Electrons, practically speaking, are the smallest, lightest particles of matter and electricity. Like light, they behave liked corpuscles guided by waves. Unlike light, however, they ravel in a straight line in a vacuum where, subject to the action of electric and magnetic fields, their behavior coincides with the laws and principles set down by Sir William Hamilton who, more than a century ago, demonstrated the existence of a close analogy between the path of a light ray through refracting media and that of a particle through conservative fields of force.

We know that these negatively-charged particles, the electrons, revolving about in their various orbits in the atom, serve to maintain the balance of the atom while the nucleus exerts the "positive" force which holds it together; and we also know that when this balance is upset, due to gain or loss of electrons, we think of the atom as "charged," since it is this circumstance which causes the tiny particle to attract or

(Page 104, Journal of the Franklin Institute 237(2):103-130 (1944))

repel other electrons according to the state of its unbalance. And Science has succeeded in unbalancing the atoms to such an appreciable extent that the negative electricity may be withdrawn and harnessed for use in such instruments as the Electron Microscopes.

The fact has long been established that atoms are in a constant state of vibration in a heated body and that the greater the heat of the body, the greater the agitation of the atoms. According to the electron theory of metals, electrons circulate about a three-dimensional network, or lattice, of positive ions, some of the electrons being comparatively free, that is to say, the attractions of the ions are practically cancelled by the repulsion of the other electrons. It does not necessarily follow, however, that the same electrons consistently remain free. Moreover, there is a critical value of speed above which the electrons are able to rise in metals and thus escape from their restraining positive charges, though at ordinary temperatures the proportion of them moving rapidly enough to do this is relatively small. However, as the heat applied to the metal is increased, not only is the thermal agitation of the electrons increased also, but the proportion among them possessing sufficiently high speeds to enable them to leave the metal.(sic)

Thus is heat applied to the electron source of the Electron Microscope which, in the case of most instruments of this kind, is a tungsten filament surrounded by a guard cylinder. After leaving the filament, or cathode, the electrons enter an electric field wherein are large accumulations of charge which serve to steadily speed up the motion of these freely-moving particles. Since the electrons travel in vacua, none of the kinetic energy gained in crossing the field is lost, the total kinetic energy, or energy of motion, gained in passing through this region being proportional to the voltage applied. We may deduce, therefore, that since increase of charge in an electric field means a proportional increase of kinetic energy of these electrons, the higher the voltage applied, the greater the speed of the electrons---all of which has been calculated mathematically and confirmed experimentally.

After traversing the electric field and passing through the anode, the electrons are concentrated on the specimen under examination by the first of three magnetic fields which are created by currents flowing through coils enclosed in soft iron shields, molded so as to concentrate the magnetic fields on a short section of the microscope's axis. Whereas in the ordinary light microscope glass lenses serve as the refractive media through which light rays are deflected, in the Electron Microscope it is these magnetic fields of rotational symmetry which are the refractive media and serve as the "lenses" which deflect the beams of electrons. The first of these, the condenser lens coil, corresponding to the substage condenser of the ordinary light microscope, concentrates

(Page 105, Feb., 1944)

the beam of electrons upon the specimen. The convergence of the beam falling on the specimen is controlled by varying the current through this condenser lens. Now, having passed through the specimen, the objective coil, similar in effect to the objective lens, focuses the electrons, and an intermediate image enlarged about one hundred diameters is formed. Finally, the projection coil, corresponding to the projection lens or ocular, produces a further magnified image on a large fluorescent screen. In some of the Electron Microscopes, there is a periscope-like attachment by means of which it is possible to locate and adjust for study the most interesting portion of the specimen, or that which it is desired should be examined, before the projection lens coil forms the final magnified image upon the screen, since it is sometimes difficult to accomplish this at high magnification. Also, if it is desired that a photographic record be made, the screen can be removed and a photographic plate substituted.

(n.b. The Plates displayed in this HTML version of this article, are derived from plates printed in the Smithsonian Annual Report version of the same article. In the Smithsonian version, 5 Plates followed the end of the article. In the Franklin version, three plates were embedded in the article.)

The specimen itself is supported on a thin nitro-cellulose membrane less than one-millionth of an inch thick, and clamped in the tip of a cartridge which is inserted between the pole pieces of the objective coil. The membrane is suspended across the opening of a fine mesh screen, and a plate, serving as the movable stage, supports the cartridge. The image is projected onto the screen according to the density and atomic weight of the specimen. In other words, whereas in the ordinary light microscope the image is seen due to refraction of the specimen or differences in absorption, in the Electron Microscope the image is seen due to scattering of the electrons, and since the electrons travel in a straight line in a vacuum, it stands to reason that even a fairly thin specimen will prove sufficient to deflect such particles. Electrons which strike a thick or solid portion of the specimen will, of course, not continue on in a straight line to the screen but will be either completely absorbed by the specimen or scattered too far out of the beam, thus failing to enter the narrow aperture of the objective, so that that portion of the screen corresponding to the thick portion of the specimen will remain dark. However, those electrons which are able to escape complete absorption or too great deflection because they do not happen to come in contact with too solid a portion of the specimen and either pass along on all sides of it or penetrate the thinner portions where it is possible they may encounter only a single heavy nucleus for considerable scattering (the angle of deflection being proportional to the square root of the thickness), continue on to the screen where they impinge and cause the chemically-treated screen to fluoresce, thus providing a study in light and shadow.(n.b.:!) If the atoms of a particular substance are heavy, they will also deflect more electrons than if they were light. It may be readily seen, therefore, that the thinner the specimen and its mounting, or the greater the variations in density of the specimen, the more internal structure and detail which may be seen, since too great density

(Page 106, Journal of the Franklin Institute 237(2):103-130 (1944))

tends to absorb or interrupt the straight-line progress of too many of the electrons.

Focusing of the image is accomplished by varying the strength of the fields and thereby altering the focal length of the "lens" coils at will, so that the need of changing the specimens position in relation to a fixed optical system as would be the case in an ordinary light microscope, is avoided. Thus, magnification in an Electron Microscope can be continuously varied.

Some specimens may be mounted directly on the fine mesh screen while others may be embedded in a collodion, sealed between films of collodion, or suspended in a gelatin film, itself supported on collodion film. The supporting films beside being very thin must be homogeneous lest an artefact be created. For the most part, no staining of bacteriological specimens is done since usually they exhibit sufficiently high contrast in density to readily reveal flagella and other details without any preparation except that of suspending the specimen in distilled water or other liquid and allowing a drop of the suspension to dry on the film surface which method is also utilised for specimens of colloidal particles, pigments, and other chemical preparations. At times, however, as Dr. L. Marton of Stanford University has mentioned in his article on the Electron Microscope (written for The Journal of Bacteriology, March 1941, when he was associated with the R.C.A. Research Laboratories), virus particles may show decided low contrast. One method which Dr. Marton mentioned for overcoming this is to secure a number of electron micrographs at various focuses and simply select the best one for study. Or the virus may be permitted to absorb colloidal gold which would result in an image of high contrast. Dr. Marton points out that there may be future need for staining in density and that already osmic acid has been tried and used for this purpose.

In this microscope, voltages of between 30,000 and 60,000 are used. It has been previously stated that the higher the voltage, the greater the speed of the electrons. This might now be augmented to read, the higher the voltage, the greater the speed of electrons; hence, the shorter the wavelength. An explanation of this may be approached through a brief discussion of short-wave diffraction as considered by Dr. Karl K. Darrow of Bell Laboratories in his book, "The Renaissance of Physics." In order to obtain convenient angles of refraction with the ordinary diffraction grating, it is necessary that the wavelengths of light be smaller, but not many times smaller, than the spacing between wires or grooves. Naturally, a limit of measurement is reached in the region of ultra-violet light since it is impossible to further lessen the spacing of these gratings. However, this limitation was overcome when von Laue conceived the idea of substituting a crystal for an artificial grating since the atoms in a crystal are a thousand

(Page 107, Feb., 1944)

times more closely set together than are the wires or grooves of a grating and are arranged in precise regular order or "lattices," and, like gratings, are unable to diffract waves which are longer than the spacings between their atoms. Von Laue suggested that if a beam of light were directed across a crystal and made to strike a photographic plate, there would appear a spray of narrow rays each composed of a single wave train instead of the broad fan-like arrangement of the grating, and a pattern of star-like spots where the rays come in contact with the plate instead of the dark irregular blot when a grating is used. Of course, the rays are disposed according to the spacings of the atoms in the lattice and according to the character of the lattice. Von Laue confirmed this idea for waves short enough to be so diffracted and then advanced the theory that this principle might hold true for x-rays as well, which theory was almost immediately confirmed by Friedrich and Knipping. Shortly after Schroedinger began to develop De Broglie's wave theory of electrons, Elsasser conceived the idea that possibly these tiny particles might also be diffracted by crystals, and Doctors Davisson and Germer of the Bell Telephone Research Laboratories, using as part of their apparatus an electron gun, set out to test and to prove this theory. Due to their experiments and those of G. P. Thomson, it was established beyond a doubt that electron beams are diffracted just as x-ray beams. However, it was also demonstrated in the course of these experiments that electrons of slow speeds and feeble kinetic energies are unable to penetrate the crystals. It was Thomson who utilized faster electrons and demonstrated that not only are electrons diffracted like x-rays, but like x-rays also they make an imprint upon a photographic plate at increased speeds. These three men, together with others, then measured the wavelengths which they compared with the momenta of these electrons by their diffraction. To these experiments and measurements were then applied the following Rules of Correlation: "Energy (E) is proportional to frequency (v), and momentum (p) is inversely proportional to wavelength (lambda), the same constant (h) appearing in both relations. (Frequency is interpreted as the velocity (V) of the waves divided by their wavelength.)" These Rules can be applied mathematically to the Electron Microscope to better illustrate the principles of its operation. In making use of the first Rule, however, it is necessary to substitute "voltage" for "frequency," and in so doing, therefore, the Rules of Correlation explain the increase of energy in relation to the increase of voltage as well as the increase of speed of electrons in relation to the decrease or shortening of wavelength when we say--the higher the voltage, the greater the speed; hence, the shorter the wavelength of electrons. It is interesting to note in passing that a 150-volt electron has a wavelength of one angstrom unit, this being more than 10^-3 times smaller than the wavelength of visible or ultra-violet light.

(Page 108, Journal of the Franklin Institute 237(2):103-130 (1944))

Because the wavelengths utilized in an Electron Microscope are so much shorter than those employed in an ordinary light microscope, it is possible to obtain greatly increased resolution and magnification. As a matter of fact, resolution up to 20,000 or 25,000 diameters may be realized, and increased magnifications beyond this point up to 100,000, even 200,000 diameters, can be obtained, such magnifications, however, constituting enlargement of the image. (Definitions of "Resolution" and "Magnification" discussed under "The Ordinary Microscope.") This high magnification is greatly desirable since otherwise they eye would be unable to distinguish the fine detail of internal structure at a resolution of the order of 25,000. As a result of this increase in resolution and magnification over that of the ordinary light microscope which is between 1,600 and 2,500 diameters and in the ultra microscope between 2,500 and 5,000 diameters, many surface cells and much intricate internal structure hitherto unsuspected, or at least undetected by ordinary microscopes, have been revealed. To cite a few examples:

The streptococcal cells appear, not as individual cells, that is, separate and apart from one another, but as chain-like groups, the cells in each chain being bound together apparently by the strong rigid membrane or outer cellular wall which extends over a number of these cells and which is so plainly evident under the Electron Microscope. Subjected to sonic vibration, these cells suffer a loss of proto-plasmic material from their interior, causing them to become mere "ghost" cells, which makes them more transparent to electron beams. That there exists considerable difference between the surface structure and internal composition of these cells has also been determined and demonstrated.

Using the Electron Microscope, Dr. Harry E. Morton of the Department of Bacteriology of the University of Pennsylvania Medical School and Dr. Thomas F. Anderson of R.C.A. Research Laboratories were able to demonstrate that in at least one instance where chemical reaction is induced by bacteria this reaction takes place "inside" the cells. The fact that diphtheria bacilli reduce potassium tellurite to metallic tellurium has been known for some time, but whether this reaction occurred inside the cell or on the cell surface or both had never been definitely shown until the Electron Microscope was made available. Then, securing unstained preparations of Corynebacterium diphtheria grown on blood infusion agar, Drs. Morton and Anderson demonstrated that the typical polar granules appear as dense spherical masses, or possibly plates, of a very black color and that in unstained preparations of this same Corynebacterium diphtheria grown on potassium tellurite chocolate agar, not only the polar granules are in evidence but also the tiny needle-like crystals inside the cell which disappear along with the black color of the cell masses when a drop of bromine water is added to 1 cc. of a suspension of the cells on potassium tellurite chocolate agar.

(Page 109, Feb., 1944)

From this the experimenters were able to deduce that tellurium metal occurs in the form of needles and is the cause of the black color, and that this reaction occurs within the cells since the crystals have never been observed to lie totally outside the cell wall, although at times there is some distortion of the wall.

The Electron Microscope also affords such study and observation as that carried out by Dr. W. M. Stanley of the Rockefeller Institute for Medical Research and Dr. Thomas F. Anderson in their recent investigation of plant viruses. By means of electronmicrographs, they were able to judge the exact manner and extent of attack made on the tobacco mosaic virus by the protein antibodies in the blood stream of rabbits in which an artificial immunity to the virus had been produced.

Structures like that of the spirochete of Weil's disease, typhoid flagella, unusual internal structure of pertussis organisms, tubercle bacilli, the isolation and recognition of the influenza virus, the spores of trychophyton mentagrophytes, spirochaeta pallida with its accompanying flaggelar appendages, and colloidal particles are but a few of the interesting revelations of the Electron Microscope for medical science. Industrial science, too, has found this new research tool of great value in the study of metals, alloys, and plastics, as well as in the study of size, shape, and distribution of particles in chemical compounds and elements.

The Electron Microscope herein described is that manufactured by the Radio Corporation of America. There are, of course, variations in construction of the different instruments of this kind but all types are built along similar lines and upon the same general principles. In the Electron Microscope there is some aberration plus the additional disadvantages of having the specimen in a vacuum, not to mention the probable protoplasmic changes induced by the terrific bombardment of electrons, and finally, what is perhaps the greatest disadvantage insofar as medical science is concerned--that of being unable to view living organisms. Nevertheless, the disadvantages of the microscope are far overshadowed by its increased resolving and magnification powers which have combined to make it an invaluable research tool.

We have stated that the resolving power of the ordinary light microscope is restricted to between 1,600 and 2,500 diameters and that of the ordinary ultra microscope to between 2,500 and 5,000 diameters, resolution in any microscope being the ability of the instrument to reveal the most minute of component parts of a specimen so that each may be seen as a distinct and separate image. For instance, let us suppose an object is examined through which run two very fine parallel lines closely set together. If the two lines are visible under the microscope and are revealed as two separate images, then, appar-

(Page 110,Journal of the Franklin Institute 237(2):103-130 (1944))

ently, no limit of resolution has been reached; but if the two lines are merged or revealed as only one, and upon further magnification the image merely becomes enlarged without separation of the lines, then a limit of resolution apparently has been reached and additional magnification would constitute only enlargement. Assuming now that the object is a point object in which case the images of the points would be diffraction disks, the disks should likewise be sufficiently resolved so that each may be distinguished as a single image. If, when these disks are seen to overlap, additional magnification fails go extend the distance between them, their size simply increasing in proportion to the increase of magnification, or, if they are all but completely merged and the image becomes just a spurious disk of light, it is evident that a definite limit of resolution has been attained and that further magnification would be useless. Resolution, in a broad sense, then, is the ability of the microscope to bring out or reveal internal structure and detail of a specimen, the shortest distance it is possible to separate two component parts, according to Abbe, being not less than the wavelength of light by which the specimen is illuminated divided by the numerical aperture of the objective lens plus the numerical aperture of the condenser lens, or, about one-third the wavelength of light utilized.

The several factors which are generally acknowledged to be responsible for the limitation of resolving power are inter-related. Now when light passes from one medium into another of different density, in the instance which we are considering that of light refracted by the specimen and passing from air into glass, the light rays are deviated from their straight-line course; that is to say, that when they come to within a very short distance of this denser medium, they are acted upon by a very powerful force in such a manner that they execute a short rapidly curving motion, or an angle, and are pulled into the medium of greater density. When the rays of light undergo such a force, the momentum of the corpuscles is increased and the speed of the waves decreased, resulting, of course, in a shortening of the wavelengths. Here, again, we may make use of the second of the Rules of Correlation---"Momentum (of corpuscles) varies inversely as wavelength (of waves)." Once well inside the new medium, however, the light rays straighten themselves out again (unless the medium is so constructed that it possesses gradation of density in which case they follow a curved path). They do this in spite of the fact that the same forces are still acting upon them, although now these forces issue from all sides of them and so cancel each other out, the momentum of the photons or light corpuscles continuing to increase while the speed of the waves is proportionately retarded. If the light is refracted normally to the surface, however, it does not bend, but tends to cause a shortening of the optical path although the wavelength is shortened regardless. It is only when it is refracted obliquely to the surface that the light is bent, the greater

(Page 111, Feb., 1944)

being the obliquity of the incident ray and the denser the medium, the greater the bending of the angle of the cone of light and the shorter the wavelength. It might therefore seem desirable to obtain as great an angle of refraction as possible. However, shortening of the wavelength is not in exact proportion to the amount of bending except in the case of the diffraction grating. And regardless of how great a change there is in its angle, the numerical aperture of the light, or angular aperture as it is more properly called, remains constant.

In order, then that the cone of light be large enough to supply the aperture of the objective with sufficient light to produce an accurate, bright, and enlarged image of the specimen, it is first necessary that the specimen be refracting or emitting light of an adequate quantity, since both magnification and resolution are largely dependent upon the amount of light which the objective utilizes and receives into the tube of the microscope and since such light as the objective does receive should be only that emitted by the specimen. It is obvious, therefore, that it is of primary importance for the specimen itself to be amply illuminated. This would seem to depend entirely on the actual light source, yet no matter how powerful a light source is employed, it is of little avail unless the condenser is of sufficient quality and aperture dimensions to accommodate the light which it receives from the source. If, for instance, the numerical aperture of the objective is 1.25, the width of the cone of light emanating from the specimen should completely fill this aperture in order for the fullest powers of the microscope to be realized. Now, since the condenser supplies the light to the specimen, it stands to reason that it, also, should have a numerical aperture of at least 1.25. However, if the condenser and specimen slide are separated by air, the condenser can provide light of only 1.00 N.A. to the specimen since, according to a law of optics, no aperture greater than 1.00 N.A., (this being the refractive index of air), can pass from a denser medium into air. To remedy this situation, an immersion fluid is placed between the top of the condenser and the lower side of the specimen slide as well as between the specimen and the objective lens.

Since no optical medium has an index of refraction greater than three and no immersion fluid an index of refraction greater than 1.7, to further increase resolving power, then, might it not be feasible to widen apertures of the objective and condenser lenses, thus affording additional illumination for utilization by both specimen and objective? This idea would be entirely practical except for the fact that such enlargement of the lenses would increase aberration, both spherical and chromatic, and apparently present-day lenses are now as highly corrected as it i possible for human ingenuity and skillful workmanship to make them. Spherical aberration, caused by the paraxial rays coming to a focus at the center of the lens before those rays near the

(page 112, Journal of the Franklin Institute 237(2):103-130 (1944))

principle axis, is corrected by using concave and convex lenses of different material and, consequently, of different refractive index. In this manner spherical aberration of a convex lens, for instance, can be overcome, without its converging action being altered, by adding to the optical system a concave lens in which there is an equal and opposite aberration. Chromatic aberration, occurring when more than one wavelength of light is used to illuminate the specimen, is due to the fact that the shortest waves of the spectrum are refracted most and the longest waves least, thus causing the blue-violet waves to come to a focus ahead of the red waves and resulting in a series of colored foci all along the axis. Now since, as we have said, the shortening of the different groups of wavelengths is not in exact proportion to their bending and since this circumstance varies according to the substance the light rays pass through, it is possible to combine lenses or lens systems ins such a way that white light may be obtained. For instance, a small concave flint-glass prism produces the same amount of dispersion as a large convex crown-glass prism. Thus, if these two prisms are placed with their edges opposite, the crown-glass will bring together the spectrum produced by the flint glass and white light will be the result. However, the rays of white light will not extend parallel with the original direction but will bend toward the base of the crown glass since the mean refraction of the crown glass is greater than that of the flint glass. Achromatic objectives, corrected spherically for one color, chromatically for two; semi-apochromatic objectives, possessing moderate refractive indices and very small dispersion, in which a lens of fluorite is substituted for one of the glass lenses; apochromatic objectives, corrected spherically for two colors, chromatically for three; and also certain monochromatic lenses for use with light of one wavelength only are available for overcoming, at least in part, one of the conditions which tends to interfere with better resolution. Condensers, also, can be corrected for both spherical and chromatic aberration and must be achromatic-aplanatic if the light which enters the objective is to come only from the specimen, for condensers with spherical and chromatic aberration are unable to direct their entire cone of light upon the specimen.

In addition to being highly corrected as possible and possessing a large numerical aperture, an objective should also be capable of adequately magnifying the image, being aided in this by the ocular which also serves at times to compensate for the defects in chromatic magnification which cannot be managed conveniently by high-power objectives, the magnification of the final image being the product of the magnification of the objective multiplied by the magnification of the ocular. An amplifier is sometimes inserted between the objective and ocular which causes the rays of light from the objective to diverge to a greater extent, thus doubling the size of the image. Magnification may also

(Page 113, Feb., 1944)

be improved by increasing the tube length, by increasing the distance from which the image is projected, and by altering the positions of the various lenses in an adjustable objective. In general, the greater the magnification, the smaller will be the specimen field, but, as has been stressed, high powers of magnification should always be accompanied by equally high powers of resolution.

As we have seen, resolution in the ordinary light microscope is definitely restricted by a number of inter-related elements. Even when monochromatic light is employed, there is always present some spherical aberration with which to contend. True, better visibility of specimens is provided by dark-field microscopy in which the specimen is viewed by the high contrast of its own scattered or reflected light against a dark field, although in this type of illumination objects in the field must be well separated. Much fine detail and brilliant color of specimens can be observed by means of the polaraization of light. Further, it is possible to illuminate the specimen with shorter and shorter wavelengths of light, the shorter the wavelength of light used, the more of the fine detail of the specimen which can be seen, but a limit is reached here, also, for ordinary glass lenses are not transparent to ultra-violet rays. However, in the ultra-violet microscope, having a resolution twice that of the instruments using "visible light," the condenser, objective, and ocular are all made of quartz and, by substituting the photographic plate for direct observation, many excellent micrographs of numerous varieties of organisms and cellular structures can be made. But when viewed directly, noting of the nature or structure of the specimen can be ascertained; only the light scattered by the specimen is distinguishable, the size of the specimen being roughly estimated by the amount of light refracted.

These seemingly unsurmountable obstacles of the ordinary microscopes would appear to indicate that Abbe's law and the contention of physicists that "any object which is smaller than one-half the wavelength of light by which it is illuminated cannot be seen in its true form or detail" are destined to remain undefied.

But Dr. Francis F. Lucas of the Bell Telephone Research Laboratories and Doctors Louis Caryl Graton and E.C. Dane, Jr., of the Department of Geology, Harvard University, have very convincingly demonstrated a reduction in these theoretical limits or resolution and visibility with their instruments, designed for use in the visible light region of the spectrum.

(Page 114, Journal of the Franklin Institute 237(2):103-130 (1944))

great stress. Any type source, such as the carbon arc, metallic arc, incandescent filament, Point-O-Lite, Mercury Vapor, or any of the special forms of monochromators, can be used for illuminating the specimen with direct and dark-field transmitted, vertical and oblique reflected, or polarized light. The image beam itself follows a straight-line path in passing from the objective, the objective ranging anywhere from the shortest to the greatest in working distance, through the tube to the ocular, as few lenses as possible being placed in its way. The spiral-cut rack and pinion which moves the stage and sub-stage assembly in longitudinal tracks or guides can be operated by hand or by an electric motor and is independent of the fine adjustment, also motor driven, which moves only the objective and the carriage carrying the objective. Whereas manual operation of the fine adjustment which is one hundred times more sensitive than that of the ordinary instruments necessitates five hundred turns of the knob to move the objective a distance of but one millimeter, (an adjustment calculated to require a time period of twenty-five minutes), by means of the motor it is possible to move the objective at the rate of 0.01 mm. per second or 0.004 mm. per second, depending upon which of the two speeds is desired, rapid motion being used when the image appears considerably out of focus and decreased speed being used when the image seems to be reaching a point of perfection.

Resolution up to 6,000 diameters and magnification up to 50,000 diameters have been achieved with this high precision microscope which photographs or enables observation of both opaque and transparent preparations; in fact, polishing scratches measuring, in width, but one-tenth the wavelength of light used have been clearly distinguished. It is the opinion of both Dr. Graton and Dr. Dane that some present-day lenses are really capable of better resolution than claimed form them by their manufacturers, it having been their experience to use objectives exhibiting superior qualities of resolution over those of identical medium and numerical aperture, proving that not only that already available lenses surpassed their theoretical limits of resolution, indicating that it might be possible to design objectives with still greater numerical apertures, but that the accepted theory regarding this resolution is sadly in need of revision. Dr. Lucas's microscope utilizing an objective a numerical aperture of 1.60, for instance, in combination with monochromnaphalene immersion fluid, also yields resolution up to 6,000 diameters being, like the Graton-Dane scope, a high precision instrument constructed with the idea of maintaining absolute stability of parts. Dr. Lucas also has expressed doubts as to the complete validity of the generally accepted theory of resolution.

In working with a high precision ultra-violet micro-camera, into which a tri-color filter system has been incorporated, which he has just recently perfected, Dr. Lucas is able to obtain a minimum magnification

(Page 115, Feb., 1944)

of 30,000 diameters and a maximum magnification of 60,000 diameters. With this instrument it is possible to view living cells and organisms, no staining or killing of organisms being necessary, and Dr. Lucas has succeeded in obtaining excellent photomicrographs (both still and motion pictures). Of special significance to industry, for instance, is the ability of this scope to demonstrate the size, shape, and reactions in motion and affinity of the tiny particles of which rubber is composed under varying conditions of temperature, etc., while its ability to reveal living rat and mouse sarcoma and carcinoma cells and to demonstrate the development and behavior of the syphilitic organism is of far more than average interest to medical science.

England's Dr. J.E. Barnard has succeeded in obtaining resolution up to 7,500 diameters with his ultra-dark-field scope in which he uses a combined illuminator. In this, an outer system of glass acts as the immersion dark-field illuminator while the inner immersion system of quartz makes possible the passage of a transmitted beam of light through the specimen. Both condensers have the same focus, one for visible light, the other for ultra-violet radiation, and both can be stopped out at will. When, for instance, bacteria are being observed, immersion contact is made between the condenser and quartz slide, the dark-field illuminator being used, thus revealing the bacteria with visible light. When the dark-field illuminator is closed, however, a beam of ultra-violet light may be directed up through the quartz condenser and focused on the bacteria. The object-glass, of course, has to be adjusted since it does not possess the same focus for ultra-violet that it does for visible light. Staining of specimens is thus unnecessary, making it possible to secure photomicrographs of living minute organisms.

In addition to these four microscopes, a fourth, belonging to the Canadian Department of Mines and located at Ottowa, and almost identical in principle and construction to that of Doctors Dane and Graton, has demonstrated ability to attain equally high resolution. This, like the scopes of Doctors Dane, Graton, and Lucas, is fitted with a tube for visual observation although intended mainly for microphotographical work in the field of metallurgy. It is Dr. Graton's belief, however, that his instrument and that of Dr. Dane might also be adaptable to the purposes of biological research. Referring, in the description of their "Precision, All Purpose Microcamera" (Journal of the Optical Society of America), to the necessity or "desirability" of "re-examining the classical conception of the limit of useful magnification," Doctors Dane and Graton have this to say:

"So long as the makers accepted the conventional limit as valid and had already attained it, there was little incentive towards progress. But with that limit apparently surpassed, there is no present knowledge as to how far ahead the true limit may lie. If present-day objectives do substantially better than the 'limit' for which they were designed,

(Page 116, Journal of the Franklin Institute 237(2):103-130 (1944))

is it not reasonable to suppose that effort to do better still may conceivably be rewarded?"

To such an inquiry there can be but one logical answer--an agreement to which, while perhaps not concurred in by all, must, for those stimulated to more intense interest and effort by the possibilities of uncovering new facts, pose further questions; for, if the improvement of one part results in the improved performance of the whole, is it not

also reasonable to suppose that additional changes of additional parts, yes, even changes with respect to principle and method might likewise bear fruit?