The Fluorescence microscopy

Fluorescence microscopy

Luminescent microscopy is an optical examination of microobjects stained with special dyes (fluorochromes) that emit glow when exposed to ultraviolet rays. For luminescent microscopy, special optical devices and microscopes are used, the main part of which is a source of ultraviolet rays and a system of filters to it.

Fluorochromes, as a rule, fluoresce differently depending on the chemical composition of the structures with which they interact. Some of them have an affinity for certain cellular structures. For example, acridine orange dye stains cell nucleoproteins, auramine - a wax-like substance contained in mycobacteria. Some microobjects do not require preliminary staining with fluorochromes and are studied with fluorescent microscopy without staining. See also Luminescence Analysis, Luminescence.

Luminescent microscopy (fluorescence microscopy) is a special type of microscopy based on the use of own (primary) or induced (secondary) photoluminescence of microscopic objects. The visible luminescence (see) of the drug is excited either by blue-violet light or by ultraviolet rays.


The luminescent microscope is basically a conventional biological microscope equipped with two light filters: one passes only exciting blue or ultraviolet rays (it is placed in front of a light source), the other absorbs these rays and transmits only the longer wavelength luminescence light of the drug (it is installed in a tube or on an eyepiece microscope). The sources of light are mercury quartz lamps of ultrahigh pressure (type DRP1) or incandescent lamps of point type. The bright color glow of objects on a dark background provides a high contrast. Optical-mechanical industry produces special fluorescent microscopes and individual illuminators.

Only a few biologically significant substances have a pronounced intrinsic luminescence in the visible region of the spectrum. These include some pigments (chlorophyll, porphyrins, lipochromes), vitamins A and B2, alkaloids (berberine, quinine, etc.), antibiotics (tetracyclines, etc.), chemotherapeutic and toxic substances. The penetration of these substances into organs and cells, their distribution and transformations can be traced by means of intravital fluorescence microscopy. More often in luminescent microscopy luminescent "coloring" is used with special substances (fluorochromes), the ability to luminesce (luminescent cytochemistry), which gives thin structures of the cell and tissues. So, for example, acridine orange is used for contrasting nuclear structures, detecting nucleic acids, mucopolysaccharides, for detecting microbes and large viruses, for cytodiagnosis, including recognition in smears of cancer cells; auramine 00 serves to identify acid-fast bacteria (tuberculosis, leprosy), rickettsia and some viruses; primuline - for fluorochroming the elementary bodies of viruses and distinguishing between living and dead cells; phosphin ZK, Nile blue and benzpyrene - for the localization of lipids in cells.


Luminescence-immunological methods are of particular importance in luminescent microscopy [A. Coons et al., 1942, 1950], based on the use of luminescent labeled specific sera (antibodies). More often fluorochrome isothiocyanate fluorescein serves as a taps. The complex "antibody-fluorochrome" allows to quickly detect, identify and localize even insignificant amounts of corresponding antigens, including viruses, rickettsia, bacteria against the background of extraneous microflora, and also to identify specific proteins, enzymes, polysaccharides in cells and tissues. Along with visual observations and photography in luminescent microscopy, objective registration of intensity, spectra and luminescence output is increasingly used.

A new type of luminescent microscopy is developing in the USSR-the so-called ultraviolet luminescence microscopy, which uses its own ultraviolet invisible object luminescence (E. M. Brumberg), which reflects subtly the features of their physiological state.

Luminescent microscopy of cells and tissues . With luminescent microscopy, one can study the primary (tissues and organs of man and animals have unsharp whitish, blue or blue luminescence) and secondary luminescence of cells and tissues. The study of the secondary luminescence of living and fixed cells and tissues (after their "coloring" with fluorochromes) has become widespread. In the study of living cells, fluorescent substances are used in very small quantities that do not cause toxic effects. In cytological studies, luminescence microscopy is used in the diagnosis of malignant neoplasms in scrapings, punctats, sputum, washing waters. This method allows you to quickly get a brightly colored preparation, in which atypical cells are distinguished by a bright glow, shades of color and structure. Luminescent microscopy is also used in histochemistry. The use of acridine orange allows the detection of nucleic acids, while DNA gives green, and RNA - red fluorescence. The same fluorochrome in unfixed sections helps to reveal mucopolysaccharides, and when this method is modified, mucins. Phosphine 3R, rhodamine B, benzpyrene, etc. reveal in the sections lipids.

See also Microscopy.