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Microscopy. II. Electron Microscopy: A Review

( Sloan-Kettering Institute for Cancer Research, New York, N.Y.)

The manner in which the electron microscope affords a higher resolving power than any other method of direct structural analysis is described. Survey of the literature relating to its first decade of availability for biological research shows that much of this time has been spent in developing the special methods required for preparing specimens sufficiently thin, sufficiently dry, and with sufficient contrast for observation in this new instrument. Methods devised for this purpose include surface replicas, metal-shadowing, freezedrying, critical-point drying, and thin-sectioning. Specimens have also been successfully prepared by cell-fractionation, cell-smear, and tissue culture. In the short time which has elapsed since the development of these methods, much submicroscopic tissue structure with widespread chemical and physiological significance has already been revealed.

The most striking contributions are those which extend the microscopist's vision to the molecular domain by describing such simple structures as muscle fibers, collagen fibers, and bacterial flagella in macromolecular dimensions. The essential simplicity of design in biological, as in other, matter is illustrated by the common lamellar structure of the nerve myelin sheath, retinal rods, and chloroplasts. This design is already, in the case of nerve, identified with a certain chemical composition. Although the structural integrity of the mitochondrion was in doubt until only recently, it has now been shown to possess a limiting external membrane and a system of internal membranes whose dimensions suggest a direct analogy to the protein-lipid structure of other membranes. The methods are now at hand for determining the ultrastructure of other cellular components (cilia, cell and nuclear membranes, brush borders, submicroscopic cytoplasmic inclusions, chromosomes, etc.) and thus achieving further correlations of cellular form with function.

The high resolving power of the electron microscope has also been useful in explaining certain phenomena of optical microscopy. The dependence of cell morphology upon the pH and chemical composition of the fixative and its change upon removal of the embedding have been clearly shown and indicate that it is the coarsened or vacuolated cell structures after acid or basic fixation and removal of the embedding that produce the best image under the lower resolving power of optical microscopes. For example, spindle fibers appear under the light microscope only when, after acid fixation, the cytoplasm precipitates very coarsely in a certain orientation. The electron microscope shows that, after neutral fixation, only chromosomal fibers appear, while the rest of the spindle exists only as an orientation of fine submicroscopic particulates.

Since virus particles are visible only in the electron microscope, some of the most striking contributions of this instrument have been in the field of virology. Most stages of bacteriophage-bacteria interaction have now been shown morphologically, and studies with other viruses, notably vaccinia, molluscum contagiosum, and herpes are rapidly progressing in this direction. Although influenza, Newcastle disease, and the encephalitis viruses are more difficult to study because of the existence of normal cell components ("microvilli" and microsomes) with similar morphology, the structural effects of these viruses are also becoming understood.

Following the description of normal cell ultrastructure, studies of tumor cell ultrastructure have already led to positive contributions to cancer research. Distinctions in the submicroscopic cytoplasmic inclusions found in normal and neoplastic cells have been observed by several investigators in tissue cultures, tissue sections, and blood cell smears. Studies of comparable cells in other pathologic conditions are already underway and should reveal the true specificity of these tumor cell characteristics. Chemical and metabolic studies of these structural distinctions can then follow. With the develop nent of thin-sectioning technics for routine use, such submicroscopic cellular criteria as those which have already been revealed could be applied to many practical problems in tumor classification and therapy evaluation beyond the scope of optical microscopy and pathology.

One of the first applications of the electron microscope to cancer research was the visualization of cytoplasmic deposits of virus-like particulates in tumors of proved viral etiology. The fact that such particulates have not yet been observed in other tumors points out, as clearly as other methods, that these tumors possess a different growth mechanism. The way is now open for following the development of viral and nonviral cellular components with the passage and growth of the tumor and so, with primarily morphological technics, further elucidating the proliferative mechanism of these cells.

^* This work was done during the tenure by the author of a post-doctoral research fellowship from the National Institutes of Health, Public Health Service. Additional support was obtained from the Lillia Babbit Hyde Foundation and the American Cancer Society.

Received 3/14/53.