Fluorescent microscopy

Fluorescence microscopy - optical study of micro-objects, painted with dyes (fluorochrome)emitting glow when exposed to ultraviolet rays. For fluorescence microscopy, special optical devices and microscopes, main part of which is the source of ultraviolet rays and system of filters to it.
Fluorochrome, as a rule, flyuorestsiruyut differently depending on the chemical composition of the structures with which they interact. Some of them have affinity to specific cellular structures. For example, acridine orange dye colors the nucleoprotein cells, auramine - wax-like substance found in mycobacteria. Some objects do not require painting fluorochrome and studied using fluorescent microscopy without color. Cm. also Luminescent analysis, Luminescence.

Fluorescence microscopy (fluorescent microscopy) - a special type of microscope, based on the use of their own (primary) or induced (secondary) photoluminescence of microscopic objects. Visible luminescence (see) drug excited or blue-violet light or UV rays.
Fluorescent microscope in principle - normal biological microscope, equipped with two filters: one allows only the exciting blue or ultraviolet rays (it is placed in front of the light source), another absorbs these rays and allows only longer-wavelength light luminescence of the drug (it is installed in a tube or on the eyepiece of the microscope). As light sources serve mercury-quartz lamps high pressure (type DRP) or incandescent lamp spot type. The brightly-colored glow of objects on a dark background provides high contrast. Opto-mechanical industry produces special luminescent microscopes and private lightboxes.
Only a few biologically important substances have expressed their own luminescence in the visible spectrum. These include certain pigments (chlorophyll, porphyrins, lipochrome), vitamins a and B2, alkaloids (berberine, quinine and other), antibiotics (tetracycline and others), chemotherapeutic, and toxic substances. The penetration of these substances in the organs and cells, their distribution and transformation can be traced with the help of in vivo fluorescence microscopy. More often in fluorescence microscopy use fluorescent "color" special substances (fluorochrome), selectively giving thin structures of cells and tissues ability luminesce (fluorescent cytochemistry). For example, the fluorochrome acridine orange is used to contrast nuclear structures, identification of nucleic acid mucopolysaccharides, for the detection of microbes and large viruses to cytodiagnostics, including recognition in smears of cancer cells; auramine 00 serves for detection of acid-fast bacilli (tuberculosis, leprosy), Rickettsia, and some viruses; pemulen - to fluoropolimerowe elementary Taurus viruses and distinguish between living and dead cells; phosphine WC, blue Nile and benzpyrene - for localization of lipids in the cell.
Of particular importance in fluorescence microscopy is attached fluorescent-immunological methods [Kuna (A. Coons) with al., 1942, 1950], based on the use of fluorescent labeled specific serums (antibodies). The tap is often the fluorochrome isothiocyanate of fluorescein. The resulting complex "antibody-fluorochrome" allows you to quickly detect, identify and locate even trace amounts corresponding antigens, including viruses, Rickettsia, bacteria on the background of foreign flora and identify specific proteins, enzymes, proteins in cells and tissues. Along with visual observations and photography in L.M. increasingly used objective registration intensity spectra and yield of luminescence.
In the USSR is developing a new kind of L. M. - the so-called ultraviolet L.M., which investigated own UV invisible under normal conditions luminescence objects (E. M Bromberg), subtly reflect their physiological state.
Fluorescence microscopy of cells and tissues. With fluorescent microscopy, you can examine the primary (the tissues and organs of humans and animals have a vague whitish blue or blue luminescence) and secondary luminescence of cells and tissues. A study of secondary luminescence of the living and the fixed cells and tissues (after their "color" fluorochrome) is widespread. In the study of living cells fluorescent substances used in very small quantities that do not cause toxic effects. Cytological research L. M. used in the diagnosis of malignant tumors in the scrapings, punctata, sputum, lavage. This method allows to receive bright colored product, in which abnormal cells are highlighted in a bright glow, shades of color and structure. L. M. applies and histochemistry. The use of acridine orange allows to detect nucleic acids, with DNA gives green, and RNA - red fluorescence. The same fluorochrome in non-fixed sections helps to identify mucopolysaccharides, and in the modification of this method is the mucines. Phosphine 3R, rhodamine, benzpyrene and other identified in sections lipids.
Cm. also Microscopy.