Geometrical Optics for Electrons Quite Similar to the Optics of Light

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THE ELECTRON MICROSCOPE A NEW ToOL FOR BACTERIOLOGICAL RESEARCH L. MARTON Research Laboratories, RCA Manufacturing Company Received for publication August 1, 1940 The science of bacteriology could hardly exist without the microscope, and it is almost providential that pathogenic bacteria are within the limits of visibility of the present-day microscope. However, the limits of microscopical observation have been severely felt since early in the development of bacteriological research, and many attempts have been made to extend the range of- observation. These attempts brought the realization that the sizes of micro-organisms extended far beyond the limits of visibility of light microscopes, and therefore the need has been constantly felt for a better instrument which would give more detail. Such an instrument is provided in the electron microscope which, in its present-day development, extends the observation range by a factor of about 50 to 100, with possible further extensions in the future. Electron microscopy is based on the discovery of geometrical optics for electrons quite similar to the optics of light. To understand the term "geometrical optics" let us first consider the action of an electric or magnetic field on an electron beam. It is well known that an electron beam is deflected by such fields, and we can therefore compare their action on the beam to the action of a refractive medium on a light beam. A lens is nothing but a refractive medium of special symmetry-in this particular case of rotational symmetry. If we create an electric or magnetic field of rotational symmetry, such a field acts on an electron beam as a lens, i.e., the electron beam is concentrated or made divergent in the same way that the light beam is acted upon 397 398 L. MARTON by a glass lens. It has been proved mathematically that the laws of geometrical optics can be fully applied to such systems, and experimentally that we can obtain electronic images which can be made visible, for instance on a fluorescent screen. An image can be formed of any self-emitting object, as would be the case with light if we observed the image of the source itself, or we can illuminate an object in the same way as we illuminate

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PEI. 1. SCHEMATIC DiGRA.AU or THE ELCRON MICROSCOPE IN COMPARISON WITH THE LIGT MCROSCOPE one in a light microscope, and observe the image of the object with the help of the illuminating beam. After this discovery, the obvious next step was the building of compound optical systems, and, in particular, a system corresponding to the compound microscope. Such an instrument is built up of different elements for which we can use the same nomenclature as in a light microscope (fig. 1). The light source of the light microscope is replaced by an electron source, the electron beam being concentrated on the specimen by means of the first field in the same way that the light beam is concentrated THE ELECTRON MICROSCOPE 399 by means of a condenser lens. The field is created by a coil through which current flows and produces inside the coil the necessary magnetic field of rotational symmetry. Two more similar coils are used in place of the objective and eyepiece lenses of the light microscope. We may call the first one the objective coil and the second one the projection coil, because the image is not viewed by applying the eye to an eyepiece, but by projecting it on a fluorescent screen. The function of both lenses is exactly the same as in the familiar light optics. The objective coil produces a first stage magnification which is re-enlarged by the projection lens. The above described microscope system has some very special properties. In the first place, the electrons travel without hindrance only in vacuum, and therefore the whole microscope must be pumped out to a high degree. This means that the specimens and photographic plates must also be in the vacuum. The operation of an instrument of this kind is necessarily different from the operation of a light microscope. Focusing, for instance, is done in a quite different way. In light optics the optical components of the system are fixed by the construction, and focusing is done by changing the distance between the specimens and the optical system. In electron microscopes the focal length of a lens is given by the strength of the applied field, and therefore the focusing is done by varying the optical constants of the lens similarly to the way in which the focal length of the human eye is varied by changing the curvature of the lens. The distance between the object and the optical system therefore remains constant, and the magnification can be changed continuously from the lowest to the highest magnification, instead of the step- by-step variation obtained in a light microscope by exchanging eyepieces. The great advantage of an electron optical system lies in its highly increased resolving power. The practical microscopist knows that the resolving power, i.e., the smallest distance sepa- rately shown by an optical system, is about one-half of a wave- length for the best light optical systems. Since the fundamental discovery of DeBroglie, we know that the electron behaves for 400 L. MARTON some applications as a corpuscle, and for other applications as a wave, the wave-length of which depends on the speed of the electron; and, for the speeds generally used in electron microscopy, it is about one 100,000th of the wave-length of visible light. Under identical conditions, this would mean 100,000 times better resolving power, or 100,000 times higher useful magnification for the electron microscope than forthe light microscope. This, however, cannot be realized, the reasons for the limitations being manifold. In the first place, the lenses of electron optics exhibit all of the optical defects known to light optics, plus a few more, and at the present state of our knowledge we are not able to correct very far the electron optical aberrations. For instance, spherical aberration is about 1,000 times worse than in light microscopy. We do not have any means of correcting the chromatic defect. The result is that the numerical apertures of electron optical systems are much reduced over the numerical apertures of light optical systems, and as the resolving power is not only proportional to the wavelength but also inversely proportional to the numerical aperture, we come to the conclusion (represented in fig. 2), that for the present-day practical apertures of about 0.001, we should expect the best resolving power to be about 10 Ang- strom Units, or a corresponding useful magnification of about 200,000 times. Incidentally, the use of extremely small apertures has another advantage: as in light microscopy, the smaller the aperture the greater is the depth of focus, and the resulting depth of focus for the herein described electron microscope is at least ten times greater than the depth of focus of the highest power light microscopes. Another peculiarity of electron microscopy is the mechanism of image formation. In light microscopes we see the image due to differences in absorption, or refraction in the specimen. In electron microscopes the image formation is not due to either of these effects, but to scattering of the electrons. As mentioned before, electrons do not travel in straight lines except in vacuum, and even a few molecules suffice to deflect an electron beam. This means that we do not have any material transparent to THE ELECTRON MICROSCOPE 401 electrons in the same sense as to light, and therefore we only have transmission of an electron beam when the substance through which the beam passes is extremely thin. This means that, first, the glass slides of light microscopy must be replaced by extremely thin films as specimen holders, and, second, that

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105 103 10 volts Electrons FIG. 2. RESOLVING POWER IN FUNCTION OF THE W)AN-ELENGTH FOR DIFFERENT NUMERICAL APERTURES what we observe in an electron microscope image are only differences in thickness and in density of a specimen. The practical application of these principles can be best ex- plained by describing~~~~~Wavelegtan RCA electron microscope (fig. 3). The electrons are generated by an "electron gun," similar in construction to an X-ray tube. The main tube contains the various 402 L. MARTON optical and mechanical elements of the microscope-three coils which act as lenses, the "object chamber," and the "photographic chamber." The lenses, as mentioned above, are coils consisting of a large number of turns of copper wire, which are housed in

FIG. 3. FRONT VIEW OF THE RCA ELECTRON MICROSCOPE iron enclosures. The function of the iron enclosures is to con- centrate the field between the gaps in the enclosure, and to give the necessary shape to the field. The object chamber has two important functions (fig. 4). The first is to introduce and withdraw the specimen to and from THE ELECTRON MICROSCOPE 403 the main tube without breaking the vacuum in the microscope, and the second is to permit motion of the specimen in front of the objective, replacing the mechanical stage of a light microscope. The former is carried out by means of an air lock somewhat similar in construction to the escape chamber of a sub- marine, whereby the specimen is introduced first into a lateral compartment separated from the main body of the microscope by means of a small gate. When the gate is closed, the lateral compartment can be opened, and the specimen replaced. After

FIG. 4. INTRODUCTION OF THE SPECIMEN INTO THE OBJECT CHAMBER the lateral compartment has been closed it can be pumped out by means of an independent pumping system to a good vacuum, after which the gate can be opened, and the specimen introduced into the microscope. The specimen itself is clamped between two "blades" which are inserted in a ring shaped holder which can be moved hori- zontally by means of two micrometric adjustments, 120 degrees apart. Another adjustment is provided for the vertical motion of the specimen. 404 L. MARTON The photographic chamber (fig. 5) is built in a somewhat similar fashion. At the bottom of the instrument an aperture can be closed by means of a gate which separates the main body from the photographic chamber. This gate can be replaced by a photographic plate, using a carriage-like system. A fluorescent

FIG. 5. INSERTION OF THE PLATE HOLDER INTO THE PHOTOGRAPHIC CHAMBER screen is mounted above the aperture so that it can either cover the photographic plate for visual observation of the image, or be swung away to leave the photographic plate open for exposure. Observation of the fluorescent screen is done through two large windows. A special periscope-like system is built into the microscope for the observation of the intermediate image (first stage THE ELECTRON MICROSCOPE 405 magnification) on a fluorescent screen located on top of the projection coil. The reason for this arrangement is due to the fact that at high magnifications (10,000 to 20,000) it is sometimes difficult to find the most interesting part of a specimen, and orientation is much easier at the low magnification of the objective lens (about 100 times). The periscope-like system allows observation of this intermediate image from the same place from which the highly magnified image is observed, and makes the operation of the microscope very convenient. For the same reason the adjustments of the "stage" and the focusing adjustments of the microscope are all concentrated within easy reach of the observers' position. Particular care has been taken to avoid blurring of the image by mechanical vibrations or by any electrical or magnetic fields. MICROSCOPE TECHNIQUE Due to the described characteristics of electron microscope image formation, a new kind of technique had to be developed for investigating specimens. The most important conditions are given by the fact that electron scattering is, in a first approximation, proportional to the thickness and density of the substance through which the beam is passing. This means that for very transparent object holders the supporting films must not only be extremely thin, but very homogeneous as well, as the slightest inhomogeneity would give a definite contrast, and might be the source of an artefact. Such films can be prepared by spreading out one or more drops of diluted solution of collodion in amyl acetate (11 to 2 grams in 100 grams of amyl acetate) over a distilled water surface. After evaporation of the solvent a thin film remains on the water surface and can be taken off on suitable holders. In order to have the film very homogeneous, the water surface should be perfectly quiet and free of dust and gas bubbles. It has been found, also, that good homogeneous films cannot be prepared without previously saturating the water with amyl acetate. As holders for the films, small apertures have been widely used, but we have found it more advisable to replace the small apertures by very fine wire screens, punched out to little disks, which can be easily inserted in the microscope. The 406 L. MARTON advantage of such wire screen holders is that there is more than one aperture free for the inspection of the specimen, and still enough support for the extremely fragile films. The usual technique of preparing bacteriological specimens for electron microscopy is to suspend them in distilled water and

FIG. 6. GROUP A HEMOLYTIC STREPTOCOCCUS, 1048 MUCOID, PREPARED FROM AN 18 HOUR AGAR PLATE CULTURE, STORED FOR SEVERAL DAYS IN REFRIGERATOR Magnification 20,000 diameters. Specimen Dr. Mudd, Dr. Lackman allow a small drop of the suspension to dry on the film surface, A good film, together with a bacteriological specimen prepared in the correct way, is shown in figure 6, compared with figure 7, which shows a film full of holes, and figure 8, a biological specimen THE ELECTRON MICROSCOPE 407

FIG. 7. FAULTY FILMI SHOWING HOLES Magnification 10,000 diameters

FIG. 8. SALT CRYSTALS 'MASKING THE SPECIMEN I\Lagnification 10,000 dialneters 408 L. MARTON which was not thoroughly washed-the salts contained in the solution crystallized around the bacteria, giving therefore the outlines of the crystals, instead of the image of the specimen. The same technique must be applied when working with virus particles (fig. 9). In the latter case sometimes difficulties arise due to low contrast, and the focusing becomes difficult. Different

FIG. 9. TOBACCO MOSAIC VIRUS Magnification 18,000 diameters. Specimen Dr. Stanley methods can be devised for overcoming this difficulty. One is to take a series of pictures of some specimen at different focusses, and select the best one. Another method, which has been used in Germany, is the adsorption of colloidal gold on the virus particles. Focusing is facilitated in this way due to the high contrast of the gold particles. THE ELECTRON MICROSCOPE 409 Of course the technique just described is not the only bacteriological technique which can be used. Some attempts have been made to incorporate the bacteria into the film material before preparing the film, and spreading them out together on the water surface. Another attempt was made to reproduce, in a way similar to light microscope technique, the so-called "india ink technique" by making a film of higher density than the bacteria itself, which consequently appear black on the electron micro-

|l--W...... s.~~~~~~~~~~~~~~~~~~~~~...... |llNNW PERTUSSIS A FIG. 10. HAEMOPHILUS PHASE 1 PREPARED FROM CULTURE GROWN ON BORDET-GENGOU~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.....MVEDIUM, KEPT DRY ON A FILM.% FOR Two M\ONTHS Magnification 15,000 diameters Specimen Dr. Flosdorf graphs. Up to the present time, however, the first-described technique seems to be the best one. Generally speaking, no staining has yet been applied in electron microscopy. The reason for this is twofold. First the stains of light microscopical technique do not show any advantage in electron microscope work, and second most of the bacteria show sufficient contrast for electron microscope work without staining or even fixing. Different details of structure as shown are prob- 410 L. MARTON ably due to the different concentrations of the intracellular material, which does not change very rapidly after drying of the specimen. Here, however, some reservations should be made for a picture of a freshly prepared specimen compared with a picture of the same kind of specimen kept dry for about two months, as shown in figure 10. Flagella and similar details can be taken without any staining, as shown in figure 11. The foregoing does not exclude a possible need which may arise later

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FIG. 11. EBERTHELLA TYPHOSA KILLED WITH FORMALIN The straight lines are wrinkles in the film. Magnification 7,000 diameters. Specimen Dr. Polevitsky. for the development of a new staining technique for electron microscopy. Such a technique would need to be a staining in density, which means selective introduction of high density materials into the bacteriological cell. Some knowledge already exists about such materials, and the known technique of fixing with osmic acid might be mentioned in this connection. The actual limits of resolving power and magnification are perhaps best shown in figure 6. One corner of this micrograph shows a hole in the nitrocellulose film which developed a small THE ELECTRON MICROSCOPE 411 crack, shown again in enlargement in figure 12. The distance from edge to edge is 10 millimicrons, and the edges are suffi- ciently sharp to estimate from them a resolving power of about 5 millimicrons, which would give a limit of useful magnification

FIG. 12. PARTIAL ENLARGEMENT OF FIG. 6 of between 60,000 and 80,000 times. The resolving power is not necessarily the same for a detail free in space as these edges and for particles on the film, but even for such particles a survey of figure 6 shows that particles of about 12 to 15 millimicrons are clearly visible. 412 L. MARTON

REFERENCES ELECTRON OPTICS BRUCHE, E., AND SCHERZER, 0. 1934 Geometrische Elektronenoptik. J. Springer, Berlin. HENRIOT, E. 1935 Optique Electronique des Systemes Centres (Premierc approximation). Rev. d'optique, 14, 146-158. MARTON, L. 1935 Le Microscope Electronique et ses Applications. Rev. d'optique, 14, 129-145. RAMBERG, E. G., AND MORTON, G. A. 1939 Electron Optics. J. Applied Phys., 10, 465-478. ZWORYKIN, V. K., AND MORTON, G. A. 1940 Television. John Wiley and Sons, New York, pp. 89-127. ELECTRON MICROSCOPES (CONSTRUCTIONAL DETAILS) MARTON, L. 1934 La Microscopie Electronique des Objects Biologiques. Bull. acad. roy. m6d. Belg., 20, 439-446. MARTON, L. 1935 La Microscopie Electronique des Objets Biologiques. Bull. acad. roy. med. Belg., 21, 600-617. MARTON, L. 1940 A new electron microscope. Physiol. Revs., 58, 57-60. PREBUS, A., AND HILLIER, J. 1939 The construction of a magnetic electron microscope of high resolving power. Can. J. Research, 17, 49-63. RUSKA, E. 1934 tber Fortschritte im Bau und in der Leistung des magnetischen Electronemikroscops. Z. Physik, 87, 580-602. VON BORRIES, B., AND RUSKA, E. 1938 Vorlaiufige Mitteilung uber Fortschritt3 im Bau und in der Leistung des tUbermikroskopes. Wiss. Veroffentl. Siemens-Werken, 17, 99-106. VON BORRIES, B., AND RUSKA, E. 1939 Aufbau und Leistung des Siemens- tbermikroskopes. Z. wiss. Mikroskop., 56, 317-333. MECHANISM OF IMAGE FORMATION, RESOLVING POWER MARTON, L. 1936 Quelques considerations concernant le pouvoir separateur en microscopic electronique. Physica, 3, 959-967. VON ARDENNE, M. 1938 Die Grenzen fur das Auflosungsverniogen des Elektro- nenmikroskops. Z. Physik, 108, 338-352. VON BORRIES, B., AND RUSKA, E. 1939 Versuche, Rechnungen und Ergebnisst zur Frage des Auflosungsvermogens beim tobermikroskop. Z. tech. Physik, 20, 225-235. MARTON, L. 1934 Electron microscopy of biological objects. Nature, 133, No. 3372, p. 911. MARTON, L. 1935 La microscopic electronique des objets biologiques. Bull. acad. roy. m6d. Belg., 21, 553-564. MARTON, L. 1936 La microscopic electronique des objets biologiques. Bull. acad. roy. mid. Belg., 22, 1336-1344. MARTON, L. 1937 La microscopic electronique des objets biologiques. Bull. acad. roy. mod. Belg., 23, 672-675. VON BORRIES, B., RUSKA, E., AND RUSKA, H. 1938 Bacterien und Virus in tUbermikroskopischer Ausnahme (mit einer Einfuhrung in die Technik des tbermikroskops). Klin. Wochschr., 17, 921-925. THE ELECTRON MICROSCOPE 413

PICKARSKI, G., AND RUSKA, H. 1939 tUbermikroskopische Darstellung von Bakteriengeisseln. Klin. Wochschr., 18, 383. RUSKA, H. 1940 Die Sichtbarmachung der bakteriophagen Lyse im tbermikro- skop. Naturwissenschaften, 28, 45. RUSKA, H., VON BORRIEs, B., AND RUSKA, E. 1939 Der Bedeutung der tUber- mikroscopie fur Virusforschung. Arch. Virusforsch., 1, 155-169.

BIOLOGICAL APPLICATIONS KAUSCHE, G., PFANKUCH, E., AND RUSKA, H. 1939 Die Sichtbarmachung von Pflanzlichem Virus im tUbermikroskope. Naturwissenschaften, 27, 292-299. VON ARDENNE, MANFRED. 1940 Elektronen-TUbermikroskopie Physik-Technik- Ergebnisse. Julius Springer. Berlin.