plates (18),vihere its very great working distance has proved very useful. Bouwcrs (27, 28) has described a simple reflecting microscope, which is already used in some European schools. INCIDENT LIGHT
A combined vertical and concentric opaque illuminator has been described by Benford ( 2 1 ) . The reflector consists of glass coated with titanium dioxide on one side and magnesium fluoride on the other. A siving-out dark-field stop allons the light to travel either down the objective nhen the customary bright-field vertical illumination is obtained, or down a plastic paraboloid surrounding the objective, which gives the so-called dark-field type of incident illumination. Hauser and leBeau (70, 71) have described an important application of the concentric opaque illuminator. Vsing a Leitz I7tropak they adapted the electron microscopic technique of
41
mounting specimens on wire gauze for the study of lyogels in the vulcanization of rubber, and applied the method to the study of structural changes of natural and synthetic rubbers during mastication and the subsequent recovery process (87). Hauser’s work emphasizes the value of rendering an object self-luminous as a means of obtaining the highest possible resolving power of the microscope. DARK-FIELD ILLUMINATION
Attention has been attracted by the method of “optical staining.” According t o this technique, which is described by 46) and Dodge ( i s ) , the material to be studied is Crossmon (44, mounted in a liquid having a much higher dispersive power but preferably the same refractive index as the material for some specific wave length of light. The preparation is examined by dark-field illumination. Substances of different refractive index may be readily differentiated by this means. One of the examples given is t h a t of corundum mised with other mineral grains, with met,hylene iodide as an immersion liquid. Under dark-field illumination, corundum appears bright yellow xvith purple borders, sharply contrasting with the whites and blues of the other mineral grains. Ultramicroscopy in color has been described by Hauser (69). PHASE CONTRAST MICROSCOPY
There has been considerable activity during the past few years in the relatively new field of phase contrast microscopy. The general principle is described by Zernike (149) and in a recent trans!ation of the paper by Kohler and Loos (82).
An annular diaphragm is placed below the substage condenser to provide a hollow cone of illumination. The internal and external diameters of the annulus are such that the image of the annulus in the rear focal plane of the objective estends from approximately one half to approsimately three quarters of t,he full aperture of the objective. h disk is placed at. the rear focal plane of the objective, which is referred to as a “diffraction plate” or “phase plate.” I t has an absorbing annulus xhich cuts down the intensity (or amplitude) of light which is not diffracted or deviated by the object through which it has passed; in addition, a phase difference is introduced between this direct beam and the inner and outer diffracted, or deviated, beams by adding a transparent coating of appropriate thickness either to the absorbing annulus or to the clear inside and outside zones. The light through the annular zone and that through the rest of the objective interfere when combined in the microscope image t o give either bright or dark detail. A variety of effects, both bright contrast and dark contrast, can be obtained by changing the intensity and phase relationships between the direct and deviated light. Burch and Stock (31) described a phase contrast microscope constrJcted in 1942. Richard3 ( 1 1 4 ) described progress being made a t the American Optical Company plant. Linfoot (89) referred to the work of Burch and mentioned that the phase platos were being made with evaporated coatings which could be cut with a stylus t o give zones of accurate dimensions. Bennett,, Jupnik, Osterberg, and Richards ( 2 2 ) described further advances being made by the American Optical Company, and experiments with phase plates having various amplitude and phase relationships. Brice and Keck (29) also studied the question of the most favorable absorption of t,he phase plate. Considerable attention has been devoted to trying to produce a variable-phase contrast system for microscopy whereby it would be possible to change from bright to dark contrast a t will, and t o change the phme relationship also. Hartlcy (67) described a combination of a quarter-wave mica and polarizing device. A device utilizing right- and left-handed quartz was described by Taylor and Payne (109, 132, 138). A device employing polarized light which gives both variable phase and variable amplitude has been described by Osterberg under the name of the Polanret microscope (107). A device using a combination of half-wave and quarter-wave plates is described by Kastler and Montarnal ( 7 7 ) . Osterberg (108) has also studied the application of the principle of the multipupil in phase contrast microscopy, and has
ANALYTICAL CHEMISTRY
42
concluded that phase contrast accessories could be placed at optical equivalents of the rear focal plane of the objective, whereby a simplification in construct'ion might be effected. Bennett, Woernley, and Kavanagh (23) have described preliminary work with an ultraviolet phase contrast microscope which employs a 16-mm. reflecting objective. Other optical parts, such as the condenser and diffraction plate, are made from fused silica. Phase contrast microscopy is proving of considerable value in the study of transparent objects having a small refractive index difference from that of their mountant. Among application6 described i n recent literature are textile fibers (112), viruses (U), cell cultures (160), and mineralogy (128). I t is also possible t o apply phase contrast methods t o metallography with vertical illuminat,ion (46, 7 6 ) . Genaral discussions of phase contrast microscopy have been given by 1,achenaud (83), Ilichel ( l o o ) , Salmon ( l a l ) ,and Martin ( 9 5 ) . IhTERFEREYCE MICROSCOP1
A number of new contributions to the interferometric study of crystals have been made by Tolansky (136). Merton (98, 9 9 ) has described a promising new method of interference microscopy in which the specimens are mounted between half-platinized surfaces. The optical system of the microscope comprises a zone plate beneath the condenser and a corresponding zone plate in the rear focal plane of the objective. The zone plates must have the same order of interference as the half-platinized cell. Either a single wave length of monochromatic light or two wave lengths bearing a simple arithmetical relationship are used. This method is not the same as phase contrast microscopy, and may yield information which cannot be obtained by the latter. Zone plates are said to be available from A. Hilger of London. Ambrose (2) has described a n interference microscope for the study of inhomogeneous media. Kayser (78) has described an interference band method of checking surface finishes. Jelley (74) has described an interference mcxthod for tho comparison of crystalline substances. FLUORESCENCE AND PHOSIWORESCENCE MICROSCOPY
A simple fluorescence microscope and its use in the detection of bacilli which stain with fluorescent, dyes have been described by Richards (115). Jailer (73) has used the fluorescence microscope in the study of the physiological distribution of atabrine. Strugger (131) has described the present status of fluorescence staining of bacteria with particular reference to the use of Acridine Orange and Brilliant Sulfoflavin. A very good photomicrograph sho\Ting the specific staining of the spores of B. inycoides is given. 4 method of photographing very weak fluorescent spectra has been described by Scheminzky (123). Harvey and Chase (4)have used the principle of the Becquerel phosphoroscope with a microscope for the ptudy of the phophoreseenccs of biological matwials. ULTRAVIOLET m C n o s C o i * Y
A simple construction for a quartz ultraviolet microscope illurninant has been described by Lavin (86),in which a Hanovia lamp is enclosed in a Transite box under the microscope. il translation of a Soviet paper on the use of the ultraviolet microscope in the study of living and dead cells has just appeared (85). Foster and Thiel (55) have described a fused-silica fluorite achtomatic ultraviolet objective having a focal length of 2.5 mm. and a numerical aperture of 0.85. Considerable progress has been made in England by Burch (36') M ith his ultraviolet reflecting microscope. A description of the system used by Burch together with some surprisingly good photomicrographs taken with the instrument are given by Barer (16) The research laboratories of the Polaroid Corporation are developing a reflection microscope for tlie studv of ultraviolet
absorption of cell components. The principle of "color translation" is used in the presentation of the photomicrographs in which photomicrographic negatives obtained with three different wave lengths in the ultraviolet, are printed in register with the three subtractive colors uscd in ordiriary color photography. This gives an interpretation in color of diffcrential absorption in the ultrxviolt~t(84). I N F R ~ R E Dmcmscow
13ailly has described the use of infrared-sensitive photocells i n the identification of opaque minerals (13, 14). The use of the now-familiar infrared imagr tube in seeing through the microscopci with infiwed light is dmight t w of use as a means of identification. Subsequently, Svuei,burg (105) fount1 that tlic arcidental birefringence of objective? a!t(ii,s the character of the figures. These reflection figures are of a w r y complex nature and do not in any way correspond to tht: convvrgerit polarized light intorferencc figures seen with transparent crystals. h brief account of some applications of opt,ieal crystallography has been given by Hartshorne ( 6 8 ) , who is preparing a new edition of his “Crystals and the Polarising Microscope” (42). Three books of interest to chemical microscopists h:tvo bwri published rccc.ntly (7.5, 191, 1.$0). .VlISCELLANEOUS XZETHODS AND 4Pl’LI
