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.
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?