Today, one of the most exciting research frontiers in biology is the once-invisible region beyond the limit of the conventional microscope—the level at which proteins, carbohydrates, fats, minerals and water are organized in the pattern we call life. Investigation of the macromolecular structure of cells and their components has become increasingly rewarding with the development of electron microscopy—a biophysical technique which, in a few short years, has answered many old questions and raised an equal number of new ones.
The electron microscope is not a new research instrument (the first successful ones were produced in the early 1930’s). but the application of this "supermicroscope" to biology has recently taken a giant stride ahead with the refinement of techniques for preparing specimens for study at high magnification. This development of "ultra-microtomy"—or thin-section techniques—grew out of the need to overcome some of the limitations posed by the very nature of the electron microscope. For, while the electron microscope can magnify an object many thousands of times more than can a light microscope, the instrument makes certain demands of the microscopist. A comparison of the light and electron microscope will be helpful in making this clearer.
In the light microscope, magnification of the image is achieved by transmitting light from a source through glass lenses. Details in the image are visible because of the light absorbed or scattered by the object being viewed. In electron microscopy, a beam of electrons is substituted for the conventional microscope’s beam of light; magnetic “lenses” take the place of glass; and most of what is visible is seen because of the scattering of electrons by the material of the specimen.
In other words, the specimen is cut so thin that those portions which appear white in the electron micrograph have not retarded or deflected the focused beam of electrons at all, while those portions of cells, such as the nucleus, which appear darker in the electron micrographs seem so by virtue of the density of the material originally present in that area. In the case of a completely black area, the specimen under study has completely retarded the electron beam and has, in this way, prevented it from reaching the exposed photographic film.
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Why is there a limitation to what can be seen with the light microscope? Both theory and experiment have shown that it is the wave length of the light which sets an absolute and unalterable lower limit to what can be seen. Because it is impossible to form a correct optical image of objects smaller than about half the wave length of the observing light, it is impossible to see anything smaller than about two-tenths of a micron (a micron, one one-thousandth of a millimeter, is equal to 0.00004 inch) with the conventional light microscope. But the wave length of the associated electrons in the beam of a 50 KV electron microscope is many hundreds of thousands of times smaller than the wave length of visible light. Theoretically, the electron microscope should be able to resolve objects as small as 0.05 Angstrom units. Since an Angstrom unit is one ten-millionth of a millimeter in length, this theoretical resolution comes to .000,000,000,197 (1.97x10-10) inch.
In practice, however, any microscope’s resolving power depends not on the wave length of the observing “light” alone, but on the lens system and on the aperture of the objective lens. In electron optics, in order to keep “lens” aberrations to a minimum, limitations are imposed on the size of the aperture. This, of course, leads to a reduced value of the actual resolving power of the electron microscope from what theoretically might be possible. In a properly trimmed electron microscope, it is actually possible routinely to achieve 30 Angstrom unit resolution—which means the difference between being able to observe a virus particle and merely theorizing its existence.
It is important to remember, in this connection, that the resolution of a given electron micrograph depends not only on the performance of the electron microscope but also on the nature of the specimen viewed. For example, living matter, thus far, has not been observed. It would be necessary to confine a living specimen to a chamber the walls of which would not scatter or absorb the electron beam: even then, it seems likely that the rapid vibration of microscopic particles in a wet preparation would probably obscure the hoped-for detail.
Another problem is that of specimen thickness. Because the beam of electrons is more easily scattered than a beam of light, it is necessary to use an extremely thin support for specimens. For a while, the electron microscopist was caught between the Scylla of too thick and dense films and the Charybdis of films so thin that they tore when subjected to the electron beam. But, with practice and experimentation, appropriate supporting films and nets have at last been developed.
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A third and more complex difficulty—but one fundamental to successful high resolution—is the problem of “contrast.” This is as important, in a way, as the problem of resolving power, since an object can be seen (if it is larger than the resolving power of the microscope) only if has enough mass to scatter electrons and if, at the same time, the supporting film does not scatter the electrons to the same degree. Many early attempts at demonstrating particles of macromolecular dimensions failed because this contrast between the specimen and the supporting film was not sufficient. It was like trying to see a black bear in the middle of a dark night.
Before the development of methods for thin sectioning, this problem of contrast was partially solved by an ingenious method devised by R. Robley Williams—the preferential attachment of atoms of a heavy metal to the surface of the object under study. One of the ways of doing this was a “shadowing” technique: the specimen was placed in a vacuum jar and the metal placed beside a tungsten filament. At the moment the filament’s heat vaporized the metal, the metal atoms traveled in straight lines in all directions: some of them were deposited on the specimen. Since the greatest amount of deposited metal was on the specimen surface facing the filament, the specimen appeared in the electron microscope as if it were casting a shadow. Such structures as collagen fibers are admirably suited to this shadowing technique, as Schmitt, Hall, Jakus and Gross have demonstrated at M.I.T.
Another approach to the problem, before today’s sectioning techniques were developed, was put forward by Dr. Keith Porter at the Rockefeller Institute. Taking advantage of the spreading characteristics of some cell types, Porter allowed cells in tissue culture to spread out on thin films. He then fixed the cells and transferred these whole mounts to the electron microscope. Despite such techniques, however, the absence of any method for sectioning meant that it was not possible to look at a cross-section of a cell’s surface membrane, to examine the relation of the nucleus to the cytoplasm, to compare the submicroscopic morphology of different cell types or to hope to define the macromolecular structure of such organelles as mitochondria or the Golgi apparatus.
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The development of ultramicrotomy grew from contributions by many different groups of workers. In 1949, Newman, Borysko and Swerdlow at the National Bureau of Standards in Washington, D.C., described a method for embedding tissue in a plastic—methacrylate—rather than in the paraffin used routinely by light microscopists. This acrylic plastic is relatively transparent to the electron beam; moreover, it does not change from a solid to a gas as readily as does paraffin under electron bombardment. More important is the ease with which the plastic may be sectioned, as thin as three or four hundred Angstrom units. Thinness of section is extremely important, since the electron beam has a comparatively limited penetrating power, and the great depth of focus makes for superimposition of particles in different planes of the section, which tends to obscure very fine structures.
In order to obtain sections thin enough for successful high resolution microscopy, much effort was spent on designing new microtomes. Two of the most successful and widely used microtomes are the Sjöstrand microtome and the Porter-Blum. In both methods, the specimen to be sectioned is mounted on the end of a metal rod which passes a cutting edge once during each cycle. By timing the rate of revolution and controlling the heating element (in the Sjöstrand microtome), the specimen can be sectioned to such thinness that the ribbon of sections is practically invisible. The thickness of these sections, as they float in the collecting basin, may be estimated by the reflected color of incident light—the thicker ones appearing yellow or red, while the thinnest seem bluish-white.
With these developments, it has now become possible routinely to section specimens from all parts of the plant and animal kingdoms. Naturally, much more attention must be paid to optimal fixing, embedding and sectioning techniques than in the case of light microscopy—since the very high magnifications at which specimens are ultimately viewed exaggerate any and all artifacts of technique. A typical procedure is to take the specimen for study as quickly as possible (to prevent post mortem changes), at low temperature (to slow enzymatic activity) and immerse a minute quantity of tissue in a fixative (the smaller the quantity the better, in order to permit fixative penetration). After fixation for an appropriate period, the specimen is rinsed and dehydrated in graded alcohols until it is free of water. It is then allowed to rest in liquid plastic until the plastic has completely penetrated the tissue: then the plastic is hardened and the specimen is ready to be trimmed and oriented for sectioning.
The procedure of trimming requires an intimate knowledge of the anatomy of the tissue, as well as delicacy and patience, since the final result depends largely on precise orientation of the desired cells to the plane of section. The specimen is usually trimmed in the form of a pyramid—with the base about 0.5 millimeter on a side. The sections which are cut from the trimmed block may be about 0.1 millimeter square and can only be seen with the help of a dissecting microscope. When they float from the microtome’s knife-edge onto the fluid surface of the collecting basin, they are collected on a grid which is then placed in the microscope for scrutiny.
The first major discovery with this new technique of sectioning was the demonstration in 1952 in (by G. Palade and F. Sjöstrand, independently) that the cellular respiratory centers, the mitochondria, have a highly organized structure—quite in keeping with the biochemical evidence of their complex function, which allows the many steps involved in the transfer of energy to take place in an orderly manner. While appearing in whole cells as long rods, mitochondria—studied at high resolution in sectioned material—were shown to have well-organized systems of internal membranes, bounded by an outer limiting membrane. As evidence accumulates from studies on different tissues, it becomes apparent that the mitochondria of all cell types do not look exactly alike. Today, there is much discussion among biologists about the true structure of the mitochondria. and some experiments have shown that mitochondria may be altered from “normal” by various means. It seems likely that well-designed experiments may show that these alterations in morphology are coincidental with alterations in function. But it is imperative that the “normal” appearance be thoroughly investigated first.
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The study of cell sections in the electron microscope has demonstrated many variations which the cell surface membrane can assume. Ordinary epithelial cells—cells covering an outer surface or lining an internal cavity—most often have small, finger-like processes projecting from their surface. A study of epithelial cells in the developing hen’s egg showed that, when the cell was experimentally infected with an influenza virus, filaments of virus particles could be seen in all stages in and out of the finger-like processes. This could mean that new virus particles are liberated from the surface of these cells and thence are delivered as infectious agents to the world at large.
Other studies have shown that virus particles not only have distinct shapes, sizes and submicroscopic structures, but that at least some viruses are apparently produced in the nucleus of cells and others in a particular component of the cytoplasm that has already been identified as a probable site of protein production.
The brush border of the absorbing cells in the kidney and intestine has been shown in the electron microscope to consist of longer and thinner fingers of the cell surface. In the intestine, these cells themselves are arranged in tiny projections, or villi, that give the inner surface of the intestine the appearance of velvet. These villi are a device for increasing the absorbing surface of the tube many times. Studies on many thousands of sections with the electron microscope have made it clear that there are probably six or seven hundred finger-like processes at the surface of each cell, increasing the surface area of the cell itself at least fifteen times. So we have adaptations on adaptations, all to allow a better absorption of what we eat.
The cilia of cells, in the animal kingdom, are another kind of modified cellular extension and are used in one way or another as transport mechanisms. It is a puzzle how these thin, small processes can wave back and forth so vigorously, even when separated from major portions of the cell. Electron microscope studies on cross-sections of cilia show that their submicroscopic organization (and presumably the component necessary for their motion) is nine pairs of cylinders arranged in a circle within the cytoplasm. In the center of these nine paired cylinders is another pair. It is a problem in geometry to relate these cylinders at their termina to one another and to see whether they are in truth continuous. Now, the tail of the spermatozoan is organized in a similar manner and looks quite identical. We can imagine that, as in the cilia, the mitochondria of the sperm middle piece supply the energy necessary to allow the tadpole-like cell to wiggle its tail until it has moved far enough to reach and fertilize an egg.
Furthermore, electron microscopy on sections of the retinal rods of eyes (the dim vision receptors) shows that there is a small portion of the outer segment which connects the stacks of plates with the inner segment. This region, which must in some way transmit the energy of light to cells nearer the brain, has a similar submicroscopic structure to that of the cilia and the sperm tail. Is this another demonstration of Nature putting the same arrangement of living matter to different uses?
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The examination of such similarities can lead us still further. Studies of sections of such retinal rods have confirmed Dr. Fritiof Sjöstrand’s earlier work (on isolated fragments) and shown that the stacks of plates, when sectioned in the proper plane, appear as very regular, platelike lamellae. This arrangement of the photoreceptors has also been confirmed as the structure of the chlorophyll-containing grana, or granules, of the chloroplast, where plants manufacture their starch. The very regular lamellar construction of photoreceptors—in animal and plant alike—is a striking feature that bears further investigation.
Long ago, an ingenious investigator showed that if electrodes are placed on two spots of a green leaf, and one of the electrodes is shaded from the sun, the electrode in the light becomes electronegative to the electrode in the shade. This reaction can also be obtained when electrodes are placed on the green and white parts of a variegate leaf—the green part is negative to the white when light falls on both. Since the white part of the leaf has no chlorophyll, this electrical evidence has been interpreted as a demonstration that chlorophyll is necessary for the leaf’s perception of light. Similar electrical patterns can be recorded from the retinal rods of animals when the retina is stimulated by a flash of light; and we know, as well, that vitamin A is essential for the function of these rods. Recently, some electron microscope studies on chloroplasts from white leaves (the plants were grown in the dark) showed that the usual lamellated appearance of the grana was not present. When the plants were put in the light and recovered their green color, the lamellae of the grana also reappeared. It was suggested, therefore, that chlorophyll is in some way necessary for the appearance of the lamellae of the grana. If the lamellae are really uniformly arranged molecules of protein and fat, as has been suggested, we now have to answer this question: was it the light, or the chlorophyll, which caused the very regular appearance of the layered structure?
Trying to answer such a question is something like trying to find out what the shade of an unexposed bit of photographic paper is. If one turns on the light to see, paper’s shade may be changed by the action of the light needed to observe it. Perhaps one way of getting at this problem would be to look at the photoreceptors of animals raised in the dark. If their retinal rods showed a lamellated structure, then perhaps some essential metabolite—such as vitamin A—might be removed from their diet. If vitamin A-deficient animals raised in the dark showed a nonregular structure of the light receptors, then some animals could be given the metabolite to see whetherit would cause the reappearance of the lamellae. Others could merely be allowed to see light, to determine what effect this energy had in aligning the molecules.
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Another interesting structure within cells which has been the subject of electron microscopic studies is the Golgi complex. The Golgi complex has been the matter of controversy for fifty years—some investigators bitterly contend that all evidences for this juxtanuclear apparatus are artifact, to which others retort that the only artifacts present were those caused by the complaining experimenters themselves because they are not using the technique correctly. With the help of careful correlation between living and fixed cells studied with the light microscope, and electron microscope studies on the same material, it seems that the controversy is at least partially resolved. A system of membranes, granules and vacuoles has been seen near the nucleus of almost every cell type examined in the electron microscope, and it seems justifiable to conclude that this structure must be the basis for what was described earlier by light microscopists.
Apart from the similar appearance of the membrane component of the Golgi complex in many different cell types, it is becoming obvious that the other components, the vacuoles and granules, are different in different cell types. In the absorbing cells of the kidney tubule, there are often large vacuoles between the membranes; in the absorbing cells of the intestine, these same vacuoles often contain small black granules. Could they represent some difference in hat the Golgi complex is metabolizing? The same large vacuoles are present in the zymogen-secreting cells of the pancreas, but they do not exist in the epithelial cells of the cornea, which functions only as a transparent covering for our eyes, and so far as we know does not produce or absorb any foodstuffs for our general use. In these same cells of the pancreas, large granules of the digestive enzyme zymogen are produced, and these granules seems to be most intimately associated with the Golgi membranes. In fact, a whole range of different-sized granules can be seen in and around the Golgi complex. We wonder if these large and small granules represent various stages in the development of the enzyme, which is then discharged in this form to where it helps digest foodstuffs.
Interpreting electron micrographs can lead the most careful investigators astray, if they are not aware of the many factors to be considered before they can draw summary conclusions. One of the most difficult problems is to reconstruct what we know are dynamic processes from the static pictures we obtain in the electron microscope. Also, before any study is complete, it is necessary to know something of the variations which can be manifested even in supposedly “normal” biological material. This means that any comprehensive investigation must include a study of many samples from different organisms. Only then is it time to alter conditions experimentally in an effort to see how infections, injury or different functional states may be reflected by changes in the structure of the tissue.
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It has often been remarked that the best experiment is the simple experiment. Once systematic electron microscope investigations of “normal” tissues have been completed, it should be possible to begin experiments where, in so far as possible, only a single variable has been introduced. Using specific enzyme poisons or removing an essential metabolite (such as a vitamin) should cause changes to appear which, while they are not easily seen with the light microscope, would be obvious at the macromolecular level. Such approaches should yield new information in which morphological and biochemical knowledge can be correlated. Dr. Lindestrom-Lang, working at the Carlsberg laboratories in Copenhagen, showed very clearly how such correlations could be achieved at the level of the light microscope when he cut alternate sections from a frozen cylinder of tissue and did chemical studies on one slice and microscopy on the next. A similar study with the electron microscope could help to identify the dense and non-dense structures seen in electron micrographs. Another technique for correlating biochemical properties with morphological appearance may be found in applying the principle that different substances absorb light of different wave lengths. Professor Caspersson, in Stockholm, has devised methods which allow him to make not only qualitative but quantitative determinations on particles as small as one-tenth of a square micron. All of these techniques offer new possibilities to the biologist who would better understand the complex nature of life.
In the future, the electron microscope will be used on an infinite number of problems—ranging from what may be the causes for cancer to what is the structure of an enzyme. But, at the moment, the most inviting problems seem to lie in correlating some of the new information we have already gleaned about biological material with the functional properties of these submicroscopic structures. The most difficult thing to do is to ask biological questions in such a way that they can be answered. We hope the electron microscope may be used as a new key with which to unlock some of these still-closed doors.