LUMINESCENT MICROSCOPY

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LUMINESCENT MICROSCOPY (Latin lumen, luminis _ light; grech, mikros small + skopeo to consider, investigate; synonym fluorescent microscopy) — the microscopic technique allowing to observe primary or secondary luminescence of the microorganisms, cells, fabrics or separate structures which are their part. Luminescence (see) it is excited by a short-wave (blue-violet) part of visible light or ultraviolet rays with the wavelength close to visible light. Color of a luminescence, i.e. wavelength of emitted light, depends on a chemical structure and on a physicochemical condition of a mikroskopiruyemy object, as causes a possibility of use of L. m for microbiological and cytologic diagnosis, for differentiation of separate components of a cell. Primary luminescence is inherent in a number of biologically active agents, such as aromatic amino acids, porphyrines, a chlorophyll, vitamins A, B2, B1, some antibiotics (tetracycline) and chemotherapeutic substances (quinacrine, Rivanolum). Secondary, or induced, the luminescence results from processing of mikroskopiruyemy objects fluorescent dyes — flyuorokhroma-m. Some of these dyes are diffuzno distributed in cells (e.g., flyuorestsein), others — selectively contact certain structures of a cell or even certain chemical substances. This ability of flyuorokhrom to selective coloring allows to carry out luminescent tsitol. and luminescent and cytochemical researches.

In the history of development of L. m allocate several stages connected with improvement of a technique:

1) proof And. Köhler of a basic possibility of creation of a luminescent microscope; 2) creation in 1911 of a luminescent microscope which was used by the Russian botanist M. S. Tsvet for studying of a luminescence of a chlorophyll of plant cells; 3) use of strongly diluted solutions of the flyuorokhrom which are selectively contacting certain structures of cells [Haytinger (M. of Haitinger, 1933 — 1935)], and first of all acridic orange [Haytinger, S. Strugger, 1940]; 4) development of a method of excitement of a luminescence incident light through a lens of a microscope with use of an interferential svetodelitelny plate (E. M. Brumberg and G. N. Krylova, 1953) and release by the domestic industry of the luminescent microscopes and devices based on this principle (ML-1, ML-2, OI-17); 5) creation of a method immunofluorescence (see), found broad application in microbiology, immunology and other areas medico-biol. researches [Koons (A. N. Goons), 1942, 1950].

In the USSR the significant contribution to development and distribution of L. m in a medico-biol. researches it is made M. N. Meysel.

For carrying out L. m apply either special luminescent microscopes, or the prefixes to usual biol, microscopes allowing to use them for observation of a luminescence of microobjects (see. Microscope ). The device of luminescent microscopes is based on some physical. laws of a luminescence. One of them — Stokes's law, according to Krom at most of a luminescent spectrum is displaced to the long-wave area in relation to a range of exciting light. It allows to use for L. m the principle of the crossed light filters which is that the short-wave light radiation (ultra-violet, blue-violet) exciting a luminescence is allocated with the exciting light filter placed before the lighter of a microscope. After passing of drug, in Krom the luminescence is excited, this light is completely absorbed by the locking light filter passing more long-wave light of a luminescence.

The luminescent microscope is supplied with a powerful source of lighting with a big superficial brightness, the maximum of radiation to-rogo is in short-wave area of a visible range, system of light filters, and also the interferential svetodelitelny plate (or a set of such plates) applied at excitement of a luminescence by incident light. This system of excitement of a luminescence incident light via the opak-illuminator used in domestic luminescent microscopes (and recently and in the luminescent microscopes released by foreign firms), has a number of essential advantages: 1) the interferential svetodelitelny plate with the layers of dielectrics applied on it selectively reflects more than 90% of light exciting a luminescence on drug and almost completely passes more long-wave light of a luminescence that allows to increase brightness of a luminescence; 2) the lens of a microscope serves at the same time as the condenser of lighting system; therefore during the use of high-aperture immersion objectives with big increase illumination of drug and respectively brightness of a luminescence increase in proportion to the fourth degree of an aperture of a lens; 3) the luminescent microscopy can be combined with phase and contrast and interferential during the lighting from below through a substage condenser. Sources of lighting for L. m more often are mercury-quartz lamps of ultrahigh pressure, and also xenon lamps and quartz and halogen filament lamps. As the light filter apply the optical glass painted in weight or use the interferential light filters having the best spectral characteristics.

For excitement of a luminescence at L. m use also optical quantum generators — lasers which radiation has high intensity and monochromaticity. At the same time need for use of exciting light filters disappears.

As for excitement of a luminescence at L. m usually use long-wave ultra-violet, blue-violet, and sometimes and a green spectral range, in a luminescent microscope apply the usual glass optics and usual slide and cover plates which are passing radiation in this part of a range and not possessing own luminescence. The immersion and concluding environments also shall conform to these requirements.

As the concluding environments for drugs buffer solution of glycerin, and also not luminescing polymers can be used (polystyrene, polyvinyl alcohol, etc.).

Along with a visual estimate it is luminescent - the microscopic image apply its microphotography (see. Mikrofotografiya ). Luminescent microphotographing has a number of features. On the one hand, the insufficient brightness of a luminescence demands long exposure, with another — under the influence of exciting light intensity of a luminescence quickly decreases, drugs fade, living cells are damaged, perish and therefore it is impossible to register dynamics of the processes happening in cells. For overcoming these difficulties it is necessary to use photographic materials with high general and selective spectral response, high-aperture lenses, eyepieces with the minimum own increase, however sufficient for transfer of details of an object, low-format photomicrographic attachments. Also processing of drugs the substances reducing fading is possible (hydrochinone etc.).

M. Ya. Korn and M. M. Butslov with sotr. (1968) the equipment and a technique color luminescent microphoto and filmings with use of the electron-optical amplifiers of brightness allowing to reduce exposure and to carry out registration of dynamics of the processes happening in living cells by several orders is developed.

To quantitative registration of intensity of a luminescence of structures of microobjects — tsitoflyuorimetriya — apply preferential photo-electric methods with use of the photoelectronic multiplier (FEM) as a sensitive recorder (see. Photo multipliers ). Use also a method of registration of intensity of a luminescence of cells and their structures in certain sites of a range — a tsitospektroflyuorimetriya. Advantages of a tsitoflyuorimetriya before absorbing cytophotometry — its higher sensitivity, lack of influence of nature of distribution of substance in a cell of a pla to structure of a cell on results of measurements.

One of options of quantitative registration of a luminescence of microobjects is the method of a pulse tsitoflyuorimetriya (a tsitoflyuorimetriya in a flow) and «sortings» of cells according to luminescent characteristics. These methods allow to analyze intensity of a luminescence of tens of thousands of cells a minute and to carry out their division according to character of a luminescence. Among methods of luminescent studying of microobjects the greatest distribution was gained a direct flyuorokhromirovaniye — coloring of a flyuorokhromama and an immunofluorescence.

The domestic industry releases various equipment for L. m. The microscope of ML-2 which is released in several options is most widely used. Also a series of the unified luminescent microscopes Lyumam, and also luminescent lighters with quartz halogen lamps (OI-28, OI-30) is made.

Luminescent microscopes of the Lyumam series: 1 — a working luminescent microscope «Lyumam R-1»; 2 — a research luminescent microscope «Lyumam I-3».

Microscopes of the Lyumam series consist of the unified nodes which various combinations allow to receive three working models (P1 — RZ) and three research (I1 — I3), differing from each other in a complete set and opportunities of use (fig).

Lighters of OI-28 and OI-30 establish on usual biological microscopes; they are intended for illumination of objects from above via the opak-illuminator by light exciting a visible luminescence.

The lighter of OI-30 differs in the fact that contact lenses are included in its package.

L. m widely apply in virology, microbiology, hematology, a wedge, cytodetection (especially in oncology to detection of malignizirovanny cells), in cytogenetics to studying of chromosomes. For this purpose throughout a long time use flyuorokhry acridic orange; apply also flyuorokhroma bromic ethidium (etidium bromide) and iodide propidiya (pripidium iodide), it is preferential for a tsitoflyuorimetriya of DNA, and also luminescent options of reaction of Feylgen. Acridic orange gained distribution in luminescent microscopy of nucleoproteids thanks to the fact that the complexes formed by it flyuorokhromy from double-helix DNA possess a green luminescence, and complexes from RNA and one-spiral DNA — a red luminescence. Also luminescent and cytochemical methods of identification of proteins and lipids are known. For high-quality and quantitative studying of localization of proteins in cells use protsionovy dyes, flyuoreskamin, and lipids — 3,4 benzpyrene, ZR phosphine, etc. Tetracycline and its derivatives apply to luminescent and microscopic studying of a bone tissue and some changes in cells at a malignancy.

L. m in combination with a direct flyuorokhromirovaniye of the fixed drugs use at bakterioskopichesky diagnosis for detection of acid resisting mycobacteria, gonokokk, causative agents of diphtheria, causative agents of malaria in blood smears, etc. Advantages of this method consist in its higher sensitivity in comparison with usual methods of coloring (e.g., colourings across Tsil — to Nelsen). Flyuorokhromirovaniye is applied also in a sanitary bakteriol. researches for detection and calculation of microorganisms in water and the soil. One of methods of lyuminestsentnomikroskopichesky diagnosis is detection of microcolonies on membrane filters after a short-term podrashchivaniye and a flyuorokhromirovaniye.

In 1940 Shtrugger suggested to use acridic orange for differentiation of live and dead bacteria, however the subsequent researches showed insufficient reliability of this method. In this regard for definition of viability of cells (in particular, at impact on them of cytotoxic factors) use diacetate of a flyuorestsein or its combination to bromic ethidium. Not luminescing ether of a flyuorestsein is split by esterases of a viable cell with release of the flyuorestsein which is brightly luminescing green, and bromic ethidium causes a red luminescence only of dead cells.

For studying physical. - chemical conditions of membranes of cells use so-called hydrophobic fluorescent tests. For this purpose cells process substances which do not luminesce in solution, but begin to luminesce, contacting hydrophobic sites of cell membranes, and intensity and color of a luminescence depend on a chemical structure and physical. - chemical conditions of structures, with to-rymi are connected these flyuorokhroma. To one of the most widespread substances such is 1-anilino-S-naphthalene-sulfonic to - that (1,8 AHC).

By method of studying physical. - chemical conditions of macromolecules, with to-rymi are connected flyuorokhroma in various cellular structures, also polarization luminescent microscopy is.

Fig. 1 — 3. Microphotos it is intravital flyuorokhromirovanny peritoneal macrophages in cellular culture (processing by acridic orange): fig. 1 — not englobing macrophage (a kernel — green, cytoplasmatic granules lysosomes — red); fig. 2 — the englobing macrophage in whom the fagotsitirovanny bacteria shining in red color, a kernel — green are visible; fig. 3 — the englobing macrophage in whom the fagotsitirovanny bacteria shining in red color, a kernel and kernels are visible — green (the microphoto is made from the screen of the electron-optical amplifier of brightness, exposure 0,5 sec.). Fig. 4 — 8. Microphotos of the cellular cultures infected with viruses (processing by acridic orange): fig. 4 — the culture of cells of VPK-21 infected with a virus of the Crimean hemorrhagic fever (the RNA-containing cytoplasmic inclusions shine in red color); fig. 5 — the same culture after processing by ribonuclease (the red luminescence of the RNA-containing structures of a cell disappeared, contents of virus inclusions are steady against effect of enzyme); fig. 6 — a red luminescence of the RNA-containing inclusions in cultural cells of the embryos of the Syrian hamster infected with Dkhori's arbovirus; fig. 7 — a weak luminescence of intranuclear inclusions in cultural cells of the embryos of the Syrian hamster infected with Dkhori's virus; fig. 8 — cytopathic changes in primary culture of cells of kidneys of the green monkey infected with a virus of an entsefalomiokardit. Fig. 9. The microphoto of culture of the cells of amnion of the person infected with a virus of measles; zones of localization of a viral antigen in structures of a symplast shine in green color; indirect method of fluorescent antibodies, marked FITTs.

Essential advantage of L. m before other methods of microscopic examination — an opportunity intravital (tsvetn. fig. 1 — 3) and a supravital flyuorokhromirovaniye with use of very low low-toxic concentrations of flyuorokhrom. At the same time various flyuorokhroma can contact different structures of cells. Acridic orange, e.g., collects in lysosomes of living cell, and they begin to luminesce red light. The same luminescence is got by fagotsitirovanny bacteria in phagosomas. Tetracycline contacts mitochondrions of cells or their analogs at bacteria and luminesces flavovirent light, and intensity of a luminescence (amount of the communicated tetracycline) depends on sensitivity of bacteria to this antibiotic.

Perhaps also flyuorokhromirovany cells and in situ fabrics for their studying by means of contact L. m.

The luminescent microscopy of viruses is applied at a lab. to diagnosis of viral diseases for identification of a viral antigen in cells, studying of chemical structure of intracellular virus inclusions, definitions of relative concentration of viral antigens and nucleinic to - t on intensity of specific fluorescence etc. Depending on research objectives as objects use smears, prints, scrapings of fabrics or drugs of cellular cultures.

Two methods L are most widespread in modern virology. m: 1) identification and differentiation nucleinic to - t of viruses in the infected cells and the purified virus suspensions by means of flyuorokhrom of aminoacridines; 2) identification of viral antigens by means of an immunofluorescence.

From aminoacridines apply acridic orange more often, giving to molecules double-helix nucleinic to - t (as a rule, DNA) green, and one-spiral nucleinic to - t (as a rule, RNA) ruby-red fluorescence (tsvetn. fig. 4 — 8). The method is applicable both on native drugs, and after fixing in acetone, liquid Carnoy, etc. Results of coloring considerably depend on concentration (usually 1: 10 000 — 1: 100 000) and pH of a flyuorokhrom.

The method of an immunofluorescence is used for identification of viruses in cells, by studying of dynamics of accumulation of a viral antigen in a cell, definitions of intracellular localization of accumulations of a virus, clarification of the nature of virus inclusions, studying of an antigenic structure of viruses, differentiations of closely related viruses, titration of viruses in cellular cultures, identifications of antigens of oncogenic viruses in fabrics and cells, studying of a pathogeny of viral diseases, control of virus contamination of cellular cultures, researches hron, viral infections etc. The method is applicable both in a straight line, and in indirect modifications. For the correct interpretation of results of a research it is important to consider concentration and purity of the applied antibodies and their conjugates about flyuorokhromam, terms and temperature of fixing of drugs (usually acetone) and processings by their antibodies. Most often for a tag of antibodies use the isothiocyanate of a flyuorestsein (FITTs) giving a characteristic green luminescence (tsvetn. fig. 9), and the sulphochloride of B 200 lissamin-rhodamine shining orange-red light.

The method of an immunofluorescence allows also identification in cells and fabrics of antibodies to viral antigens.

L. m of viruses apply to diagnosis of such infections as smallpox, herpes, epidemic parotitis, etc. Special value has L. m in express diagnosis of respiratory viral infections when prints from a mucous membrane of a nose of patients (rinotsitogramma) process by means of the methods called above for the purpose of identification of antigens or definition of type nucleinic to - t. Thus carry out differential diagnosis between the infections caused by influenza viruses of A2 or B, a parainfluenza, adenoviruses, a respiratory and syncytial virus or combinations of the called viruses. Also diagnosis by L is possible. m of the cellular cultures infected with material of patients. The final diagnosis in all cases is made on a combination of these L. m with results virusol, and serol, researches of patients.

Luminescent microscopy of bodies and fabrics — one of modern methods of a research applied in normal and patol, histology. Main advantages of L. m are high sensitivity (more sensitively usual cyto - and gistokhy, methods not less than by 1000 times), ease of quantitative measurement of maintenance of various chemical components of fabric and cells, availability of the equipment. For L. the m of bodies and fabrics use primary and secondary luminescence. Primary luminescence (the luminescent luminescence arising without pretreatment of drugs) with sufficient intensity some substances which are a part of cells and fabrics possess: vitamins (hepatoflavin gives a flavovirent luminescence, oryzamin in alkaline solution passes in trikhry and gives a blue luminescence, carotene luminesces flavovirent light, vitamin A at radiation in the UF-range has a blue-white luminescence), hormones (estrogen, adrenaline give a flavovirent luminescence, serotonin, noradrenaline during the processing of drugs in couples the concentrated chamois to - you have a yellow luminescence), lipopigments (lipofuscin gives a red luminescence, tseroid — bluish), etc. The principle of primary luminescence is the basis for cytochemical, quantitative studying of maintenance of various components of cells (first of all, proteins) by means of a method of a luminescence in UV rays.

The secondary luminescence of bodies and fabrics is reached by means of processing of drugs flyuorokhromam (see). Acridic orange is applied to diagnosis of cancer in tsitol, and gistol, drugs. The same dye is used for determination of early terms of a myocardial infarction (sites of ischemia have a greenish-yellow luminescence). Korifosfin and acridic orange apply to identification of acid mucopolysaccharides. Such flyuorokhroma as caffeine 5 and rhodamine, can be used for definition of a glycogen. Phosphine 3P is applied to definition of lipids, with the same purpose use solution 3,4 of benzpyrene in saturated solution of caffeine (lipids have a bluish-white luminescence). Thioflavin T. S. paints amyloid (a green luminescence) therefore it is widely applied to diagnosis of an amyloidosis of internals. In alcohol define calcium in fabrics (a green luminescence) by Morin's solution. During the processing of drugs solution of solochrome black possible to reveal aluminum (a yellow-orange luminescence). In lungs define surfactant (an orange luminescence) by rhodamine 6Zh.

By means of a method of an immunofluorescence it is possible to reveal hormones, antigens and antibodies (tsvetn. fig. 9), various products of exchange to identify gistogenetichesk unripe tumors, various inf. diseases, etc. Development of immunochemistry expanded possibilities of this method even more. There was an opportunity to define by artificial haptens nonprotein substances in fabrics and cells.

See also Microscopic methods of a research .



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M. Ya. Korn; V. A. Varshavsky (stalemate. An.), I. E. Hesin (vir.).

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