MICROSPECTRAL ANALYSIS — the spectral analysis of microobjects, the sizes to-rykh do not exceed 0,2 — 200 microns (separate cells of plants or animals, intracellular organoids, single microorganisms, accumulations of cells, and also crystal or amorphous microparticles in the form of powders or various inclusions). M and. it is used for obtaining information on molecular or element structure of microobjects. There are several types of M. and.
Absorbing microspectral analysis is based on existence at molecules of any substance of characteristic strips of absorption in the ultra-violet or visible range of a range — so-called absorption spectrums (see. Spectral analysis ). It is most often used for identification in cells or their organoids nucleinic to - t, proteins, pyridine nucleotides, flavoproteins, carotinoids, tsitokhrom, hemoglobin and a myoglobin in different forms, a chlorophyll and other substances having characteristic absorption spectrums.
Registration of absorption spectrums is carried out by means of microspectrophotometers.
In the USSR MUF-5, MLI-2 microspectrophotometers, etc. are issued. More MARCH universal microspectrophotometer, allowing to carry out spektrofotometrichesky is developed (see. Spektrofotometriya ) and spektropolyarimetrichesky (see. Polarimetry ) researches of a microobject, including also registration of the first derivative of a range.
The microspectrophotometer contains in the structure a microscope (usually on the ultra-violet and visible range of a range), the monochromator and system of two beam registration with a possibility of record on the recorder.
As an example of the data obtained by means of absorbing M. and., in fig. 1 ranges of easing (i.e. set of absorption spectrums of all substances which are present at this site of a cell, and ranges of their selection light scattering) large (the size apprx. 2 — 2,5 microns) lipofustsinovy granules (pyramidal neurons of a cerebral cortex of cows) indicating presence at structure of lipofustsinovy granules of carotinoids (maxima of absorption of 450 — 480 nanometers in a curve 1) and haemo proteins are given (a maximum of absorption of 420 — 430 nanometers in a curve 2).
The simplified systems of absorbing microspectral analysis are the multichannel microphotometers measuring the optical density of the site of a cell at the same time in two and narrower spectral intervals. Are used as a basis of devices for the automatic analysis of cell populations (usually blood cells) on previously stained preparations. The Hematrac, Coulter dif-3 and ADC-500 devices intended for automatic (or semi-automatic) the analysis of a leukocytic blood count can be an example of this kind of qualifiers of cells.
Luminescent microspectral analysis is based on registration and the analysis of characteristic luminescent spectrums in the ultra-violet and visible spectral ranges inherent to a number of chemical compounds (see. Luminescence ). As examples of the intracellular connections possessing own luminescence proteins, the recovered pyridine nucleotides, the oxidized flavoproteins, porphyrines, a chlorophyll, vitamins A, In, etc. can be called, luminescent spectrums to-rykh have the characteristic arrangement of maxima of radiation allowing to identify these connections in the respective sites of living cell or in its organoids. A specific luminescence, in particular, mitochondrions, chlorolayers, lipofustsinovy granules etc. possess.
In addition to own luminescence of intracellular connections, pretreatment of cells is often used by the substances causing a luminescence of the intracellular components connecting them. The simplest examples of such connections are flyuorokhroma acridic orange, flyuorestsein and its derivatives, etidium bromide, an auramine (see. Flyuorokhroma ).
The device for luminescent M. and. represents a combination luminescent microscope (see), spektroanaliziruyushchy system and chart recorder. In practice various mikrospektroflyuorimetra are used. Can be an example developed in Ying-those biological physics of Academy of Sciences of the USSR mikrospektroflyuorimetr, in Krom the standard nodes released by the industry of the USSR (fig. 2) are used. The basis of the device is made by the luminescent microscope connected to a spectral photometric nozzle, at the exit the cut is established photo multiplier (see). The output signal of the photo multiplier arrives on the recorder. Excitement of a luminescence is carried out by the superstable mercury arc lamp fed by a source of the stabilized tension.
Important feature of a luminescent mikrospektroflyuorimetriya is the possibility of use of the «probe» system of observation representing a mirror with an opening — the photometric probe — in the center. This mirror is established in a path of rays of a luminescent microscope in the plane of the image and therefore, having combined the probe with the interesting site of the luminescent image of drug, it is possible to observe at the same time both the luminescent image and the photometric probe (fig. 3).
Thus, there is an opportunity simultaneous morfol, and a spectral analysis of a cell (or its parts).
Other feature of a mikrospektroflyuorimetr is use of excitement of a luminescence the radiation «falling» on an object. At the same time the same microlens is applied also to excitement of a luminescence, and to registration of luminescent radiation of a microobject. There was an opportunity to investigate single cells not only on the smears or thin sections (frozen or paraffin), but also directly on a surface of body — in situ.
For identification of intracellular substances on characteristic luminescent spectrums use also overseeing by change of spectral characteristics of an object in time at action for an object of these or that chemical or physical. factors.
Laser issue microspectral analysis is based on use of characteristic ranges of optical radiation of atoms of various chemical elements excited by electric discharge. Is a version issue spectral analysis (see) it is also intended for qualitative and quantitative test of element structure of microsamples with a diameter within 20 — 40 microns. Installation for this type of M. and. includes a microscope, a laser source of excitement, the generator of a spark and the spectrograph. By means of a microscope find the necessary site of a microobject, the impulse of laser radiation evaporates and partially excites substance in the explored microsite of drug. The electric spark in addition excites the characteristic radiation of atoms of various elements which were a part of the evaporated site of a microobject. This radiation is analyzed by the spectrograph, and the radiation spectrum is fixed on a photographic plate. The analysis of a range allows to establish element structure of the evaporated site of a microobject. Apply the MSL-2 installation (USSR) and a laser microspectrum analyzer of LMA-10 (GDR) to laser issue microspectroscopy.
X-ray luminescent microspectral analysis — uses characteristic ranges of x-ray emission of the atoms which are a part of the studied microsite of a cell or fabric for quantitative and qualitative test of its element structure. Is a kind of a X-ray spectral analysis. Initiation of x-ray emission of atoms is made by a narrow bunch of the accelerated electrons. Installation for X-ray fluorescence analysis consists of the supermicroscope connected to a spectrometer of x-ray emission. The sizes of the analyzed site in practice make 1 micron and more. The method is applied in biology and medicine to identification of distribution of elements in various intracellular organoids, cells and fabrics.
Microspectral analysis in the medicolegal relation. In court. - the medical practician M. and. it is applied to establishment of availability of blood by transfer of hemoglobin in its derivatives. In court. - medical laboratories the method of the microspectral absorbing analysis (see above), is more often used by M. and. it is especially effective at trace amounts of the studied material.
During the carrying out a research scraping from the analyzed object is placed on a slide plate, process 1 — 2 drops 33% of solution of alkali, and then a reducer and covered with cover glass. At the same time there is an education hemochromogen (see). Also other methods of formation of hemochromogen, including phenolic reaction are offered. For this purpose on a slide plate place particles of the studied substance and phenol in the ratio 1: 10, add any salt of iron (or metal iron), heat on a flame of a torch, then add 50% solution of hydrazine hydrate in glycerin. Phenolic reaction is applied most often at a research of «old» spots. In the prepared drug under a microscope choose the site suitable on color. Then to the place of an eyepiece insert a nozzle (microspectroscope). Existence of a range of hemochromogen forms the basis for a conclusion about presence at a spot of blood. At a negative take it is recommended to increase time of observation, to choose other sites of drug for a research or to prepare it again. The microspectral research of drug is made also by transfer of the blood rest in haematoporphyrin (see. Gematoporfirinovaya test ).
For production of such drug the particle of the studied substance (scraping, fiber of fabric) is placed on a slide plate, add 2 — 3 drops konts. a chamois to - you also subject to microscopy, finding the site painted in red-violet, lilac or gray-green color. Then the chosen site is subjected to microspectral analysis. On a characteristic arrangement of h establish to features of strips of absorption existence of a range of haematoporphyrin in acid solution; at a negative take the research is repeated. Negative repeated researches demonstrate that blood in a spot is absent (unless blood in a spot was affected by high temperature — to degree of a carbonization). In doubtful cases recommend a research in UV rays with use of a special luminescent microscope or luminescent lighters that allows to reveal the sites containing haematoporphyrin on their purple-red luminescence on drug; in these sites by means of M. and. availability of haematoporphyrin is investigated. For detection of very trace amounts of blood the microspectrographic method of a research allowing to establish existence of absorbing properties of hemochromogen at a research of particles to dia is offered. apprx. 5 — 10 microns, and a range of haematoporphyrin — at particles to dia, to 50 microns.
Bibliography: L. S.'s agroskin and Father yan G. V. Cytophotometry, L., 1977; Barenboim G. M., D about m and nanosecond to and y A. N. and Turove'Rov of K. K. Lyuminestsention of biopolymers and cells, M. — L., 1966; Bronnikova M. A. and and r to and in and A. S. Technique and technology of forensic medical examination of material evidences, M., 1963; Karnaukhov V. N. Fluorescence spectral analysis of a cell, M., 1978; T mind and N about in A. K. Bases of forensic medical examination of material evidences, M., 1975; Microprobe analysis as applied to cell and tissues, ed. by T. Hall a. o., L — N. Y., 1974.
V. P. Karnaukhov; V. M. Smolyaninov (court. medical).