Microtechnique


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Microtechnique

 

(in biology), the aggregate of the methods and procedures used to study the structure, vital activity, development, chemical composition, and physical properties of cells, tissues, and organs by means of optical and electron microscopes. Such techniques include the preparation of living specimens for microscopic examination; the act of examination; the preparation of permanent (inanimate) specimens; microchemical, histochemical, and cytochemical studies; and special methods of preparing specimens for the electron microscope.

Observations in vivo. Observations in vivo in transmitted light are carried out on protozoans, small eggs, cultured cells and tissues, and transparent parts of metazoans (for example, blood vessels in the nictitating membrane of the frog). The superficial structure of the cell, tissue, and organ can be studied in reflected light under a microscope. Staining in vivo, which provides an indication of the pH of the cell and its organoids, and also of the physiological condition of the living specimen, is used for cytophysiological observations. Equipment required for in vivo observations includes a heated stage (that is, a special thermostat that can be adjusted to a particular temperature within a broad temperature range) and glass, plastic, quartz, metal, or other chambers with a constant or flow-through medium of the required composition. The object to be observed (often cells of monolayer cultures) may remain normal for long periods if adequately supplied with nutrients and oxygen.

One of the tasks of microtechnique for living specimens is to increase image contrast. A phase-contrast device, for example, is used for this purpose. In addition, interference microscopy provides data on the thickness of the object, the concentration of dry matter, the water content, and the index of refraction. In vivo observations are also conducted in a dark field (ultramicros-copy) with a special condenser; in this process the object is illuminated from the side, but the background remains dark. A dark-field device makes it possible to see extremely small particles (such as colloidal particles). Specimens (or parts of specimens) that have optical anisotropy can be studied with a polarization microscope. Luminescence microscopy is used to study both living and nonliving biological specimens, and particularly to study the secondary fluorescence that arises when cells and tissues are stained with weak concentrations of fluorochromes (such as acridine orange, erythrosin, and rhodamine). Differences in the fluorescence of certain chemical substances (nucleic acids and lipids) make it possible to study their localization and the dynamics of changes and even to determine the quantity of a substance. The combination of a protein with a fluorochrome (fluorescein isocyanate) and the binding of this substance with antibodies make it possible to elucidate the localization of antigens, the fate of antibodies, and other problems of immunology. Microscopy of living and nonliving objects in ultraviolet light, using special quartz optics, has recently come into use. Observations of living specimens are documented by motion-picture photomicrography, especially slow-motion photography.

To obtain permanent specimens, the organisms are fixed—that is, killed in such a way that they retain their structure to the greatest degree possible. The most widely used fixatives are Formalin, alcohol, and osmium tetroxide, as well as combination fixatives (mixtures of substances). Fixation is also done by lyophilization and by the drying of smears (such as blood) or impressions, especially in the case of electron microscopy. Glass or mica plates on which a single layer of cells is placed are used in work with cell cultures. In other cases sections produced by a microtome are used for microscopy. Here the specimen is dehydrated and immersed in paraffin, pyroxylin, or gelatin, or it is frozen. Material for electron microscopy is usually fixed with osmium tetroxide and immersed in acrylic monomers, which are polymerized by the corresponding catalyst, or in epoxy resins.

Microchemical, histochemical, and cytochemical studies. Dyes that selectively stain different cell structures are used to increase the contrast of preparations observed under an optical microscope. Dyes are particularly widely used in histochemistry. Histochemical reactions are based on the formation by certain substances of insoluble and sometimes stained precipitates that can be seen under a microscope. Enzymes are observed in cells on the basis of their activity upon exposure to certain substrates that are present in the tissue or are added from without. The intensity of histochemical reactions is often studied and assessed visually. Quantitative methods of assessment, such as counting the number of cells with a certain staining intensity and the number of precipitate grains, as well as autoradiography and cytophotometry, are more advanced.

Electron microscopy. In the electron microscopy of viruses, microorganisms, and ultrathin sections of larger specimens, the contrast is heightened by shadowing with metal particles. For negative contrasting the specimen is placed in a solution of a denser substance (for example, phosphotungstic acid) that fills the gaps between the particles under study, which appear light against a dark background. Contrast is also enhanced by using “electron dyes” (such as osmium tetroxide and uranyl), which selectively bind to certain sites on the specimen. When ferritin is used, its grains, which contain molecules of iron, are observed within the cell structures.

REFERENCES

Meisel’, M. N. “Liuminestsentnaia mikroskopiia.” Vestnik AN SSSR, 1953, no. 10, pp. 3–10.
Romeis, B. Mikroskopicheskaia tekhnika. Moscow, 1954. (Translated from German.)
Brumberg, E. M. “O fluorestsentnykh mikroskopakh.” Zhurnalobshchei biologii, 1955, vol. 16, no. 3, pp. 222–37.
Sovremennye metody i tekhnika morfologicheskikh issledovanii [Collection of articles]. Edited by D. A. Zhdanov. Leningrad, 1955.
Roskin, G. I., and L. B. Levinson. Mikroskopicheskaia tekhnika, 3rd ed. Moscow, 1957.
Appelt, G. Vvedenie v metody mikroskopicheskogo issledovaniia. Moscow, 1959. (Translated from German.)
Zubzhitskii, lu. N. Metod liuminestsentnoi mikroskopii v mikrobiologii, virusologii i immunologii. Leningrad, 1964.

S. IA. ZALKIND

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