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POLARIZING MICROSCOPY

Physical Encyclopedic Dictionary. - M.: Soviet Encyclopedia. . 1983 .

POLARIZING MICROSCOPY

- see Art. Microscopy.

Physical encyclopedia. In 5 volumes. - M.: Soviet Encyclopedia. Editor-in-Chief A. M. Prokhorov. 1988 .

See what "POLARIZING MICROSCOPY" is in other dictionaries:

POLARIZING MICROSCOPY- microscopy, based on the ability of different components of cells and tissues to refract polarized rays. In a polarizing microscope, you can examine objects that are characterized by birefringence ... Glossary of botanical terms

A set of methods (and devices that provide these methods) designed to observe and study objects under a microscope that change in any respect the polarization of light (See Polarization of light) that passes through objects ... ...

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M. when illuminating an object with polarized light; is used to detect and study objects or their structures that have the properties of birefringence ... Big Medical Dictionary

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Introduction to Quantitative Cytochemistry, . A summary of the quantitative methods of studying the cell and the optical equipment used for this. The focus of the book is on the most reliable methods for quantifying…

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Introduction

Light microscopy

electron microscopy

Polarizing microscopy

Annex 1

Light microscopy

Light microscopy is the most ancient and at the same time one of the most common methods of research and study of plant and animal cells. It is assumed that the beginning of the study of the cell was precisely with the invention of the light optical microscope. The main characteristic of a light microscope is the resolution of a light microscope, which is determined by the wavelength of the light. The resolution limit of a light microscope is determined by the wavelength of light, an optical microscope is used to study structures that have a minimum size equal to the wavelength of light radiation. Many constituent cells are close in their optical density and require pre-treatment before microcopying, otherwise they are practically invisible in a conventional light microscope. In order to make them visible, various dyes with a certain selectivity are used. Using selective dyes, it becomes possible to study the internal structure of the cell in more detail.

For example:

hematoxylin dye stains some components of the nucleus blue or purple;

after treatment successively with phloroglucinol and then with hydrochloric acid, the lignified cell membranes become cherry-red;

Sudan III dye stains corky cell membranes pink;

a weak solution of iodine in potassium iodide turns starch grains blue.

When conducting microscopic examinations, most of the tissues are fixed before staining.

After fixation, the cells become permeable to dyes, and the cell structure is stabilized. One of the most common fixatives in botany is ethyl alcohol.

During the preparation of the preparation for microcopying, thin sections are made on a microtome (Appendix 1, Fig. 1). This appliance uses the principle of a bread slicer. Slightly thicker sections are made for plant tissues than for animals, since plant cells are relatively larger. The thickness of sections of plant tissues for - 10 microns - 20 microns. Some fabrics are too soft to cut straight away. Therefore, after fixing, they are poured into molten paraffin or a special resin, which impregnate the entire fabric. After cooling, a solid block is formed, which is then cut on a microtome. This is due to the fact that plant cells have strong cell walls that make up the framework of the tissue. Lignified shells are especially durable.

Using the filling during cooking, the cut raises the danger of violating the structure of the cell, to prevent this, the method of rapid freezing is used. When using this method, fixing and pouring are dispensed with. Frozen tissue is cut on a special microtome - a cryotome (Appendix 1, Fig. 2).

Frozen sections better preserve the features of the natural structure. However, they are more difficult to cook, and the presence of ice crystals breaks some of the details.

phase-contrast (app. 1, fig. 3) and interference microscopes (app. 1, fig. 4) allow you to examine living cells under a microscope with a clear manifestation of the details of their structure. These microscopes use 2 beams of light waves that interact (superimpose) on each other, increasing or decreasing the amplitude of the waves entering the eye from different components of the cell.

Light microscopy has several varieties.

Bright field method and its varieties

Bright field method in transmitted light used in the study of transparent preparations with light-absorbing particles and details included in them (thin colored sections of animal and plant tissues, thin sections of minerals). In the absence of the preparation, the beam of light from the condenser, passing through the lens, gives a uniformly illuminated field near the focal plane of the eyepiece. In the presence of an absorbent element in the preparation, partial absorption and partial scattering of the light incident on it occurs, which causes the appearance of the image. It is also possible to use the method when observing non-absorbing objects, but only if they scatter the illuminating beam so strongly that a significant part of it does not enter the lens.

Oblique illumination method is a variation of the previous method. The difference between them is that the light is directed at the object at a large angle to the direction of observation. Sometimes this helps to bring out the "relief" of the object due to the formation of shadows.

Bright field method in reflected light used in the study of opaque light-reflecting objects, such as thin sections of metals or ores. Illumination of the preparation (from an illuminator and a translucent mirror) is carried out from above, through a lens, which simultaneously plays the role of a condenser. In the image created in the plane by the lens together with the tube lens, the structure of the preparation is visible due to the difference in the reflectivity of its elements; in a bright field, inhomogeneities are also distinguished, scattering the light incident on them.

Dark field method and its varieties

Dark field method in transmitted light used to image transparent, non-absorbent objects that cannot be seen using the bright field method. Often these are biological objects. The light from the illuminator and the mirror is directed to the preparation by a condenser of a special design - the so-called. dark field condenser. After leaving the condenser, the main part of the light rays, which did not change its direction when passing through a transparent preparation, forms a beam in the form of a hollow cone and does not enter the objective (which is located inside this cone). The image in the microscope is formed with the help of only a small part of the rays scattered by the microparticles of the drug located on the glass slide inside the cone and passed through the lens. In the field of view on a dark background, light images of the elements of the preparation structure are visible, which differ from the environment in the refractive index. For large particles, only bright edges are visible, scattering light rays. Using this method, it is impossible to determine by the appearance of the image whether the particles are transparent or opaque, whether they have a higher or lower refractive index compared to the environment.

electron microscopy

The first electron microscope was constructed in 1931 by Knoll and Ruska in Germany. It was only in the 1950s that methods were developed for making sections with the necessary qualities.

The complexity of electron microscopy lies in the fact that special processing of preparations is necessary for the study of biological samples.

The first difficulty is that electrons have a very limited penetrating power, so ultrathin sections should be made, 50–100 nm thick. In order to obtain such thin sections, the tissues are first impregnated with resin: the resin polymerizes and forms a hard plastic block. Then, using a sharp glass or diamond knife, the sections are cut on a special microtome.

There is another difficulty: when electrons pass through the biological tissue, a contrast image is not obtained. In order to obtain contrast, thin sections of biological specimens are impregnated with salts of heavy metals.

There are two main types of electron microscopes. In a transmission (transmission) microscope, an electron beam passing through a specially prepared sample leaves its image on the screen. The resolving power of a modern transmission electron microscope is almost 400 times greater than the light one. These microscopes have a resolution of about 0.5 nm.

Despite such a high resolution, transmission electron microscopes have major drawbacks:

have to work with fixed materials;

the image on the screen is two-dimensional (flat);

when treated with heavy metals, some cellular structures are destroyed and modified.

A three-dimensional (volumetric) image is obtained using a scanning electron microscope (EM). Here, the beam does not pass through the sample, but is reflected from its surface.

The test sample is fixed and dried, after which it is covered with a thin layer of metal, an operation called shading (the sample is shaded).

In a scanning EM, a focused electron beam is directed onto a sample (the sample is scanned). As a result, the metal surface of the sample emits low-energy secondary electrons. They are registered and converted into an image on a television screen. The maximum resolution of the scanning microscope is small, about 10 nm, but the image is voluminous.

Varieties of electron microscopy:

Amplitude electron microscopy- Methods of amplitude electron microscopy can be used to process images of amorphous and other bodies (particle sizes of which are less than the distance resolved in an electron microscope), scattering electrons diffusely. In a transmission electron microscope, for example, the contrast of the image, i.e., the difference in the brightness of the image of neighboring sections of the object, in the first approximation is proportional to the difference in the thicknesses of these sections.

Phase electron microscopy- To calculate the contrast of images of crystalline bodies with regular structures, as well as to solve the inverse problem - to calculate the structure of an object from the observed image - methods of phase electron microscopy are used. The problem of the diffraction of an electron wave on a crystal lattice is considered, the solution of which additionally takes into account the inelastic interactions of electrons with an object: scattering by plasmas, phonons, etc. In transmission electron microscopes and high-resolution scanning transmission electron microscopes, images of individual molecules or atoms of heavy elements are obtained . Using the methods of phase electron microscopy, it is possible to reconstruct the three-dimensional structure of crystals and biological macromolecules from images.

Quantitative electron microscopy- Quantitative electron microscopy methods are the accurate measurement of various parameters of a sample or process under study, such as the measurement of local electrical potentials, magnetic fields, surface relief microgeometry, etc.

Lorentz electron microscopy- The field of study of Lorentz electron microscopy, in which the phenomena due to the Lorentz force are studied, are internal magnetic and electric fields or external stray fields, for example, the fields of magnetic domains in thin films, ferroelectric domains, fields of heads for magnetic recording of information, etc.

Polarizing microscopy

Polarizing microscopy is a method of observation in polarized light for the microscopic examination of preparations containing optically anisotropic elements (or consisting entirely of such elements). Such are many minerals, grains in thin sections of alloys, some animal and plant tissues, etc. Observation can be carried out both in transmitted and reflected light. The light emitted by the illuminator is passed through a polarizer. In this case, the polarization reported to him changes with the subsequent passage of light through the preparation (or reflection from it). These changes are studied using an analyzer and various optical compensators. Analyzing such changes, one can judge the main optical characteristics of anisotropic microobjects: the strength of birefringence, the number of optical axes and their orientation, rotation of the polarization plane, dichroism.

Phase contrast method

Method phase contrast and its variety - the so-called. method "anoptral" contrast designed to obtain images of transparent and colorless objects that are invisible when observed using the bright field method. These include, for example, living unstained animal tissues. The essence of the method is that even with very small differences in the refractive indices of different elements of the drug, the light wave passing through them undergoes different changes in phase (acquires the so-called phase relief). Not perceived directly by either the eye or the photographic plate, these phase changes are converted by a special optical device into changes in the amplitude of the light wave, i.e. into changes in brightness ("amplitude relief"), which are already distinguishable by the eye or fixed on the photosensitive layer. In other words, in the resulting visible image, the distribution of brightness (amplitudes) reproduces the phase relief. The resulting image is called phase contrast.

A typical scheme of the method operation: an aperture diaphragm is installed in the front focus of the condenser, the hole of which has the shape of a ring. Its image appears near the back focus of the lens, and the so-called. a phase plate, on the surface of which there is an annular protrusion or an annular groove, called a phase ring. The phase plate is not always placed at the focus of the objective - often the phase ring is applied directly to the surface of one of the objective lenses.

In any case, the rays from the illuminator that are not deflected in the preparation, giving the image of the diaphragm, must completely pass through the phase ring, which significantly attenuates them (it is made absorbing) and changes their phase by l / 4 (l is the wavelength of light). And the rays, even slightly deflected (scattered) in the preparation, pass through the phase plate, bypassing the phase ring, and do not undergo an additional phase shift.

Taking into account the phase shift in the specimen material, the total phase difference between the deflected and non-deflected beams is close to 0 or l/2, and as a result of light interference in the specimen image plane, they significantly enhance or weaken each other, giving a contrast image of the specimen structure. The deflected beams have a much smaller amplitude compared to the non-deflected beams, therefore, the attenuation of the main beam in the phase ring, bringing the amplitude values closer together, also leads to a greater image contrast.

The method makes it possible to distinguish small elements of the structure, which are extremely weakly contrasted in the bright field method. Transparent particles, relatively small in size, scatter light rays at such small angles that these rays pass through the phase ring together with those that are not deflected. For such particles, the phase-contrast effect takes place only near their contours, where strong scattering occurs.

infrared observation method

Method observations in infrared(IR) rays also require the conversion of an image invisible to the eye into a visible one using photography or using an image intensifier tube. IR microscopy makes it possible to study the internal structure of those objects that are opaque in visible light, such as dark glasses, some crystals and minerals, etc.

Method of observation in ultraviolet rays

Method observations in ultraviolet (UV) rays makes it possible to increase the maximum resolution of the microscope. The main advantage of the method is that the particles of many substances, which are transparent in visible light, strongly absorb UV radiation of certain wavelengths and, therefore, are easily distinguishable in UV images. Many substances contained in plant and animal cells (purine bases, pyrimidine bases, most vitamins, aromatic amino acids, some lipids, thyroxine, etc.) have characteristic absorption spectra in the UV region.

Since ultraviolet rays are invisible to the human eye, images in UV microscopy are recorded either photographically or using an image intensifier tube or luminescent screen. The drug is photographed in three wavelengths of the UV region of the spectrum. Each of the resulting negatives is illuminated with visible light of a certain color (for example, blue, green and red), and they are all simultaneously projected onto one screen. The result is a color image of the object in conditional colors, depending on the absorption capacity of the preparation in ultraviolet.

Microphotography and microfilming is the acquisition of images on light-sensitive layers using a microscope. This method is widely used in conjunction with all other methods of microscopic examination. For microphotography and microcine filming, some restructuring of the optical system of the microscope is required - different from visual observation of the focusing of the eyepiece relative to the image given by the objective. Microphotography is necessary when documenting studies, when studying objects in UV and IR rays invisible to the eye (see above), as well as objects with a weak glow intensity. Film microfilming is indispensable for studying processes that unfold over time (the vital activity of tissue cells and microorganisms, the growth of crystals, the occurrence of simple chemical reactions, etc.).

Interference contrast method

The method of interference contrast (interference microscopy) consists in the fact that each beam splits into two, entering the microscope. One of the obtained beams is directed through the observed particle, the other - past it along the same or additional optical branch of the microscope. In the ocular part of the microscope, both beams reconnect and interfere with each other. The condenser and lens are equipped with birefringent plates, of which the first splits the original light beam into two beams, and the second recombines them. One of the beams, passing through the object, lags in phase (acquires a path difference compared to the second beam). The value of this delay is measured by the compensator. This method makes it possible to observe transparent and colorless objects, but their images can also be multi-colored (interference colors). This method is suitable for the study of living tissues and cells and is used in many cases precisely for this purpose. The interference contrast method is often used in conjunction with other microscopy methods, in particular, observation in polarized light. Its use in combination with ultraviolet microscopy makes it possible, for example, to determine the content of nucleic acids in the total dry mass of an object.

Research method in the light of luminescence

Method studies in the light of luminescence It consists in observing under a microscope the green-orange glow of micro-objects, which occurs when they are illuminated with blue-violet light or ultraviolet rays not visible to the eye. Two light filters are introduced into the optical scheme of the microscope. One of them is placed in front of the condenser. It transmits radiation from the illuminator only at those wavelengths that excite luminescence either of the object itself (intrinsic luminescence) or special dyes introduced into the preparation and absorbed by its particles (secondary luminescence). The second light filter, which is installed after the lens, passes only luminescence light to the observer's eye (or to the photosensitive layer). In fluorescence microscopy, illumination of preparations is used both from above (through an objective, which in this case also serves as a condenser), and from below, through a conventional condenser. The method has found wide application in microbiology, virology, histology, cytology, food industry, soil research, microchemical analysis, and flaw detection. Such a variety of applications is explained by the very high color sensitivity of the eye and the high contrast of the image of a self-luminous object against a dark non-luminescent background.

Replica method

The replica method is used to study the surface geometric structure of massive bodies. An imprint is taken from the surface of such a body in the form of a thin film of carbon, collodion, formvar, etc., which repeats the surface relief and is examined in a transmission electron microscope. Usually, a layer of heavily electron-scattering heavy metal is deposited onto the replica in vacuum at a sliding (small to the surface) angle, shading the protrusions and depressions of the geometric relief.

Decoration Method

The decoration method investigates not only the geometric structure of surfaces, but also microfields caused by the presence of dislocations, clusters of point defects, growth steps of crystal faces, domain structure, etc. According to this method, a very thin layer of decorating particles (Au atoms) is first deposited on the surface of the sample. , Pt, etc., molecules of semiconductors or dielectrics), which are deposited mainly in the areas of concentration of microfields, and then a replica is taken with inclusions of decorating particles.

Various types of centrifugation are widely used to obtain cell fractions: differential centrifugation, zonal centrifugation and equilibrium centrifugation in a density gradient. Theoretical and practical issues related to centrifugation are comprehensively analyzed in Sykes's review.

Differential centrifugation

In the case of differential centrifugation, the samples are centrifuged for a certain time at a given speed, after which the supernatant is removed. This method is useful for separating particles that differ greatly in sedimentation rate. For example, centrifugation for 5-10 min at 3000-5000 g leads to the precipitation of intact bacterial cells, while most of the cell fragments remain in the supernatant. Cell wall fragments and large membrane structures can be pelleted by centrifugation at 20,000–50,000 § for 20 min, while small membrane vesicles and ribosomes require centrifugation at 200,000 § for 1 hour to precipitate.

Zonal centrifugation

Zonal centrifugation is an effective way to separate structures that have similar buoyant density but differ in particle shape and mass. Examples include the separation of subunits of ribosomes, different classes of polysomes, and DNA molecules with different shapes. Centrifugation is carried out either in bucket rotors or in specially designed zonal rotors; to prevent convection during centrifugation, a weak gradient (usually sucrose) is created in the bucket-rotor cups or in the chamber of the zonal rotor. The sample is applied in the form of a zone or a narrow strip at the very top of the gradient column. For subcellular particles, a sucrose gradient of 15 to 40% (w/v) is typically used.

Laue method

applied to single crystals. The sample is irradiated with a beam with a continuous spectrum, the mutual orientation of the beam and the crystal does not change. The angular distribution of diffracted radiation has the form of individual diffraction spots (Lauegram).

Debye-Scherrer method

Used to study polycrystals and their mixtures. The random orientation of the crystals in the sample with respect to the incident monochromatic beam transforms the diffracted beams into a family of coaxial cones with the incident beam on the axis. Their image on photographic film (debyegram) has the form of concentric rings, the location and intensity of which makes it possible to judge the composition of the substance under study.

Cell culture method

Some tissues can be divided into individual cells in such a way that the cells remain alive and are often able to reproduce. This fact finally confirms the idea of a cell as a unit of life. A sponge, a primitive multicellular organism, can be divided into cells by rubbing through a sieve. After a while, these cells recombine and form a sponge. Animal embryonic tissues can be made to dissociate using enzymes or other means that weaken the bonds between cells.

The American embryologist R. Harrison (1879-1959) was the first to show that embryonic and even some mature cells can grow and multiply outside the body in a suitable environment. This technique, called cell culture, was perfected by the French biologist A. Carrel (1873-1959). Plant cells can also be grown in culture, but compared to animal cells, they form larger clusters and are more strongly attached to each other, so tissue is formed during culture growth, rather than individual cells. In cell culture, a whole adult plant, such as a carrot, can be grown from a single cell.

Microfigure method

With the help of a micromanipulator, individual parts of the cell can be removed, added, or modified in some way. A large amoeba cell can be divided into three main components - the cell membrane, cytoplasm and nucleus, and then these components can be reassembled and a living cell is obtained. In this way, artificial cells can be obtained, consisting of components of different types of amoebas.

Considering that it is possible to synthesize some cellular components artificially, experiments on the assembly of artificial cells may be the first step towards the creation of new life forms in the laboratory. Since each organism develops from a single cell, the method of obtaining artificial cells in principle allows the construction of organisms of a given type, if at the same time using components that are slightly different from those found in currently existing cells. In reality, however, complete synthesis of all cellular components is not required. The structure of most, if not all, components of a cell is determined by nucleic acids. Thus, the problem of creating new organisms is reduced to the synthesis of new types of nucleic acids and their replacement of natural nucleic acids in certain cells.

Cell fusion method

Another type of artificial cells can be obtained by fusion of cells of the same or different types. To achieve fusion, the cells are exposed to viral enzymes; in this case, the outer surfaces of two cells stick together, and the membrane between them collapses, and a cell is formed in which two sets of chromosomes are enclosed in one nucleus. You can merge cells of different types or at different stages of division. Using this method, it was possible to obtain hybrid cells of a mouse and a chicken, a human and a mouse, a human and a toad. Such cells are hybrid only initially, and after numerous cell divisions they lose most of the chromosomes of either one or another type. The end product becomes, for example, essentially a mouse cell, where human genes are absent or present only in small quantities. Of particular interest is the fusion of normal and malignant cells. In some cases, the hybrids become malignant, in others they do not; both properties can appear both as dominant and as recessive. This result is not unexpected, since malignancy can be caused by various factors and has a complex mechanism.

cell microscopy light

Annex 1

Figure 2. Cryotome Figure 3. Phase contrast microscope

Figure 4. Interference microscope

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Phase contrast microscopy method

Most of the cellular structures differ little in the refractive index of light, the absorption of rays from each other and the environment. In order to study such components, one has to change the illumination (with a loss of image clarity) or use special methods and devices. Phase-contrast microscopy is one such method. It is widely used in the vital study of cells. The essence of the method is that even with very small differences in the refractive indices of different elements of the drug, the light wave passing through them undergoes different phase changes. Invisible directly neither to the eye nor to the photographic plate, these phase changes are converted by a special optical device into changes in the amplitude of the light wave, i.e., into changes in brightness that are already visible to the eye or are recorded on the photosensitive layer. In the resulting visible image, the distribution of brightness (amplitudes) reproduces the phase relief. The resulting image is called phase contrast. Objects can appear dark against a light background (positive phase contrast) or light against a dark background (negative phase contrast).

Interference contrast method (interference microscopy)

The method of interference contrast is similar to the previous one - they are both based on the interference of rays that have passed through the microparticle and passed it. A beam of parallel light rays from the illuminator splits into two streams, entering the microscope. One of the obtained beams is directed through the observed particle and acquires changes in the oscillation phase, the other - bypassing the object along the same or additional optical branch of the microscope. In the ocular part of the microscope, both beams reconnect and interfere with each other. As a result of interference, an image will be built, on which sections of the cell with different thicknesses or different densities will differ from each other in terms of contrast. The interference contrast method is often used in conjunction with other microscopy methods, in particular, observation in polarized light. Its use in combination with ultraviolet microscopy makes it possible, for example, to determine the content of nucleic acids in the total dry mass of an object.

Polarizing microscopy

Polarizing microscopy is a method of observing in polarized light objects that have isotropy, i.e. ordered orientation of submicroscopic particles. A polarizer is placed in front of the condenser of a polarizing microscope, which transmits light waves with a certain plane of polarization. After the preparation and the lens, an analyzer is placed, which can transmit light with the same plane of polarization. If the analyzer is then rotated by 90o with respect to the first one, no light will pass through. In the event that between such crossed prisms there is an object that has the ability to polarize light, it will be seen as glowing in a dark field. Using a polarizing microscope, one can verify, for example, the oriented arrangement of micelles in the plant cell wall.

Polarizing microscopy

Polarizing microscopy makes it possible to study objects of study in light formed by two beams polarized in mutually perpendicular planes, i.e., in polarized light. To do this, filmy polaroids or Nicol prisms are used, which are placed in a microscope between the light source and the preparation. Polarization changes when light rays pass through various structural components of cells and tissues, the properties of which are inhomogeneous, or when reflected from them.

In optically isotropic structures, the propagation velocity of polarized light does not depend on the plane of polarization; in anisotropic structures, it varies depending on the direction of light along the longitudinal or transverse axis of the object. If the refractive index of light along the structure is greater than in the transverse direction, positive birefringence occurs, with reverse relationships - negative birefringence. Many biological objects have a strict molecular orientation, are anisotropic, and cause positive light birefringence.

Dark field microscopy

During microscopy using the dark field method, the preparation is illuminated from the side with oblique beams of rays that do not fall into the objective. Only rays enter the lens, which are deflected by the drug particles as a result of reflection, refraction or diffraction. Because of this, microbial cells and other particles appear to glow brightly against a black background (the picture resembles a twinkling starry sky).

For dark field microscopy, a special condenser (paraboloid condenser or cardioid condenser) and conventional objectives are used. Since the aperture of the immersion objective is larger than the aperture of the dark field condenser, a special tubular diaphragm is inserted inside the immersion objective to reduce its aperture.

Polarizing microscopy— one of the highly effective methods of morphological research, which has a wide range of possibilities for identifying biological structures, which, combined with accessibility and relative simplicity, determines its high value. The method allows to study not only the histological structure of the preparation, but also some of its histochemical parameters. In the 40-50s of the XX century. polarization microscopy was considered to be an ultrastructural method, since it made it possible to see the ultrastructural abilities of tissues.

Polarization microscopy is designed to study the properties of histological structures that have the ability of birefringence (anisotropy) - bifurcation of a light beam when it passes through an anisotropic medium. A light wave in an anisotropic medium breaks up into two waves with mutually perpendicular planes of oscillations of electromagnetic waves. These planes are called planes of polarization. Polarized light differs from ordinary (unpolarized) light in that in the latter, light wave oscillations occur in different planes, while in polarized light they occur only in a certain plane.

To create the effect of polarization in a polarizing microscope, two polaroids are used. The first, which is called a polarizer, is placed between the microscope illuminator and the histological preparation. The second polaroid, located between the histological preparation and the researcher's eye, is the analyzer. Both the polarizer and the analyzer are optically exactly the same polarizing filters, so they can be interchanged (if the microscope design allows it). Previously, Nicol, Ahrens, or Thomson prisms made from Icelandic spar were used for polarizing microscopy. These prisms had a limited angle of light refraction. Currently, flat polarizing filters are used instead, producing wide-field polarized light.

The relative position of the polarizer and analyzer relative to the optical axis of the microscope plays a decisive role in the creation of polarized light. If they are oriented in such a way that both transmit polarized light in the same plane, i.e. when their polarization planes coincide, both polarizing filters are capable of transmitting polarized light; the field of view of the microscope is bright in this case (Fig. 1a).

Rice. 1 Human lung preparation in bright field, OlympusCX41, 10x objective

If the planes of polarization of the polarizing filters are mutually perpendicular (this is achieved by rotating the analyzer by 90° around the optical axis of the microscope), then the polarized light does not pass and the researcher sees a dark field of view (Fig. 2).

When the polarizer is rotated 360° during its rotation, the field of view is twice completely darkened and twice completely enlightened. In the past, Bernauer compensatory filters have been used, in which the darkened field of view has a reddish tint ( U-TP530 ). When black specular filters are applied, the darkened field of view does not appear completely dark, but faintly illuminated.

Fig. 2 Human lung preparation in polarized light, 10x objective

In those cases when, with the crossed position of polarizing filters (i.e., in orthoscopies), anisotropic substances contained in a histological specimen are encountered in the path of polarized light, these substances split the polarized light into two beams with mutually perpendicular planes of light wave oscillations. Light rays with a plane of oscillation coinciding with the plane of polarization pass through the analyzer, and with a perpendicular one they are cut off, as a result of which the intensity of the light flux entering the eye of the researcher and the camera is only half the intensity of the initial light beam. As a result of the described processes, anisotropic substances located between two crossed polarizers are visible against a dark background in the form of bright luminous objects. In this case, isotropic structures that do not have the ability of birefringence remain dark.

It also affects the choice cameras for polarizing microscopy. Since the task is to capture small light signals against a dark background, a camera for bright-field microscopy may not usually be sufficient due to the low sensitivity of the camera and the large amount of noise that is generated during shooting. For shooting in polarizing microscopy you need a camera for microscopy with high sensitivity and accurate color reproduction. It is preferable to use cameras based on CCD matrices ( , VZ-CC50S), however, at the current stage, you can also use budget options for cameras based on Sony IMX series CMOS matrices ().

Biological tissues contain a sufficient number of anisotropic structures: elements of the contractile apparatus of muscles, amyloid, uric acid, collagen formations, some lipids, a number of crystals, etc.

The light rays split in an anisotropic object and passing through the analyzer are characterized by unequal wave propagation speed. Depending on the magnitude of this difference (it is also called the amount of delay of the light beam) and from differences in light absorption in the analyzer, the glow of anisotropic objects can be white or colored. In the latter case, we are talking about the phenomenon of dichroism ( double absorption I). Color effects in the study in the field of polarization give, for example, many crystals.

The process of birefringence can be enhanced by the use of certain dyes, the molecules of which have the ability to be oriented deposited on anisotropic structures. Histochemical reactions, which result in the effect of anisotropy, are called topooptical reactions (G. Romhanyi). There are two types of such reactions - additive and inverse. With additive reactions, the delay of the light beam increases, which is called positive anisotropy; with inverse reactions, it decreases - negative anisotropy.

APPARATUS AND EQUIPMENT

Polarizing microscopy is carried out using special polarizing microscopes. As an example, we can name imported microscopes,. Most modern optical microscopes are equipped with accessories for polarizing microscopy.

For polarizing microscopy, any light microscope of laboratory and research grade can be adapted. It is sufficient to have two polarizing filters, one of which, acting as a polarizer, is placed between the light source and the specimen, and the other, which plays the role of an analyzer, is placed between the specimen and the researcher's eye. The polarizer can be built into the condenser or placed below it above the field diaphragm, and the analyzer can be placed in the revolver slot or an intermediate insert.

On fig. 3 is a schematic diagram of a polarizing microscope. In addition to the components common to all light microscopes, a polarizing microscope has two polarizing filters (a polarizer, usually placed under the condenser, and an analyzer, located in the eyepiece), as well as a compensator. The analyzer must necessarily rotate, and an appropriate graduated scale is necessary to determine the degree of rotation.

A polarizing microscope uses an illumination source that provides a high light beam density. A 100 W lamp with a voltage of 12 V is recommended as such a source. For some types of research, monochromatic light is required. For this purpose, a metal interference filter is used, which is best placed above the mirror. The light-scattering frosted glass is placed in front of the polarizer, i.e. between it and the light source, but in no case after the polarizer, since this violates the function of the polarizing filter.

In the past, achromatic objectives without internal tensions were used for polarizing microscopy, but these are now rare. To date, in a polarizing microscope, only plan achromatic objectives are used, which do not have internal tensions. Apochromatic lenses can be used only in cases where normal color reproduction is required for microphotography.

Polarizing microscopes are equipped with a rotating object stage, the position of which relative to the optical axis can be changed. The angle of rotation of the table is measured using a degree scale marked on its circumference. One of the prerequisites for the effective use of polarizing microscopy is the careful centering of the rotating stage using the centering screws.

An important element of a polarizing microscope is a compensator placed between the objective and the analyzer, usually in the microscope tube. The compensator is a plate made from special varieties of gypsum, quartz or mica. It allows you to measure the difference in the path of split light rays, expressed in nanometers. The functioning of the compensator is ensured by its ability to change the difference in the path of light rays, reducing it to zero or increasing it to a maximum. This is achieved by rotating the compensator around the optical axis.

METHOD OF MICROSCOPY IN POLARIZED LIGHT

It is more convenient to carry out polarization microscopy in a darkened room, since the intensity of the light flux entering the researcher's eye decreases by 2 times compared to the original one. After turning on the illuminator of the microscope, the brightest possible illumination of the field of view is first achieved by rotating the polarizer or analyzer. This position of the polarizing filters corresponds to the coincidence of their polarization planes. The drug is placed on the object table and studied first in a bright field. Then, by rotating the polarizer (or analyzer), the field of view is darkened as much as possible; this position of the filter corresponds to the perpendicular arrangement of the planes of polarization. In order to reveal the effect of anisotropy, it is necessary to combine the plane of polarization of an anisotropic object with the plane of polarized light. Empirically, this is achieved by rotating the object stage around the optical axis. If a light microscope is used for polarizing microscopy, which is not equipped with a rotating stage, then it is necessary to rotate the histological preparation manually. This is permissible, however, in this case, it is impossible to carry out certain types of polarizing microscopy that require a quantitative assessment (determination of the sign of birefringence, the magnitude of the difference in the path of light rays).

If anisotropic objects in the studied preparation are arranged in an orderly manner (for example, anisotropic disks of striated muscle fibers), it is convenient to study them in a fixed position of the stage, at which these objects give the maximum glow against a dark background. If, however, anisotropic structures are arranged randomly in the preparation (for example, crystals), then during their study it is necessary to constantly rotate the object table, achieving the glow of one or another group of objects.

To conduct a more in-depth analysis and evaluation of topooptic reactions, it is necessary to know the method for determining the relative sign of birefringence, the magnitude of the difference in the path of rays and the index (coefficient) of refraction.

The sign of birefringence characterizes the degree and direction of displacement of the light rays passing through the analyzer. This shift is caused by topo-optical dyes, and in the event that it is directed towards a decrease in the difference in the path of the rays, one speaks of a negative sign of birefringence ( negative anisotropy), but if it contributes to an increase in the difference in the path of the rays, then the positive sign of birefringence is ascertained ( positive anisotropy). If the difference in the path of the rays disappears, then the effect of anisotropy is leveled.

The sign of birefringence is determined using a compensator. The procedure for its application is as follows. The object under study is placed in a position at which the maximum glow of anisotropic structures is achieved in the dark field of view. The plate of the RI-compensator is rotated around the optical axis at an angle of +45° with respect to the plane of polarization of the analyzer. The object, depending on the difference in the path of light rays, which can range from 20 to 200 nm, acquires either blue or yellow color. In the first case the sign of birefringence is positive, in the second it is negative. It should be borne in mind that in the case when the compensator is located at an angle of +45°, the general background of the darkened field of view has a red tint.

You can also use the λ/4 compensator (U-TP137). The procedure for its application is the same, only the field of view has a gray tint instead of red, and the object glows with a positive sign of refraction, and is darkened with a negative one.

Quantitative determination of the difference in the path of light rays, expressed in nanometers, is carried out using the Köhler Braque compensator. To do this, use the formula:

Γ=Γλ×sinφ

where λ is a constant put on the compensator by the manufacturer, φ is the angle of rotation of the compensator relative to the analyzer polarization plane.

The refractive index of an anisotropic object is determined by comparing it (under a microscope) with a test object placed nearby. Standard liquids with a known refractive index are used as test objects. The object and sample are placed side by side on the stage. If their refractive indices do not match, a bright line is visible between the object and the sample, called the Beck line. Raising the microscope tube relative to the focused position causes a shift of the Beck line towards the medium, which gives a more pronounced effect of refraction. When the coefficients of refraction of the object and the sample coincide, the Beck line disappears. Usually, the refractive index is determined in monochromatic light for the sodium line of the spectrum (at a wavelength of 589 nm and a temperature of 20 ° C). The refraction should be determined for two mutually perpendicular planes of polarization. For this purpose, the analyzer is removed and the refraction of the object is recorded in its two mutually perpendicular positions. The difference between the two refractive indices (ng - nk) characterizes the refractive power.

FEATURES OF PROCESSING MATERIAL AND PREPARATION OF PREPARATIONS

Fixation of the material for polarization microscopy in acid formalin is undesirable, since the formalin pigment formed during the interaction of tissue hemoglobin with acid formaldehyde has anisotropic properties and makes it difficult to study preparations in polarized light. G. Scheuner and J. Hutschenreiter (1972) recommend using 10% neutral formalin, Baker's calcium-formol solution, Carnoy's liquid for this purpose.

The duration of fixation in 10% neutral formalin is 24-72 hours at 4°C, in calcium-formol solution according to Baker - 16-24 hours at 4°C. Fixation in calcium formol is especially preferred in the study of lipid-protein compounds. Carnoy's fluid quickly permeates fabrics. Pieces with a thickness of 1 - 2 mm are profiled after 1 hour at a temperature of 4 ° C. For the study of lipids, fixation in Carnoy's fluid is unsuitable. In addition, Zenker's liquid is used, especially when impregnating with salts of gold and silver. After treatment with a mixture of Zenker's liquid and acetic acid, erythrocytes acquire the ability to birefringence.

When examining dense tissues (bones, teeth) in a polarizing microscope, in addition to acid decalcification, additional processing is necessary to remove collagen fibers. For this purpose, sections of such tissues are boiled for several minutes in a mixture of glycerol and potassium hydroxide (10 ml of glycerol and 2 grains of potassium hydroxide) until completely white, then the alkali is carefully drained, the section is washed in water and transferred with tweezers to the microscope stage.

For polarizing microscopy, paraffin, frozen and cryostat sections are used. Unstained frozen sections for polarized light examination are embedded in glycerol. Unfixed cryostat sections are suitable for polarization microscopic analysis immediately after preparation. Due to their high sensitivity to the damaging effect of various environmental factors, these sections are still recommended to be fixed in 10% neutral formalin or calcium-formol solution.

The results of polarizing microscopy are influenced by the thickness of histological sections. When studying thick sections, conditions are created for superimposing different anisotropic structures on top of each other. In addition, the anisotropic properties of the structures under study can change at different slice thicknesses; therefore, it is very important, especially in comparative studies, to ensure a constant slice thickness. The recommended maximum slice thickness should not exceed 10 µm.

Another prerequisite is careful deparaffinization of the sections, since unremoved paraffin residues give a pronounced anisotropy effect, making it difficult to study. Paraffin lingers especially long on erythrocytes and cell nuclei. In order to completely remove paraffin from sections, it is recommended to carry out their following processing.

Xylene 30 min
Alcohol 100% 5 min
Mixture of methanol and chloroform (1:1) at 50 °С 24 h
Alcohol 100% 5 min
Alcohol 70% 10 min Water

It should also be borne in mind that sections that are subjected to polarization microscopy should not come into contact with phenols (for example, they cannot be cleared in carboxylic acid).

More information on polarizing microscopy and the use of compensators can be found at (http://www.olympusmicro.com/primer/techniques/polarized/polarizedhome.html).

If you have any questions about polarizing microscopy, please contact the School of Microscopy.