SUBMICROSCOPY — the method of a morphological research of objects by means of a cathode rays allowing to study structure of these objects at the macromolecular and subcellular levels.
After release of the first industrial model of translucent (transmission) supermicroscope (see) E. m passed a big way of development and allowed to move to qualitatively new level of studying of matter. AA. found for m broad application in morphology, microbiology, virology, biochemistry, oncology, medical genetics, immunology. Thanks to E. the m revealed submicroscopic structure of cells, a number of unknown earlier cellular organellas, such as is open lysosomes (see), ribosomes (see), an endoplasmic reticulum, microtubules, a cytoskeleton, the structures specific to separate types of cells (see. Cell ). AA. allowed m popyat many thin mechanisms of development of diseases, including at early stages of their emergence, even before emergence accurate a wedge, symptoms.
AA. the m is more and more widely applied to early diagnosis of diseases, and also to identification of an etiology inf. processes. It is used in oncology for definition of a histogenesis tumors (see) that is important in treatment and the forecast onkol. diseases. In nephrology a research by means of E. the m of the material received at a puncture biop these allows to reveal early morfol. changes of structures of kidneys to diagnose a form glomerulonephritis (see), etc. At E. the m of punctates of a liver is possible to carry out differential diagnosis of hematite (see. Hepatitis ), hepatoses (see) and other diseases of a liver to define activity of process and quite often its etiology.
Researches of a structure of matter at the subcellular and macromolecular levels restrain opportunities of resolving power of supermicroscopes (see. Supermicroscope ). Use E. the m in combination with other methods, e.g. with an autoradiography, histochemical, immunological methods, was caused by emergence of electronic autoradiography , (see), electronic histochemistry (see), immune submicroscopy (electronic immunomorphology), etc. It allowed to expand considerably information obtained by means of E. m to observe structural expression of a course of biochemical processes in a cell that, in turn, confirmed one of the main methodological principles of modern biology — dialectic unity of structure and function.
AA. the m demands special preparation of objects of studying, from a cut possibilities of a method considerably depend. According to research objectives the technique of such preparation can be various. However an indispensable condition at any electronic microscopic examinations is fixing of fabrics or microbes with the maximum preservation of their intravital structure. There are two essentially various ways of fixing — chemical and physical; each of them has various options.
V E. m, as a rule, use chemical fixing by means of the fixers having the stabilizing properties. The fixer, universal for any fabrics, does not exist therefore depending on problems of a specific research use the corresponding fixers. At the choice of chemical fixers proceed from their ability to coagulate proteins (alcohols, acetone, nek-ry to - you, salts of heavy metals, etc.) or to stabilize lipids and gels (osmium tetroxide, glutaric dialdehyde, formalin, potassium bichromate, etc.).
For a research take biopsy material or material from a corpse of the person or animals soon after approach of death. There are optimum terms of capture of various fabrics and cells which are usually estimated for minutes. Than earlier fabric is placed in the fixer, especially reliable data are obtained about intravital structure of cells. Fixers have various speed of penetration into fabric; the possible size of an object of a research depends on it. So, osmium tetroxide and glutaric dialdehyde get into fabric on 0,1 — 0,5 mm approximately for 1 — 1,5 hour that allows to investigate a sample of fabric no more than 1 mm 3 . Time of fixing in chemical fixers averages 1 — 1,5 hour, but for nek-ry fabrics it can be increased to 4 hours or is reduced to 20 — 30 min. Fixing during 1 days is in some cases allowed. The greatest distribution was gained by fixing of material in glutaric dialdehyde with the subsequent dofiksation in osmium tetroxide. Glutaric dialdehyde is better, than osmium tetroxide, fixes proteins, but stabilizes lipids worse, as causes use of both fixers as supplementing each other.
For selective fixing of separate subcellular structures use more specific fixers (potassium permanganate, potassium bichromate, etc.). Quality of fixing substantially depends on pH and the osmotic pressure of the fixing solution. PH 7,2 — 7,4 is optimum that corresponds fiziol. to parameters. Therefore apply buffered solutions (see). Use phosphatic or kakodilatny buffers more often. Fiziol. osmotic pressure is created by addition of osmotically active agents, e.g. sucrose or nek-ry salts.
There are several methods of chemical fixing: perfused when the fixer is entered into a blood flow; fixing on site when the fixer is entered into fabric before its excision: a method of immersion of the excised pieces of fabric in the fixer. For delay of the autolytic processes proceeding in the excised pieces of fabric before their full fixing the last will be out at 2 — 5 °.
After fixing it is necessary to carry out dehydration of fabric. This process shall be rather bystry, gradual and at the same time provide the fullest elimination of water from a sample that is reached by carrying out the fabric which is subject to a research via the battery of alcohols or atseton of the ascending concentration (from 30 to 100%) within 1 hour.
The following important stage of a preparation of material for E. the m is filling (conclusion) of fabrics in embedding mediums for the purpose of receiving the block possessing the optimum combination of hardness and elasticity allowing to prepare thin section of fabric (thickness less than 100 nanometers), through to-ry there can pass the electron beam. Methylmethacrylate and butylmethacrylate were the first filling material. In a crust. time they are almost not applied since are toxic and easily sublimated under a bunch of electrons that leads to the expressed artifacts and pollution of a supermicroscope. Most widely for filling of fabrics use epoxies, generally araldite and epon, often applied jointly. Polyester resinss (vestopat W) and water-soluble filling mixes are less widespread, from to-rykh a thicket use glycolmethacrylate and durkupany. However any embedding medium is not chemically inert and to some extent exerts impact on fabric; it needs to be considered at interpretation of results mikroskopirovannya.
In recent years broad application was found by filling in so-called compounds, i.e. in mix of certain substances: fundamentals (monomer), the hardener giving to the formed polymer durability and hardness, the plasticizer providing elasticity and elasticity of polymer, the initiator dissociating with education of free radicals, the accelerator to-ry, interacting with monomer, releases additional active radicals, and the catalyst promoting the beginning of polyreaction. In practice usually use the compound consisting of a basis, a hardener, the plasticizer and the catalyst a role to-rogo the accelerator or the initiator can play. There is rather large number of ways of impregnation of fabric embedding mediums. Process of impregnation usually proceeds at the room temperature or in the thermostat at t ° 30 — 48 ° within 15 — 48 hour. Then pieces of fabric transfer to the marked gelatin capsules or special forms filled with filling mix. For polymerization of mix of the capsule on 48 hours place in the thermostat at t ° 60 ° or leave during 24 hours at the room temperature, then for 24 hours place them in the thermostat at t ° 48 ° and for 24 hours — at t ° 60 °. As a result of polymerization the block having the corresponding properties is formed.
For receiving ultrathin sections 30 — 50 nanometers thick glass or diamond knives are used. Diamond knives are more long-lasting glass, but because of high cost they were not widely adopted.
To the back side of a knife attach a special tray and a knife establish on ultravolume. In a tray pour 10% solution of alcohol or 10% solution of acetone on a distilled water. Fix the block with fabric in the mobile holder of ultravolume. Cutting of the block is carried out due to progress of a core of the holder, and rise and lowering of the holder of rather trimming blade of a knife are provided with an electronic circuit. Usually in the beginning produce to the so-called polutonena cuts 1 micron thick, studying to-rye, choose the site interesting the researcher. Having oriented appropriately semi-thin section and the block, carry out sharpening of the block so that at top of the formed pyramid there was a necessary site. Then produce ultrathin sections 30 — 50 nanometers thick. From a tray cuts transfer to metal gauzes.
Apply substances with big atomic weight to contrasting of cuts, such as the salts of heavy metals which are intensively disseminating electrons. Jonah Nek-rykh can form bonds with oxygen of these substances and join phosphatic groups nucleinic to - t; others, especially uranilatsetat, in addition, affect as universal dyes proteins; lead contacts complexes of fabric and osmic fixers. Increase contrast and fixers. Carry usually out contrasting of ultrathin sections or combine contrasting of pieces and ultrathin sections of fabric then they are studied in a supermicroscope.
Chemical methods of fixing and filling of material have a number of shortcomings. So, at their use there are chemical changes of macro-molecular structure of cells; during the fixing and dehydration of a cell and fabric lose nek-ry substances; at interaction of fabric, the fixer and an embedding medium localization of intracellular structures can change. Therefore physical methods of preparation of fabric for E are intensively developed. m and especially for electronic histochemistry (see) and cytochemistry (see). The most part of these methods is based on very snap-freezing of pieces of fabric.
During the use of a freeze-etch method — drying of a piece of fabric place in coolants (propane, isopentane or freon) cooled to t ° — 150 °, and in cells exchange processes instantly stop. At the same time in fabric crystals of ice and therefore subcellular structures do not collapse do not manage to be formed, and water passes into a vitreous state. Then in a high vacuum (10 - 6 — 10 - 7 mm of mercury.) there is a sublimation then fabric is filled in with a special way in the frozen methacrylates, araldite or vestopat W. However it is not always possible to freeze quickly enough and evenly fabric and consequently, completely to avoid formation of crystals of ice, to-rye at temperature increase damage intracellular structures. There are also other difficulties leading to emergence of artifacts. Therefore apply a kind of this method more often — freezing — substitution. After freezing the water which passed into a vitreous state is deleted, placing fabric in dehydrators at a low temperature. In these conditions alcohol and acetone influence structure of cells a little.
The method of freeze-fracture (freezing — etching) allows to avoid emergence of chemical reactions during the processing of fabric. Fragments of fabrics freeze in a coolant with a speed exceeding 1000 ° in 1 sec. An object is placed in a vacuum chamber and one way or another split or broken off. Apply a platinouglerodny covering (remark) on a surface of a chip. Then the remark is cleared of an organic residues in solution of a strong oxidizer, washed out in water and placed on a reticulum for a submicroscopy.
For a research of a surface biol. fabrics use replica techniques and otteneniye. The greatest distribution was gained by a method of preparation of remarks by a dusting in a vacuum chamber of carbon on a surface of a sample of fabric. For contrasting of the formed remark on it at an acute angle napylyat electronic and dense substances (e.g., platinum or platino-iridium alloy). At the same time the amount of atoms of metal are much more on that party of contours of a sample, edges are closer to a source of a dusting; at a research in a supermicroscope it looks low-contrast. The opposite surface of contours has few atoms of metal; in a supermicroscope it is contrast and as if shades a low-contrast surface of a contour. A method of an otteneniye length of a shadow and increase allows to calculate height of contours of the studied object since are known favors of a dusting, at Krom photography of a remark in a supermicroscope was made.
For studying of a surface of the isolated cells and fabrics serves scanning (raster) E. m. One of the main conditions of preparation of an object for scanning E. the m is need of preservation of the corresponding surface intention of cells in order to avoid their deformation. The surface of the studied fabric is washed out the balanced isotonic buffering salt solutions or protein-free culture mediums with pH 7,3 — 7,4 which are warmed up to t ° 37 °. Usually apply isotonic solution of glutaric dialdehyde with the subsequent dofiksatsiy osmium tetroxide to fixing. Fabric is dehydrated in alcohols or atsetona, and then dried up freeze-etch methods — drying or transition of a critical point. The last is based on use of such physical phenomenon as emergence under certain conditions a critical state of balance of steam and liquid. At this method fabric turns out in a gaseous fluid (i.e. dried up) what allows to avoid the damaging action of surface intention. For achievement of high degrees of permission. increase conductivity of an object, napyly on it heavy metals — gold, platinum, silver or their alloys. Also the method of ionic bombing in vacuum of a plate of metal is applied by ions of inert gas. The «beaten-out» atoms of metal accumulate on a surface of an object of a research. Apply mechanical, thermal, chemical and other methods to identification of interstitial and intracellular structures.
For E. m of microbes apply similar methods taking into account features of a structure of microbes, their sizes, osmotic pressure, etc. So, special approach is carried out at E. m of viruses. Studying of structure of viruses is complicated because of the small sizes and the weak disseminating ability of virion. At the first stages E. in m of viruses this difficulty was overcome by an otteneniye of particles at evaporation of heavy metals in vacuum. Up to the end of the 50th of 20 century the technique of an otteneniye of virus particles was the main during the studying of viruses in suspensions. More often for this technique used uranium-238, platinum, palladium or alloys of platinum with palladium. The greatest distribution was gained by alloy of platinum with palladium concerning 4:1. Knowing the set corner of an otteneniye on length of the formed shadow, determine height of a virus particle and its diameter. Otteneniye metals of the studied object at a combination to cryogenic techniques (freezing — drying) allows to obtain important information during the studying of structure of virion of isometric viruses.
The technique of negative contrasting of viruses by means of volframofosforny was widely adopted to - you are (Hzpw12o40), edges at alkalinize caustic potassium or caustic sodium (from <pH2,0 value to pH 7,0) changes and after putting drug on a virus creates a zone of high dispersion of electrons therefore come to light morfol. symptoms of a virus.
Development of knowledge of structure of virion was promoted by cryogenic techniques. One of the simplest options of such techniques consists in the following: grids with a substrate and the virus which is on it after putting the contrasting solution place in liquefied pro-shares (t ° — 150 °) or the overcooled nitrogen (t °< — 200 °); further drying of a sample at t ° — 100 ° in a high vacuum promotes preservation of the three-dimensional organization of virion. The exception in these conditions of the deforming role of surface tension forces of water led to review of the point of view that the form of virion at lipidosoderzhashchy viruses has high lability. More difficult cryogenic techniques applied in a submicroscopy (e.g., freeze-fracture), also made a noticeable contribution to development of ideas of structure of the virions which especially have a lipoproteidny cover.
The structure of a genome of viruses is studied by means of modified in 1959 by Kleynshmidt and Lang (A. Kleinschmidt, D. Lang) techniques of an otteneniye of linear macromolecules heavy metals, to-rye evaporate from V-shaped cathodes at an angle in 10 — 5 °.
The condition allowing to study nucleinic to - you, and especially one-filamentous (cross sectional dimension of 1 — 1,1 nanometer) at are their linkng with the main proteins since at the same time the formed nucleoproteid has diameter to 18 nanometers in two-filamentous structures and to 15 nanometers — in one-filamentous. As such protein use cytochrome C more often. Placed on a substrate nucleinic to - that in a proteinaceous cover has the most fancy contour, and an opportunity to see it throughout is feasible only if during an otteneniye metals make at least one turn of a grid with an object for a dusting on 360 °. The best results are achieved at a long otteneniye (up to 10 min.) alloy of platinum with palladium and repeated turning of the raised dust grid on the rotating little table.
Numerous modifications of a technique of Kleynshmidt and Lang allow to obtain data not only on length of a genome, but also to study degree of a homology of a genome of various viruses, to localize an insert of this or that gene as a part of hybrid molecules, to investigate hybrid molecules nucleinic to - t.
Extensive information on a morphogenesis of viruses is obtained by means of ultrathin sections of viruses. Technology of receiving ultrathin sections of viruses does not differ from standard.
In recent years specific weight E increases. m as an express method for diagnosis of viral infections. The role of the method of an immune submicroscopy allowing to establish patrimonial accessory of a virus is especially big.
Immune E. m played a crucial role at the first investigation phases of infectious hepatitis (hepatitis A), and also a virus gastroenteritis and made an essential contribution to studying of hepatitis B.
Theoretical bases of an immunomorphology at the svetooptichesky level are developed in 1942 by Koons (A. N. Coons) with sotr., to-rye for the first time showed that it is possible to enter nek-ry substances into a molecule of an antibody, without breaking significantly its specificity (see. Immunofluorescence ). Development of this idea allowed Zingeru (s. J. Singer) to develop in 1959 immunomorfol. the method at the electronic and microscopic level based on use of antibodies, marked by ferritin. Ferritin — protein with the high content of iron, in Krom atoms of metal are organized in 4 subunits located closely to each other that provides high dispersion of electrons at E. m and accurate identification of a molecule. Conjugation of protein with an antibody is possible by means of various bifunctional agents, the greatest distribution among to-rykh was gained 2, 4-toluyendiizotsianat and ksilenmetadiizotsianat. The immune submicroscopy by means of antibodies, marked ferritin, is effective for identification of extracellular antigens of microbes in the infected fabrics, including viral antigens on a surface of a cell, and also surface antigens of a cell. At the same time this technique in some cases is not effective, e.g. if it is necessary to investigate intracellular antigens, antigens of microbes in a cell that takes place, first of all, at viral infections. It is caused by the fact that the molecular weight (weight) of antibodies, marked ferritin, apprx. 800 000 and therefore their passing through a plasma membrane of a cell is impossible, and antibodies, marked ferritin, got through it by endocytosis, do not come into contact with a specific antigen. Therefore the ground mass of researches connected with identification of intracellular antigens by means of antibodies, marked ferritin is executed at destruction of a plasma membrane by freezing — thawing or processing by a complement. Replacement of ferritin with the low-molecular weight compounds containing mercury, iodine did not find broad application because of low specificity of a method.
Since the end of the 60th of 20 century in immunology the immunoperoksidazny technique E is applied. m for detection of antigens inside and on a surface of cells. The peroxidase of horse-radish used for a tag of antibodies allows to gain the best effect in comparison with other enzymes (phosphatases, nek-ry oxidases) thanks to lower molecular weight (apprx. 40 000) and resistance to various gistol. to procedures. Antibodies, marked peroxidase, get into a cell and contact homologous antigen. Konjyugirovany antibodies with enzyme it is carried out by a number of bifunctional agents, among to-rykh the most available glutaric dialdehyde is. After there was a communication of an antibody with the corresponding antigen, localization of enzyme comes to light after its contact with the corresponding substrate — benzidine, α-naphthol, mix of α-naphthol with a r-phenylenediamine (nadi-reagent). In the course of interaction of enzyme with substrate in the presence of hydrogen peroxide the product with more expressed disseminating ability of electrons in comparison with surrounding structures is formed.
The Immunoperoksidazny technique has direct and indirect options of modification, and with their help important information both in cytology is obtained, and at virologic researches. However and this approach is not deprived of shortcomings, among to-rykh the most essential massiveness of the formed product in the place of localization of enzyme and as a result of it small resolving power is. Proceeding from it, in 1971 Mr. E. Kurstak recommended to use a conjugate without the subsequent introduction of substrate since enzyme contains iron and after standard coloring of cuts salts of heavy metals (uranium and lead) well comes to light in the zones containing homologous antigen.
The technique immune E is most effective. m of the viruses processed by immune serum. At the same time antibodies agglutinate virions that allows to identify viruses in suspensions with low concentration of viruses. In the elementary option mix virus suspension with homologous serum. The procedure provides initial incubation to a sma of the SI at t 37 ° within 1 hour, and then during 8 — 12 hours at t 4 °. For sedimentation of the formed units, as a rule, there is enough centrifuging at 10 000 rpm. The deposit is parted in the minimum quantity of a distilled water, suspension is applied on a substrate and carry out contrasting by means of 1 — 2% phosphatotungstic to - you.
In other modification immune E. m of virus suspensions exclude centrifuging in the course of preparation of drug. In this case the grid with a positively charged substrate is placed on a drop of immune serum or immunoglobulin for 15 — 30 min. at the room temperature, excess of serum or immunoglobulin is deleted, the grid is washed out buffer solution (pH 7,2), and after that placed on a drop of virus suspension for 30 — 60 min. at the room temperature. Contrasting of drug is carried out any of the drugs-kontrasterov applied to these purposes. Immune E. the m by 100 times increases sensitivity of an electronic and microscopic method at a research of virus suspensions that is important during the studying of material with a low titre of a virus.
More and more widely in immune E. the m is used more difficult, but at the same time and more sensitive modification, at a cut originally on a substrate protein A (from a cell wall of Staphylococcus aureus), then immunoglobulin G or immune serum and only then virus suspension is adsorbed.
Bibliography: Bogolepov N. N. Methods of electronic microscopic examination of a brain, M., 1976, bibliogr.; Gayer G. An electronic histochemistry, the lane with it., M., 1974; The Histochemistry and a submicroscopy in clinical and experimental oncology, under the editorship of N. A. Krayevsky, etc., M., 1975; Goldki L. S. Bases of the histologic equipment of a submicroscopy, M., 1963, bibliogr.; Gouldsten Dzh., etc. A practical raster submicroscopy, the lane with English, M., 1978, bibliogr.; Zhdanov V. M., Azadov N. B. and Kulberg A. Ya. Marking of antibodies rtutnoorganichesky connection, Vopr. virusol., No. 10, page 110, 1962; Gunner N. S. and Kaprelyants A. S. Introduction to the elektronnomikroskopichesky equipment for medicobiological researches, Kiev, 1982, bibliogr.; A raster submicroscopy and the X-ray microanalysis, the lane with English, under the editorship of V. I. Petrov, book 1 — 2, M., 1984; Rivne Yu. A. Raster submicroscopy of normal and tumor cells. M, 1979, bibliogr.; Stoyanova I. G. and Anaskin I. F. Physical bases of methods of the translucent submicroscopy, M., 1972, bibliogr.; Uikli B. S. A submicroscopy for beginners, the lane with English, M., 1975; Ultrastructure of tumors of the person, under the editorship of N. T. Raykhlin, etc., M., 1981; Finean D. Biological ultrastructures, the lane with English. M, 1970; Shimmelg. A technique of a submicroscopy, the lane with it. M, 1972, bibliogr.; Yagubov A. S. and Katz V. A. Submicroscopy of soft tissues, Novosibirsk, 1984; Brenner S. and. Horne R. W. A negative staining method for high resolution electron microscopy of viruses, Biochim. biophys. Acta (Amst.), t. 34, p. 103, 1959; Methods in virology, ed. by K. Maramorosch a. H. Koprowski, V. 5, N. Y., 1971; Roth J. The preparation of protein A-gold complexes with 3 nm and 15 nm gold particles and their use in labeling multiple antigens on ultra-thin sections, Histochem. J., v. 14, p. 791, 1982; Singer S. J. Preparation of an electron-dense antibody conjugate, Nature (Lond.), v. 183, p. 1523, 1959.
See also bibliogr. to St. Supermicroscope .
V. S. Paukov, A. K. Kranchev; S. M. Klimenko (vir., of).