Glossary:

Polarized light is light waves that vibrate in one direction.

A light wave is an electric and magnetic radiation with a plane of oscillation perpendicular to the plane of propagation of the wave.
A polarizer (Nicol I) is a device that allows only fully or partially polarized light to pass through. Designed to transmit polarized light to (through) the transparent object under study and cut off (scatter) non-polarized light (natural light, artificial light, including radiation from a microscope illuminator). The intensity of light passing through the polarizer falls in proportion to the square of the cosine of the angle between the polarization planes of the polarizer and analyzer (Malus's law):

Where: I is the intensity before passing through the polarizer, I is the intensity of the light after passing through the polarizer, φ is the angle between the polarization planes of the polarized light and the polarizer.

Analyzer (Nicol II) - a device similar to a polarizer, but designed to analyze polarized light.

Rotation of the analyzer relative to the polarizer by an angle ϕ. Light intensity is shown by the red arrow.

The compensator is a device for determining quantitative characteristics polarization. Converts a high-contrast visible image into a color image by attenuating certain wavelengths in white light.
Linearly polarized light is light with a plane of oscillation bounded in one direction and propagating in one plane.
The phase of the light wave oscillations, from a mathematical point of view, is the argument of the light wave function, that is, ωt+φ 0 in the sin(ωt+φ 0) function. Physically, it is a certain electromagnetic state at a certain point in time.

Wavelength is the distance between two nearest points that are in the same phase.

Reflection is a change in the direction of the wave. Full reflection is called a change in the angle of refraction of the wave less than 90 °.

Refraction is a change in the direction of a wave at the boundary of two media. Birefringence is the splitting of one beam of light in an anisotropic medium into two beams.

Figure 4 - Refraction of rays in a crystal of Icelandic spar.

Dichroism is the partial absorption of light by a substance, depending on its polarization.

Interference is the change in light intensity when two or more light waves are superimposed.

The difference in the path of light rays is a value that characterizes the deceleration of the speed of light when passing through a transparent substance. The path difference is measured by the distance traveled by light in vacuum for the same time that is necessary for passage in the substance under study, at the studied points in space.

Conoscopy is a method for studying the optical properties of anisotropic objects in converging beams of polarized light. During conoscopy, changes in the interference pattern are monitored when the analyzer is rotated. Rotating the analyzer and polarizer relative to each other, the researcher observes conoscopic figures in the microscope, consisting of isogyres (these are dark bands corresponding to the direction of oscillation of light waves in the polarizer) and isochromes (these are bands of different interference colors that correspond to the directions of the rays in the crystal with the same path difference).

Orthoscopy is a method for studying the optical properties of anisotropic objects in parallel beams of polarized light.

Pleochroism is a change in the observed color of some anisotropic objects with a change in the viewing angle (change in the color of crystals when the table is rotated).

A polarizing microscope is a microscope designed to study the birefringence of polarized light passing through an anisotropic medium.

The first polarizing microscope was designed in 1863 by Henry Clifton Sorby and differed from the optical microscope we are used to by two Nicol prisms installed in the optical path. The Nicol prism transmits light through itself only in one direction and in one plane, that is, plane polarized light, the rest of the light that enters these prisms is completely reflected and scattered. These prisms structurally do not differ from each other and act as polarizers (analyzer and polarizer). When the analyzer's polarization plane is rotated by 90º relative to the polarization plane of the polarizer, the researcher observes the polarization pattern of a birefringent object, and all objects that do not have birefringence are darkened. In modern microscopes, to obtain more For information, DIC prisms (combining the relief with the polarization pattern, for studying uncolored samples), compensators (for quantitative polarization), a round table (for studying pleochroism) and simple polaroids for simple observations (for example, in biology and medicine) can be used.

Polarization is most often used in crystallography microscopes, where the properties of anisotropic objects can be determined using conoscopy and orthoscopy. Pay attention to the similarities and differences between conoscopy and orthoscopies: the light beam passes through the polarizer (1), is limited by the aperture diaphragm (2), passes through the condenser lenses (3); analyzer (which turns the researcher) (8) and compensators (7).

Figure 1 - Scheme of a polarizing microscope for: a) Orthoscopy b) Conoscopy

Legend: 1 - polarizer, 2.6 - diaphragms; 3 - condenser; 4 - preparation; 5 - lens; 7 - compensator; 8 - analyzer; 9 - Bertrand lens; 10 - focal plane of the eyepiece; 11 - eyepiece.

The observed picture consists of conoscopic figures. conoscopic figures - consist of isogyres (these are dark straight or curved stripes in which the directions of vibration are parallel to the main sections of the nicols) and isochromes (these are stripes painted in different interference colors. Each strip corresponds to the directions of the rays formed during birefringence and having the same path difference ).

Let us give an example: in the plates of a uniaxial crystal cut perpendicular to the optical axis, we will see an isogyra in the form of a cross and concentric isochrome rings, see Fig. 5.

Figure 5 - A) Conoscopic figures of a uniaxial calcite mineral B) Biaxial phlogopite mineral with an inserted compensator.

By the nature of the obtained interference pattern, the birefringence value, the angles of rotation of the polarization plane, the extinction angles, the number of optical axes and other characteristics are measured. All these characteristics make it clear which crystal the researcher is observing, its structure. Microscopes such as the BX53P and H600P have been designed for mineralogy and crystallography. They are equipped with the best stress-free optics and compensators made on modern equipment, eliminating play and gaps when they are installed in the microscope.

Birefringence is used not only in crystallography, but also in medicine, biology, forensics and metallography, because it is important for researchers to quickly and accurately isolate vitamins, acids, minerals, stresses in isotropic objects, non-metallic inclusions in the original sample, and others. For example, microscopes for histology and cytology are equipped with polarizers to detect various kinds of objects. Round objects with a diameter of about 2.4 microns, lipoids and drops, with crossed polarizers form an interference pattern of the Maltese cross. Not all substances have the same properties of refraction at different temperatures, so, for example, one can distinguish 1) substances that acquire anisotropic properties when cooled and lose them when heated: cholesterol and its esters 2) do not lose their anisotropic properties when heated: cerebrosides, phosphatides, myelins. Such variability of properties is due to the ability of a substance to maintain a crystalline structure, tk. This is what causes the birefringence. Observing anisotropic objects in a polarizing microscope and determining their concentration, one can diagnose such diseases as: arthritis, atherosclerosis, lipoiduria, cilinuria and lipidosis by the glow of lipids with crossed polarizers, as well as gout, urolithiasis, selicosis and asbestos by crystals of urea, silicon dioxide and asbestos fibers, respectively. For histology and cytology, the BX46 microscope has been developed, which is equipped with a low stage, a powerful illuminator and a height-adjustable tube, which will save the researcher's back from wicking.

Coloring different from isotropic objects in polarized light is: starch, cellulose, some acids, vitamin C, therefore microscopes for pharmacology and pharmaceutics should also be equipped with polarizers. A pharmacological microscope includes both CX43 and BX43, and other models, because there is more and more research in this area every year, and new research objects require a different approach.

In forensic science, it is important to distinguish inclusions of grains of quartz and other minerals from organics and other materials that can be found at a crime scene, so the microscope must be equipped with reflected light to view opaque objects as well. The BX53M microscope is suitable for forensics, as it is equipped not only with a powerful source of transmitted light, but also with the same powerful reflected light illuminator, and inserts to increase the working distance of the microscope will allow you to study very large objects without long preliminary preparation.

Polarizing microscopes are also used in metallography, but for such studies it is enough to know the presence or absence of anisotropic objects, as well as their spatial distribution. It is for the classification and counting of such objects that VHX6000, BX53P microscopes with Stream installed can be used in metallography.

Of all the variety of devices for microscopy, polarizing microscopes are the most technically complex. Such attention to the design of the device in terms of manufacturability is due to the need to obtain an image highest quality, which is directly affected by the design of the optical and illumination parts of the microscope. The main field of use of polarizing devices for microscopy is the study of minerals, crystals, slags, anisotropic objects, textile and refractory products, as well as other materials that are characterized by birefringence. The latter principle is the basis for image formation in such devices for microscopy, in which the sample under study is irradiated with polarization beams. In this case, the anisotropic properties of the samples appear after changing the direction of the beam. For these purposes, polarizing microscopes are designed with field filters rotating in different planes relative to each other: the analyzer rotates 180 degrees, and the polarizer rotates 360. other types of microscopes.

The study of the sample under a polarizing microscope begins with the installation of a polarizer in the illuminating part of the microscope under the condenser, next to the aperture diaphragm. At the same time, the analyzer is located between the eyepiece and the lens - behind the latter along the path of the light rays. With the correct setting of such an instrument for microscopy, after crossing the filter fields, the visible field will be uniformly dark, forming the so-called extinction effect. Upon completion of the device settings, the test sample is fixed on the stage and its study is carried out. The stages of polarizing microscopes are centered relative to the optical axis and can be rotated 360 degrees, and in similar devices for laboratory and research purposes, they also have a vernier. The optics and illumination system of the polarizing microscopes are of the highest quality and of such precision manufacturing that allows you to get the clearest image without distortion. Often, a set of devices for studying samples in polarized light includes a compensator and a Bertrand lens. The first one makes it possible to effectively study the structure of minerals, and the lens - to increase and focus the area of observation when changes in the image appear after the turn of the stage. Today, there are three main types of such devices for microscopy on the market - these are the already mentioned research and laboratory ones, as well as a working polarizing microscope.

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 nucleic acids in the total dry weight of the 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.

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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. Main characteristic of a light microscope is the resolution of a light microscope, determined by the wavelength of 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 length waves 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 in more detail internal structure cells.

For example:

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

after treatment successively with phloroglucinol and then hydrochloric acid 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 drug structure that differ from environment 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 resolution of modern transmission electron microscope almost 400 times more light. 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 by 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 scanning transmission electron microscopes high resolution get images of individual molecules or atoms heavy elements. 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 phenomena due to the Lorentz force are studied, are internal magnetic and electric fields or external stray fields, for example, 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. The polarization imparted to it in this case 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 obtained negatives is illuminated by visible light. certain color(for example, blue, green and red), and they are all projected onto one screen at the same time. 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. Microphotography and microcine photography require some adjustment optical system microscope - different compared to the visual observation of the focusing of the eyepiece relative to the image given by the lens. 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 in the study of processes unfolding in time (the vital activity of tissue cells and microorganisms, the growth of crystals, the flow of protozoa chemical reactions and so on.).

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. IN optical design microscope, two light filters are introduced. 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, under a sliding (small to the surface) angle, a layer of highly electron-scattering heavy metal, 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.

are widely used to obtain cell fractions. different kinds centrifugation: differential centrifugation, zonal centrifugation and equilibrium density centrifugation. 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 cell fragments remain in the supernatant. Fragments cell wall 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 h to precipitate.

Zonal centrifugation

Zonal centrifugation is effective method separation of structures having a similar floating density, but differing in shape and mass of particles. Examples include the separation of subunits of ribosomes, different classes of polysomes, and DNA molecules that have different shape. 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) looks like 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 - cell membrane, cytoplasm and nucleus, and then these components can be reassembled and obtained living cell. In this way, artificial cells can be obtained, consisting of components different types amoeba

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 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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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 as light rays pass through different structural components 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.

polarized microscope. Polarizing microscopes: features and principle of operation. A polarizing microscope is a microscope designed to study the birefringence of polarized light passing through an anisotropic medium.

Glossary:

A polarizing microscope is a microscope designed to study the birefringence of polarized light passing through an anisotropic medium.

Phase contrast microscopy method

Interference contrast method (interference microscopy)

Polarizing microscopy

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Polarizing microscopy

Dark field microscopy

Glossary:

A polarizing microscope is a microscope designed to study the birefringence of polarized light passing through an anisotropic medium.

Phase contrast microscopy method

Interference contrast method (interference microscopy)

Polarizing microscopy

Send your good work in the knowledge base is simple. Use the form below

Similar Documents

Polarizing microscopy

Dark field microscopy

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