PHASE-CONTRAST MICROSCOPY — the way of microscopic examination of the transparent, not absorbing light objects based on strengthening of contrast of the image.
The transparent, not painted objects (the live and fixed microorganisms, cells, etc.), different from the environment on index of refraction, do not absorb light, but change its speed and, therefore, a phase of light fluctuations. And extent of these changes depends on the size of index of refraction and thickness of structures of an object. However these changes are not perceived by an eye, are not registered photographic materials, and the studied objects at light microscopy almost do not differ from a background. Apply F to strengthening of picture contrast. - to. m. It is widely used for intravital studying of microorganisms, protozoa, cells of plants and animals. In hematology, e.g., the phase-contrast microscopy is applied to calculation and differentiation of cells, by studying of their mobility, differential diagnosis of leukoses, etc.
The way of transformation of phase changes in corresponding to them amplitude was offered in the 30th 20 century by the Dutch physicist F. Zernike. Principle F. - to. the m is that light which is not rejected by an object passes through the phase ring applied on one of lenses of a lens displacing its phase on a quarter of wavelength and weakening its intensity (to balance it with intensity of light diffracted by an object), and the diffracted (rejected) light passes by a phase ring (see the Microscope).
Passing of the direct, not diffracted objektokhm light through a phase ring is provided with the ring diaphragm which is in the condenser, the projection a cut is equal in the plane of an output pupil of a lens on diameter and width to a phase ring and shall match it completely. For each lens there is the ring diaphragm.
In the plane of the image there is an interference of the light waves which passed and did not pass through a phase ring. At the same time there are distinctions in amplitude reflecting changes of a phase depending on properties of sites of an object. Unlike phase amplitude changes of light waves are well visible an eye and can be registered.
Depending on a way of production of a phase ring the phase of the direct, not diffracted by an object light can or advance a phase diffracted, or lag behind it. At the same time arises or the most widespread positive phase contrast where particles with index of refraction big, than at the environment (more dense), look dark on a light background, or negative where the same particles give the image is lighter than a surrounding background. It should be noted, however, that this picture remains only up to the certain size of index of refraction, and after achievement of this size there is an inversion of contrast, i.e. the return patterns are observed.
The phase and contrast device (in particular, KF-4 which is released in our country) consists of lenses, the phase ring, the condenser with the turret-mounted disk containing a set of ring diaphragms and tsentriro-vochny adaptation, and also an auxiliary microscope (fig. 1) is applied on one of lenses to-rykh, with the help to-rogo in the plane of an output pupil of a lens it is possible to watch combination of a phase ring and projection of a ring diaphragm of the condenser. It ustroyst-
Fig. 1. The phase and contrast KF-4 device consisting of the condenser (I), lenses (2) and an auxiliary microscope (3).
in it can be established on any microscope.
There is a number of constructive kinds of phase and contrast devices: with one ring diaphragm for all lenses during the use of the pankratichesky condenser (e.g., in domestic microscopes of MBI-6, MBI-15), with the so-called taken-out pupil, at Krom the phase ring is located out of a lens that allows to use for F. - to. m usual lenses (such device is available in domestic microscopes of MBI-13, MBI-17). Also devices with variable phase contrast are issued (with two rings of different diameter).
One of kinds of negative phase contrast is the anoptralny (phase and darkfield) device. The Anoptralny device was created in 1953 Mr. A. Wilska and is used for studying of the objects bringing small shift of a phase. Modification of this method was offered by M. A. Peshkov and was widely used at us in the country.
A technique of preparation of drugs for F. - to. the m depends on an object of a research and duration of its studying: uncolored microorganisms can be considered in drugs the crushed drop (see), for long observation and film registration of reproduction of microorganisms special agar .mikrokamer (see) on slide plates (Fonbryun's camera, the HI-shaped camera of Peshkov) use. In single-layer cultures of fabric also use microcameras to studying of dynamics of processes (stationary and perfused). Very important factors considerably defining quality of the image are thickness of drug and distinction in indices of refraction of an object and Wednesday.
Equipment F. - to. - m it is rather simple: lenses and a substage condenser replace on special
(on domestic phase and contrast lenses there is a designation F, on foreign — Ph), establish a disk of the condenser in situation O (i.e. a through hole without ring diaphragm), on a subject little table place drug, adjust light according to Köhler (see. Microscopic methods of a research.), rotation of a disk enter the ring diaphragm corresponding to objective magnification. Instead of an eyepiece establish an auxiliary microscope. Putting forward its upper part, receive the sharp image of a phase ring and ring diaphragm. Tsentrirovochny screws of the condenser precisely combine both
Fig. 2. Combination of a phase ring with a ring diaphragm: at the left — a nepra
vilno, on the right — it is correct.
rings then instead of an auxiliary microscope establish an eyepiece. During the change of drug it is reasonable to check combination of rings (fig. 2).
Of F. - k.m. an opportunity to make intravital observations (without any processing) biol is. objects, napr, macrophages (fig. 3), and a shortcoming — emergence light (in case of positive contrast) or dark (in case of negative) an aura around an object and its structures. More complete information can be obtained at a combination of phase and contrast and luminescent microscopy at use both immunolyu-minestsentny (see the Immunofluorescence), and a luminescent and cytochemical method (see. Luminescent microscopy). During the work with the luminescing serums F. - to. the m allows to be convinced
by Fig. 3. Peritoneal macrophages in culture of fabric (intravital phase and contrast shooting); x 1100.
available a microobject if it does not connect the luminescing antibodies and also to study objects, at to-rykh an antibody are fixed on separate structures.
Especially big role in intravital cytologic studying of dynamics of various physiological and pathological processes in cellular biology, microbiology, virology was played by a combination of phase and contrast and anoptralny microscopy to microcinematography (see). This method was used for studying of cytology of bacteria and protozoa, a mitosis in various cells, a cytopathic effect of viruses and rickettsiae on cells. Features of education and development of L-forms of bacteria and mycoplasmas, action of antibiotics on a bacterium were also studied.
Bibliography: Kravchenko A. T., Milyutin V. N. and Gudima O. S. Microcinematography in biology, M., 1963; The Guide to microbiological diagnosis of infectious diseases, under the editorship of K. I. Matveev, page 5, 25, M., 1973;
G. E. Starlings, etc. Microscopes, L., 1969; Franson M. Fazovokontrastny and interferential microscopes, the lane with fr., M., 1960; Cinemic-rography in cell biology, ed. by G. G. Rose, N. Y. — L., 1963; Zernike F. Diff-raktion theory of the knife edge test and its improved form of the phase contrast method, Physica, v. 1, p. 689, 1934.
M. Ya. Korn, E. S. Stanislavsky.