LUMINESCENCE - «cold» luminescence; a type of a luminescence, in Krom emission of light is caused by other processes, than at the thermal (temperature) radiation. Depending on the factor causing a luminescence distinguish a photoluminescence, an electroluminescence, piezes about a luminescence, chemiluminescence etc. A special kind of chemiluminescence is the thermoluminescence. It is observed, e.g., if to freeze an object in liquid nitrogen (t ° — 196 °), to irradiate with UF-light and after that to heat. The reactive particles of substance formed at such radiation — free radicals (see) — during the defrosting enter the chemical reactions which are followed by a luminescence. Frequent and eurysynusic type of L in wildlife. — the bioluminescence resulting biochemical, the reactions proceeding in a live organism (see. Biokhemilyuminestsention ).
Studying of character of this or that type of L., its intensity and a range found broad application in a lab., dignity. - a gigabyte. and wedge, practice.
Mechanisms of a luminescence
In stationary conditions molecule (see) possesses the lowest of all possible levels of a potential energy of electrons and is in a so-called ground state (S0). Thermal agitation of atoms in a molecule, rotation of a molecule change a potential energy of its ground state that is characterized by so-called subtotals of a ground state. If the molecule receives from the outside a sufficient portion of energy, then it can turn into excited state: one of electrons from an external orbit moves to energetically more high level (fig. 1). Excited state of a molecule is very unstable. In time about 10 - 13 sec. an electron, having spent a part of energy for thermal fluctuations (the wavy line in fig. 1), passes to energetically lower excited subtotal. At this level the electron can be apprx. 10 - 9 sec. then the molecule can let out quantum (S1 —> S0 + hγ), having passed to one of oscillatory subtotals of the ground unexcited state (S0). L., accompanying such transition, received the name fluorescence (see). After the end of operation of the activator of a luminescence fluorescence stops.
sometimes pass to one of oscillatory subtotals of a so-called triplet state (T1). At the same time in a molecule there are not coupled electrons (see. Electronic paramagnetic resonance ). According to quantum theory (see) transitions <>of the S0-T type are improbable; in light atoms or diatomic molecules they do not take place, in heavy atoms and polyatomic molecules such transitions are possible. Time of life (τ) can make molecules in such state from 10 - 4 to 10 2 — 10 4 sec. From a triplet state the molecule can, having let out a light quantum (T — S0 + hγ), to pass into a ground state of S0. L., caused by such transition, call a phosphorescence. This phenomenon it is possible to observe a long time and upon termination of operation of the activator of a luminescence (afterglow). As transition to a triplet state is followed by loss of a part of energy, the range of a phosphorescence is displaced in more long-wave part of a range, than a range of fluorescence (fig. 2).
In addition to time of life of a molecule in wild spirits (τ), process of L. it is characterized by intensity (J), a photon yield (φ), the provision of a maximum (λmax), a range of L. and other parameters which measurement finds application in a lab. to practice. E.g., for pure substances intensity of L. (I) at excitement by monochromatic light intensity of Jv it is equal:
I = k*Jв*φ (1 — 10:-D),
where k — the constant characterizing sensitivity of a flyuorimetr, D — the optical density of a sample. D = εCl, where ε — a molar absorbtion coefficient of substance, With — molar concentration, l — thickness of a sample. At small the D intensity of L. almost linearly depends on concentration that is widely used in quantitative fluorescence analysis.
L. many substances can be result of contents in them a small amount of impurity. Therefore during the carrying out the analysis it is important to establish to what connection it is inherent to L. (fig. 3). For this purpose measure ranges of excitement of L. (see. Spectral analysis ). Ranges of excitement of L. measure also for detection of processes of migration (transfer) of energy between molecules etc.
At interaction of molecules of the luminescing substance with molecules of solvent and other substances the phenomenon of suppression of L is observed., consisting in reduction of a photon yield of L. Such suppression of L. can be a consequence of formation of not luminescing complex between the luminescing substance and substance quencher — suppression of the I row, at Krom only the photon yield decreases, and time of life of a molecule in wild spirits does not change. If the molecule of the luminescing substance loses a part of energy (due to kinetic collisions with molecules of substance quencher), suppression of the II row takes place, at Krom both the photon yield, and time of life of a molecule in wild spirits decreases.
L. biol, objects can be own (primary) or arise due to addition in the analyzed system of special substances or the chemical modifications which are available (secondary L.).
Own L. simple proteins it is caused by availability of amino acids of tryptophane and tyrosine. The maximum of fluorescence of tryptophane varies from 330 to 350 nanometers depending on localization of tryptophane in a proteinaceous molecule. In complex proteins own L. some coenzymes, napr, f avalanches, the recovered pyridine nucleotides, etc. possess. In particular, the recovered pyridine nucleotides at excitement by UF-light fluoresce in a blue spectral range (440 nanometers) oxidized — do not fluoresce. It allows to investigate molecular mechanisms of work of an electron transport chain in mitochondrions, the whole cells and even in fabrics, to study kinetics biochemical, reactions of in vivo, etc. Mikroflyuorimetricheski can register kinetics of change of maintenance of the recovered pyridine nucleotides and other fluorescent coenzymes in various areas of one cell in vivo when use of other methods is complicated. Own L. B6, E, etc., at many medicines is observed at vitamins A (quinine, griseofulvin, etc.). With its help with very high sensitivity and specificity define cancerogenic carbohydrates — benz (and) pyrene, dibenzanthracene, etc. — in air of the cities, in tobacco smoke, etc. Primary L. use in the diagnostic purposes — for detection of a fungal infection at the person and dermatomycoses at animals on the characteristic flavovirent fluorescence of the affected hair excited by UF-radiation at wavelength to 365 them. On own L. carry out quality control of foodstuff. So, at long-term storage of milk and cream Riboflavinum is oxidized in a lumichrom that is followed by discoloration of fluorescence from flavovirent to blue. The eggs infected with bacteria of the sort Pseudomanas at excitement by UF-light begin to fluoresce intensively (at the expense of a pigment of the pioverdin developed by bacteria).
Many connections which are not possessing own L., begin to luminesce after the corresponding chemical processing. By such way in biol, materials it is possible to determine redoxons, D, V 12 etc., drugs (with sensitivity to 0,02 mkg in test) morphine, heroin, etc. Secondary L. use also in diagnosis of diseases.
During the studying of structure and function biol, membranes, the structural organization of macromolecules of proteins and nucleinic to - t widely use secondary L. the connections called by fluorescent probes. As such probes choose substances, parameters L. which sharply change depending on the characteristic of the environment surrounding them: polarity, viscosity, on
verkhnostny a charge, etc. Probes happen three types: loaded (e.g., 1-anilinonaphthalene-8-sulphonate, 2-toluidinonaftalin-6-sulphonate, etc.), not loaded, but having considerable dipole moment (e.g., 4 dimethylaminochalcone, 3-metoksibenzantron, etc.), not having neither a charge, nor considerable dipole moment (e.g., perylene, methylanthracene, pyrene, Retinolum, etc.). As a rule, as fluorescent probes use molecules which in water almost do not fluoresce, and during the binding with biol, membranes or proteins intensity of their L. increases in tens of times. It was succeeded to determine by such probes, in particular, viscosity of a hydrocarbon part biol, membranes (e.g., it is shown that viscosity of membranes of disks of outside segments of sticks of a retina is equal about 1 pz that corresponds to viscosity η luccu oil). By means of probes study a relative positioning of molecular components and conformational reorganizations biol, membranes and proteins, phase changes. During the studying of structure nucleinic to - t apply acridic orange and other probes. It is shown, e.g., that at most A L. is located with double-helix, native DNA in a green spectral range (530 nanometers) whereas in one-chained DNA and RNA it is displaced to the red area (640 nanometers). Mikroflyuorimetricheski by means of probes analyze DNA directly in cells.
In the medical equipment were widely adopted phosphors (see) — the substances capable to photo, rentgeno-, cathodoluminescences etc. By the chemical nature they can be divided on organic and inorganic. Inorganic phosphors use in fluorescent lamps. At production of x-ray screens apply the tsinkkadmiysulfidny phosphors capable to roentgenoluminescence. Fluorescent probes can be carried to phosphors of the organic nature.
Fluorescence analysis — the method of a research of various objects (including biological) based on observation of their L.
Qualitative and quantitative fluorescence analysis is widely applied at a gigabyte. researches of foodstuff, for identification of some environmental pollution, in experimental hygiene by drawing up evidence-based standards, in judicial examination of living persons, corpses and material evidences. Widely use fluorescence analysis in microbiology and virology.
At medical researches measure as own, primary, fluorescence of objects, and the secondary fluorescence arising at the expense of special dyes — flyuorokhrom (see) luminescent probes. Analysis primary L. bodies and fabrics carry out most often visually by means of points light filters or special devices allowing to investigate a luminescence in various outside cavities and to carry out photographing. In such a way it is possible to determine a microsporia (by a green luminescence of hair), hypodermic hemorrhages (by suppression of fluorescence by hemoglobin), anomalies of pigmentation (by lack of a luminescence of the pigmented skin), seborrheal eczema, a hyperkeratosis and some other a dermatosis (by change of a range and intensity of fluorescence of skin), disturbance of blood circulation in skin rags at a pedicle graft (by lack of a luminescence of skin or the flyuorestsein entered into blood), an early stage of jaundice (at illumination of a mucous membrane of an oral cavity UF-light bilious pigments take a form of dark-brown spots with a brown shade), caries of teeth (on lack of a luminescence of affected areas of tooth).
Use of a flyuoroskopiya for diagnosis of malignant tumors was not repaid because of inconsistency of results yet. It is for this purpose reasonable to use more reliable luminescent tsitol. a method (see. Luminescent microscopy ).
In medical researches apply various flyuorokhroma, especially flyuorestsein. Intensity of a luminescence of this connection is so big that it is found even in very insignificant concentration. In several seconds after intravenous administration of 1 — 2 ml of 10 — 15% of solution of a flyuorestsein the bright green luminescence 'a mucous membrane of an oral cavity, lips, eyes and other body parts appears. Flyuorestsein is harmless and is brought from an organism for 50 — 70 hours Flyuorestseinovuyu use test for definition of a circulation time of blood, for a permeability survey of capillaries of skin and influence on it of medicines, for detection of zatek of cerebrospinal liquid, identification of borders of tumors for the purpose of their removal.
Fluorescence analysis helps to otdifferentsirovat tubercular granulomas from tuberkulezopodobny, found in limf, nodes at survey of pork hulks. In 3 — 5 min. it is possible to establish concentration of protein in the studied samples of milk (instead of 8 — 12 hours by Kyeldal's method), and average discrepancy with Kyeldal's method makes no more than 0,1%.
Broad application was found by flyuorokhroma in a luminescent gistol. researches. Use of luminescent methods in a wedge, and experimental biochemistry allows to increase sharply sensitivity of researches, having brought closer it in certain cases to the level of sensitivity of radio isotope analyses. Flyuorimetrichesky techniques of definition of activity of a number of enzymes, quantitative definition of amino acids, amines, vitamins, coenzymes and their metabolites, proteins, steroids, uric to - you, ammonia, glucose, Oxytetracyclinum, salicylates, ions of Ca2+, Mg2+, purines, pyrimidines, nucleinic to - t, porphyrines and other connections are developed.
For assessment physical. - the chemical processes proceeding in an organism and also as additional diagnostic test the research of chemiluminescence of a blood plasma can be applied. There are data that the superweak luminescence of blood serum decreases at malignant new growths and atherosclerosis and raises at tuberculosis. On intensity of chemiluminescence it is possible to judge the oxidation level, level of free radicals and its deviation from norm that is essential, e.g., at radiation injury.
Fluorescence analysis in hygiene found broad application for definition of damage or falsification of foodstuff, quantitative establishment of contents in them of vitamins and other biologically active agents or harmful impurity (pesticides, etc.). High quality of meat is defined, measuring the luminescence of its protein-free extract caused by presence of the water-soluble substances which are formed at damage of meat. Size L. fresh meat extract — to 30 relative units, from meat with initial signs of damage — from 30 to 50 relative units, from the spoiled meat — more than 50 relative units. High quality of meat is defined also qualitatively — but color of a luminescence. Depending on extent of damage of meat the greenish, bluish and bright red luminescence consistently appears; at initial signs of damage on fat of pork light blue points of a luminescence, on fat of beef — whitish-yellow appear. By means of fluorescence analysis establish also freshness of fish and fish products.
Define presence and concentration at air of production rooms of resinous substances, a benzatren, naphthylamines by fluorescence analysis, boric to - you, Naphthalanums, phthalates, etc., the content in water of oil products, sulfates, acetone, uranium, etc. The spectral and luminescent method of definition of polycyclic aromatic hydrocarbons in tests of water, snow and the soil is developed; benz (and) pyrene — in free air, tobacco smoke, some foodstuff.
Fluorescence analysis in medicolegal researches. High sensitivity of fluorescence analysis and an opportunity with its help to identify many substances on character of a luminescence, and also a number of bodies and fabrics do it by very important method of a research at court. - medical examination. In court. - medical practice during the carrying out fluorescence analysis use any sources of UF-light. The L preventing observation. visible light is eliminated with the corresponding light filters. L., caused by light of a visible part of a range, it is observed by naked eye and it can be recorded on a film.
Also the microscopic fluorescence analysis which is carried out by means of luminescent microscopes is widely applied. For strengthening of weak own luminescence and for identification of not possessing L. structures gistol, drugs process flyuorokhromam (acridic orange, Riboflavinum etc.).
At survey of living persons fluorescence analysis allows to establish a form and the sizes of hypodermic hemorrhages and bruises after disappearance of their external manifestations, a form of the burns which were earlier, prescription of skin hems on color of their L. (a dark-violet luminescence of fresh hems, a blue-violet luminescence of hems of an old origin), intentional administration of various substances (the oil products infected materials) under skin for the purpose of calling of phlegmons etc.
During the opening of corpses it is possible to prove by means of fluorescence analysis reception of medicines and some foodstuff (egg yolk, garlic, strong tea, mushrooms, etc.) on color of a luminescence of a mucous membrane of an oral cavity, gullet and stomach; it is possible to establish approximately age of the dead on intensity and color of a luminescence of cartilaginous tissue (e.g., at a research of the dismembered corpses). Fluorescence analysis helps to establish the term of burial, and also to differentiate on character of a luminescence of a bone of the buried and burned corpses, to accurately define borders of some patol, processes (sclerous, fatty dystrophy, malignant new growths, etc.), to confirm alleged lead poisoning, quinine and other substances (at plumbisms in extracts from urine very much the reddish luminescence of porphyrines early is defined, at poisoning with quinacrine, quinine and other substances possessing bright L., the corresponding luminescence of internals is noted).
Investigating clothes and other material evidences by means of fluorescence analysis, it is possible to establish inlet opening at a gunshot wound, and at multiple damages — their sequence. At various transport incidents fluorescence analysis helps to determine on clothes and a body of the victim a form and an arrangement of pollution by lubricating oils and on them to establish character of the damaging subject and the mutual provision of transport and the victim at the time of incident. It is possible to establish approximately existence on material evidences of blood (on brilliant orange to a luminescence of haematoporphyrin after a denaturation of hemoglobin a chamois to - that), allocations from a nose, saliva, sperm, urine, to define a sex of cellular elements on tools of a crime and other objects (by identification of Y - lame catfishes at luminescent microscopy of objects). Presence of blood on material evidences can also be proved by a spectral research L. haematoporphyrin. In court. - to medical practician methods intensively are implemented immunofluorescence (see).
Methods of the analysis and devices
the Methods of the analysis and devices based on the phenomenon of L., apply to obtaining information on structure and physical. - chemical properties of biological and other objects, measurements of concentration of substances, and also for a research of change of a state or concentration of substances in such objects. For this purpose use more often fluorescence (see), photoluminescence less often phosphorescence (see).
At the solution of a number of practical tasks in biology and medicine (e.g., definition of concentration of these or those substances in biol, liquids, their studying immunol, characteristics, gistol, indicators etc.) it is possible to be limited to measurement of intensity of fluorescence in a certain spectral region at a certain wavelength of exciting light.
Basic elements of fluorescent devices — a light source, the camera (ditch) for the analyzed object, the receiver of light and the chart recorder. In flyuorimetra usually use light sources with a line spectrum (more often mercury-quartz lamps), and in spektroflyuorimetra — with a continuous spectrum (xenon lamps). The short-wave border of initiation of fluorescence of a xenon lamp is in area of 240 nanometers, long-wave border in the area 600 — 800 nanometers. For allocation of certain sites of a radiation spectrum of a lamp (monochromatization) in flyuorimetra use light filters from stained optical glass or interferential light filters. For monochromatization of exciting light in spektroflyuorimetra use monochromators with diffraction gratings with resolving power of 10 — 12 nanometers more often. Range of operation of the monochromator of fluorescence is defined by spectral response of a photo the receiver and and purpose of the device. Working range of the monochromators used in the majority of models of spektroflyuorimetr, from 280 to 700 nanometers.
Cameras for the analyzed object represent ditches from glass of special grades, depending on appointment the having various capacity, a design etc. Volume a ditch in devices of 1 — 10 ml. In a number of devices use microditches of 2 mkl. Further improvement a ditch is connected with universal introduction a microditch, drain and flowing a ditch.
The most common receiver of light having high sensitivity are photoelectronic multipliers (see. Photo multipliers ), photo cells, and also the bolometers allowing to measure a radiation energy directly.
Chart recorders represent analog and digital schemes of registration: the micro ampermeters, peryevy recorders printing recorders, etc.
A constructive version are the mikroflyuorimetra and mikrospektroflyuorimetra intended for carrying out fluorescence analysis of microscopic objects. At measurement of ranges automatic correction of a range of fluorescence since the intensity registered by the device is defined not only by physical properties of a sample, but also spectral characteristics of a light source, monochromators and the receiver of light is of great importance. Automatic correction is reached, as a rule, by complication of an optical or electronic circuit of devices.
Studying of L. chromophores in structure biol, molecules and structures makes one of sections of researches in molecular biology. It allows to obtain information on the general structure of molecules and supermolecular structures, on localization in them of chromophores, mobility of certain sites of molecules, structural (conformational) transfomations of molecules etc. E.g., ranges and a photon yield of L. amino acids are various as a part of protein and just in solution; besides, change of protein, being reflected in a microenvironment of amino acids — flyuorofor, leads to change proteinaceous L., owing to what tryptophane and tyrosine are as if the luminescent tags which are built in protein by the nature and informing on conditions in to-rykh they are in a macromolecule. Fluorescent methods are used in the analysis of conformational reorganizations of proteinaceous molecules as a part of enzymatic systems, studying of mechanisms of photosynthetic reactions, the structural organization of membranes and so forth. Apply to determination of content of not fluorescent products flyuorokhroma (see) and fluorescent probes — fluorescent methods of identification of DNA and RNA in cytochemistry, definition of concentration of microorganisms, contents of plasminogen and fibrinogen in a blood plasma and so forth, based on linkng of a flyuorokhrom with molecules and cellular structures.
A lack of fluorescent methods of quantification is the limited selectivity connected with the fact that many substances have the big width of a range of fluorescence. The next ways of increase in their selectivity were outlined: optical or electronic modifications of the existing fluorescent methods, a combination of fluorescent spectroscopy and hromatografichesky methods, a combination of the fluorescent methods and specific biochemical, tests.
At derivative fluorescent spectroscopy measure so-called derivative ranges since in the analysis of mixes in such a way permission of minor components significantly raises. Derivative ranges can be received as an optical way, and by means of special electronic prefixes. This way is suitable, e.g., for identification of tyrosine in the protein containing tyrosine and tryptophane. The method of synchronous scanning is based on simultaneous scanning of both monochromators of a spektroflyuorimetr with the fixed spectral shift between them.
Flyuorimetrichesky scanning thin layer hromatogramm allows to divide pikogramma of substances.
For scanning thin layer hromatogramm in nek-ry cases it is more favorable to use the fosforimetrichesky analysis since many not fluorescent substances phosphoresce (see. Phosphorescence ).
A perspective way of increase in selectivity of fluorescent quantification is the combination of a flyuorimetriya and immunol. techniques, napr, differential coloring of chromosomes by means of fluorescent dye Chinacrinum yperite for the subsequent fluorescent analysis of a karyotype and the method of fluorescent antibodies used at statement highly sensitive immunol, reactions and identification of various antigenic determinants on a surface of cells (see. Immunofluorescence ), etc.
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A. Ya. Potapenko; Yu. V. Novikov (gigabyte.), B. A. Pekkel (fluorescence analysis), V. V. Tomilin (court.); P. P. Lidemang, H. P. Matveeva.