MICROSCOPIC METHODS OF THE RESEARCH — ways of studying of a microscopic structure of various objects, the sizes to-rykh are outside resolving power of an eye. M of m and. play an important role in bacterial., virusol., tsitol., gematol., gistol, and other researches; they are applied also in pharmacology, chemistry, mineralogy, a crystallography, etc. Among M. there is a m and. along with usual light microscopy widely use stereoscopic, darkfield, interferential, phase and contrast, polarizing, ultra-violet, a submicroscopy, etc.
Basis for M.'s development by m and. works to ABBA (E. K. were Abbe) but to diffraction properties of electromagnetic radiation. To ABBA define resolving power of microscopes by the theory and make the lenses deprived of chromatic and spherical aberration, lenses, diffraction gratings, lighting and drawing devices.
Diffraction grating to ABBA serves for studying of the phenomena of diffraction and consists of system of the thin transparent and opaque alternating lines, to-rye cut a special cutter in the thickness of the metallic coating applied on a glass substrate.
The lighting device to ABBA is used in microscopes to illumination of an object in a transmitted light. It consists of a mirror (flat or concave) and the condenser, by means of to-rykh a flow of light send to the plane of an object in the form of the meeting bunch of beams that provides higher illumination of drug and improves resolving power of a microscope. The condenser consists, as a rule, of two-three lenses; the lens, closest to a lens, is established so that its plane surface was parallel to the plane of a subject stage of microscope. During removal of the condenser from the plane of an object brightness of lighting decreases, however picture contrast increases.
The drawing device to ABBA serves for a sketch with gistol, drugs. It consists of the system of glass prisms located over an eyepiece of a microscope, sends edges to an eye of the researcher the light rays which passed through gistol, drug and reflected by means of a mirror from the sheet of paper lying near a microscope. Thanks to it the observer sees the combined image of drug and the hand outlining, e.g., a pencil contours of details gistol, pictures of drug.
At M.'s use of m and. the particularly important becomes the correct installation of lighting, to-ruyu carry usually out by Köhler's method. For this purpose the independent lighter, e.g. OI-19, have so that the plane of an iris diaphragm of the lighter was at distance of 15 — 25 cm from the center of a mirror of a microscope. Then through closed on 1/2 — 1/3 diaphragm project the image of thread of a filament lamp of the lighter in the center of a mirror of the microscope covered for simplification of observation with the sheet of white paper. Changing distance between a microscope and the lighter, make focusing of the image of thread of an incandescence and then a mirror of a microscope send the image to its lens. At the same time the size of the lit spot shall match diameter of an aperture diaphragm of a microscope, the sharp image a cut can be received, changing position of the condenser and the plane of a mirror. In conclusion open an aperture diaphragm of a microscope and by means of macro - and microscrews of a microscope receive a bright and sharp image of an object.
During the work with small increases in a microscope this way not always allows to receive full and uniform illumination of a field of vision. In these cases remove or take aside a frontal lens of the condenser, use the condenser with long focal length. At widely open aperture diaphragm of a microscope the image happens insufficiently contrast. In the course of diaphragming picture contrast increases and depth of sharpness increases, but resolving power of a microscope at the expense of the diffraction phenomena accruing at the same time can decrease. During the change of a lens the image should be focused in the focal plane at the closed diaphragm of the lighter again. In case of a deviation of an axis of the lighter from an axis of a lens of a microscope of an image edge can be lit unequally. That illumination of image edges became identical and uniform on all area of a field of vision, observing the image through an eyepiece, move the lighter.
Installation of lighting by Köhler's method is applied also during the studying of drugs in a so-called dark field. In this case replace the ordinary condenser darkfield and, observing in an eyepiece, slowly lift the condenser before emergence of the darkfield image.
The objects studied under a microscope can be transparent, and also opaque, i.e. changing amplitude and phase properties of the electromagnetic radiation directed to them. Depending on properties of an object physical change. properties of light — color (wavelength), brightness (amplitude of a wave), a phase, the plane and the direction of distribution of a wave that use m in M. and. Apply a light microscope to microscopic examination of the painted objects. Color of the image and distinction in coloring quite often allow to judge the chemical nature of separate structures of a body of interest, but do not give the chance to estimate its life activity (the movement, a chemotaxis, merge, etc.) since during the coloring often use the chemical or temperature fixing killing biol, an object, but providing effective coloring. Unlike a research fixed biol, objects, the vital microscopy is based on intravital coloring, as a result to-rogo many structures of living cell change under the influence of special dyes a little. The vital microscopy can be carried out also without coloring if to enter the darkfield condenser into a usual light microscope.
Independent option dark field method (see) the ultramicroscopy is, at a cut the most fine particles of a body of interest light with a side beam of light and against a dark background they look in the form of points. With the help ultramicroscope (see) it is possible to measure particles and to define nek-ry properties of bodies of interest.
Apply microscopes to phase and contrast and amplitude and contrast microscopy, in to-rykh a ray of light is exposed to diffraction depending on features of a body of interest; at the same time length and a phase of a wave of light changes. Live microscopic objects in a light microscope look transparent and almost do not change amplitude and color of a light ray and cause only shift of a phase of its wave. The rays of light which passed through a body of interest deviate the special translucent phase plate enclosed in a lens, and, thus, between beams of a background and an object there is a difference of wavelength. If this difference reaches 1/4 wavelengths, then there is an effect, noticeable for an eye, when a dark object clearly is visible on a light background or, on the contrary, depending on structure of a phase plate (see. Fazovokontrastnaya microscopy ). The plates changing only brightness and color of a background use in an amplitude and contrast or anoptralny microscope. The amplitude and contrast device can be installed also on biol, a microscope. These microscopes considerably expand possibilities of an intravital research biol, objects without preliminary fixing and coloring of drug.
The interferential microscopy is constructed approximately on the same principles, as phase and contrast, but, unlike it, gives the chance to obtain quantitative data. By means of the interference microscope it is possible to measure the difference of phases caused by various cellular structures and to determine their weight. Consecutive measurements of a difference of phases in two environments with the known indices of refraction give the chance at the same time to determine thickness of an object, concentration of nonvolatile solid, water content and allow to judge indirectly cellular metabolism, permeability of membranes, activity of enzymes. The interferential microscopy finds application in tsitol, researches, is used for quantification of cellular structures of live objects, napr, cultures of fabrics, the elementary, etc.
The polarization microscopy is based on various refraction by structural components of cells and fabrics of the polarized light. In one of them light spreads with an identical speed irrespective of the plane of polarization (isotropic structures), in others — rate of propagation of the polarized light depends on the direction it on a longitudinal or lateral axis of an object (anisotropic structures). Row biol, objects (myofibrils, ciliary cilia, etc.) has strict molecular orientation, is anisotropic and possesses double refraction. The polarized light is created by means of special polarizers — filmy polaroids or Nicolas's prisms, to-rye placed in a microscope between a light source and a body of interest. The bunch of a plane-polarized light formed by them decays on two beams polarized in mutually perpendicular planes. One of these beams passes through anisotropic structures of an object, being late concerning another. During the escaping of an object both beams appear in different phases. When the index of refraction along structure is more, than in transverse direction, speak about positive double refraction, at the return relations — about negative double refraction. The structures of a cell formed by the oriented proteinaceous molecules possess own positive double refraction. The nature of refraction of the polarized light, size of anisotropy in combination with change of these optical indicators after extraction by zhirorastvoritel allow to judge the molecular organization of structure. E.g., as a result of researches of myelin covers of nerves by means of the polarizing microscope the arrangement of molecules of lipoid substances, radial in relation to a longitudinal axis of a nerve, and an arrangement of macromolecules of protein, perpendicular in relation to them, was revealed. The similar arrangement of proteinaceous and lipoid elements is found in erythrocytes and chlorolayers. By means of the polarizing microscope in gaversovy systems of tubular bones plates with longitudinal and circular orientation of fibrilla are found. Besides, on the nature of double refraction study a form of viruses, proteinaceous macromolecules, etc.
In the polarized light it is possible to investigate as painted, and the uncolored fabric cuts prepared on the freezing microtome or which are filled in in paraffin. In particular, lipids and a myelin investigate on frozen sections whereas the research of cross-striped muscular tissue and crystals can be made as in paraffin, and frozen sections. The double refraction of collagen which clearly is shown in such environments as caprate of cellulose or pitch can be lost completely in the drugs concluded in mix glycerin-gelatin.
In a scientific and practical research widely apply luminescent microscopy (see), for a cut use ultraviolet rays or a blue-violet part of a range. Nek-ry intracellular educations, napr, lipids, possess own (primary) luminescence (see). Other components of a cell can luminesce after preliminary coloring so-called. flyuorokhromam (see).
The ultraviolet microscopy is used in tsitol, and histochemical researches. She allows to study localization, quantitative distribution in cells and fabrics of high-molecular compounds (proteins, nucleic acids) and to watch their dynamics in the course of life activity. This method gives the chance without preliminary fixing and coloring of drugs to consider the studied material, e.g., for the purpose of intravital studying of microobjects.
The ultraviolet absorbing microscopy is based on ability of the nek-ry substances which are a part of fabrics and cells transparent in visible light, to absorb ultraviolet rays with a certain wavelength.
At a research of the live or fixed uncolored objects picture contrast at the expense of selective absorption of ultraviolet rays high-molecular compounds increases. In particular, the ultraviolet microscopy is important for studying of distribution in a cell nucleinic to - the t absorbing ultraviolet radiation in the site of a range apprx. 260 nanometers. Absorption of ultraviolet radiation proteins depends on the aromatic amino acids (tyrosine, tryptophane, phenylalanine) giving a maximum of absorption in the site of a range apprx. 280 nanometers which are their part. For receiving evident idea of distribution in drug of substances the studied site is photographed in an ultraviolet light with a different length of waves. In the subsequent pictures make a copy on a color film in a chromoscope, before the picture made in short-wave beams place a blue filter in Krom, in beams of average length — green and in long-wave beams — the red light filter. These pictures by means of special adaptation combine on the screen, and the image becomes visible, transferring by conditional flowers of distinction of absorption of ultraviolet rays by separate structures of a cell.
Ultraviolet fluorescent microscopy, as well as absorbing, studying of the live or fixed uncolored objects because ranges of ultra-violet fluorescence of substances differ from each other use for cytochemical.
The infrared microscopy gives the chance to establish structure of an object on the nature of light absorption with a length of waves of 800 — 1000 nanometers. The wide spread occurance has a research in infrared light of substances, partially or completely opaque in ultra-violet and visible spectral ranges. For infrared microscopy biol, objects do not subject to additional chemical processing. By means of an infrared microscope make a research of impregnirovanny nervous tissue and capillaries in gistol, cuts, distinguish injuries of a retina and an iris of the eye of an eye.
The stereoscopic microscopy allows to investigate opaque objects and creates effect of the volume image. It is applied, e.g., to a research of section, operational and biopsy material, to work by means of a method macromicroscopy (see). In forensic medicine the stereoscopic microscopy is used for studying of bodies and fabrics of a corpse, and also for a research of various material evidences (see).
For increase in resolving power of M. of m and. create the optical systems based on electromagnetic lenses using as a source of radiation of a cathode rays, napr for submicroscopy (see) use a bunch of high-velocity electrons, and the role of lenses is carried out by electric and magnetic fields of a certain configuration. A kind of a submicroscopy is the scanning (raster) microscopy, edges gives the chance to receive the volume image of an object at the expense of the secondary electrons radiated by it.
In nek-ry microscopes smooth, stepless increase without change of a lens allows to establish within broad range the interesting details of an object, napr, to the loudspeaker biol, the processes happening in fabric cultures.
Bibliography: Appelt. Introduction to methods of microscopic examination, the lane with it., M., 1959, bibliogr.; Biophysical methods of a research, under the editorship of F. Hubert, the lane with English, M., 1956; D e Robertis E., Novinsky V. and With and at with F. Cytobiology, the lane with English, page 94, M., 1973; Ditchbern R. Physical optics, the lane with English, M., 1965; Ilyin P. Page, Fedotov G. I. and Fedin L. A. Laboratory optical devices, M., 19 66, bibliogr.; L and l of l and R. Patogistologicheskaya of the technician and a practical histochemistry, the lane with English, page 7, M., 1969; G. E. Starlings and d river. Microscopes, L., 1969, bibliogr.
N. K. Permyakov, G. M. Mogilevsky.