in microbiology: Microscope device and basic techniques of microscopation of living microorganisms

1 Features of different types of microscopy

2 Slight Microscope Device

3 Terms of work with an immersion lens

4 microscopic receptions of living microorganisms

1 Features of different types of microscopy

The main tasks of microscopy are the following:

Detection of microorganisms in various materials.

Approximate identification of microorganisms in the sample.

Study some morphological signs and the structures of microorganisms (for example, capsules, flavors, etc.).

Studying painted smears from colonies and pure crops.

To date, the most used is light microscopy.

Light microscopy Provides an increase of up to 2-3 thousand times, color and movable image of a live object, the possibility of microckering and long-term observation of the same object, evaluating its dynamics and chemicals. The image in the light microscope is formed due to the fact that the object and its various structures selectively absorb light with different wavelength (absorption contrast) or due to the change in the phase of the light wave when light passes through the object (phase contrast).

The main characteristics of any microscope are resolution and contrast. Resolution - This is the minimum distance on which there are two points, demonstrated by a microscope separately. The resolution of the human eye in the best vision mode is 0.2 mm. Contrast image- This is the difference in brightness of the image and background. If this difference is less than 3-4%, it is impossible to catch it either by an eye or a photoplastic; Then the image will remain invisible, even if the microscope allows its details. The contrast affects both the properties of an object that change the light stream compared to the background and the ability of the optics to catch the differences in the properties of the beam. The possibilities of the light microscope are limited to the wave nature of light. The physical properties of the light - the color (wavelength), brightness (wave amplitude), phase, density and direction of the wave propagation varies depending on the properties of the object. These differences are used in modern microscopes to create contrast.

Increase microscope Determined as a product of an increase in lens to an increase in eyepiece. In typical research microscopes, an increase in eyepiece is 10, and an increase in lenses is 10, 40 and 100. Accordingly, an increase in such a microscope is from 100 to 1,000. Some of the microscopes have an increase of up to 2,000. An even higher increase does not make sense, since In this case, the resolution does not improve. On the contrary, the quality of the image is worsening.

Numerical apertura Used to express the resolution of the optical system. The numerical aperture is an optical "coverage" of lenses, it is a measure of the amount of light falling into the lens. The numeric aperture of the lens is indicated on its rim. The condense aperture must match the lens numeric aperture. The numerical aperture of any lens bordering the air (that is, the "dry system") cannot exceed 1, since the refractive index of the air is equal to 1. The numeric aperture can be enhanced if you increase the refractive index between the front lens lens and the object glass, Approaching it to the refractive index of glass (1.5). To do this, a drop of fluid is placed between the frontal lens of the lens and the test object with a refractive index greater than the refractive index of the air, for example, a drop of water (n \u003d 1,3), glycerol (n \u003d 1.4) or cedar (immersion) oil (N \u003d 1.5). For each of the above liquids, special lenses are manufactured, which are called immersion.

Light microscopyincludes ordinary translucent microscopy (light, dark-axis), phase-contrast, luminescent. Recently, other methods of microscopy and microscopes have been developed - inversion and Confocal laser scanning microscopy.

Sveta microscopy Allows you to explore objects in the transmitted light in a light field. This type of microscopy is designed to study the morphology, cell size, their mutual location, structural organization of cells and other features. The light microscope has a maximum resolution of 0.2 μm, which ensures a high-precision increase in the microscope up to 1500x.

Phase-contrast microscopy Allows you to more clearly observe live transparent objects that have refractive indexes close to the refractive indices of the medium. The action of the phase-contrast microscope is based on the interference of light in the plane of the image due to a phase shift (when using a phase ring in aperture diaphragm). In phase-contrast microscopy, biological microscopes with reverse optics are used - inverted microscopes. In such microscopes, the lenses are located below, and the condenser is from above.

With the help of phase-contrast microscopy, the shape, dimensions, the relaxation of cells, their mobility, reproduction, germination of microorganisms, etc., thanks to the use of this method of microscopy, the contrast of living unpainted microorganisms sharply increases and they look dark on a bright background (positive phase contrast ) or bright on a dark background (negative phase contrast).

Damnopol microscopy Based on the illumination of the object with oblique rays of light. With such lighting, the rays do not fall into the lens, so the field of view looks dark. Such lighting of the drug is achieved by using a special dark-axis condensor. Damnopoly microscopy is a very simple, but efficient method and well suited for obtaining images of living and unpainted biological samples. Given the simplicity of installation, the quality of the obtained images is very good.

When microscopation in a dark field, you can see objects whose value is measured by hundredths of the micrometer, which is beyond the limits of the resolution of a conventional light microscope. However, observation of objects in the dark field allows you to explore only the contours of the cells and does not make it possible to consider their internal structure.

Luminescent (fluorescent) microscopy It is based on the ability of a number of substances of biological origin or some dyes to glow when they are illuminated by invisible ultraviolet or blue light. When using ultraviolet light, the microscope resolution can reach 0.1 μm.

The cells of microorganisms are treated with special dyes - fluorochromas (acridine orange, primulin, rhodamine, etc.) in the form of strongly diluted aqueous solutions: 1: 500-1: 100,000. Such solutions are weakly toxic, which makes it possible to study intact cell. Depending on the chemical composition, the cellular structures are in different degrees adsorb dyes and luminescent differently. In addition, fluorochromes are not detaine adsorbed by alive and dead cells. This allows you to use this species Microscopy for cytological and immunological studies, determining the viability of cells, etc.

Electronic microscopy. Allows you to detect objects that are not allowed when using light or ultraviolet rays. In theory resolution translucent electron microscope is 0.002 nm; Real resolution of modern electronic microscopes approaches 0.1 nm. In practice, permission for biological objects reaches 2 nm.

The short wavelength of the electron allows you to distinguish objects with a size of 0.5-1.0 nm. In modern electronic microscopes, an increase of 5000-200,000 is achieved on the screen. Due to such a high resolution, it becomes possible to identify the details of bacterial structures. For example, by spraying salts of heavy metals surrounding bacteria and penetrating surface irregularities, contrasts are obtained by the differential delay of electrons. This effect was called negative contrast .

An electron microscope in which the image is formed due to the passage (translucent) of electrons through the sample, called translucent (or transmission ).

IN scanning electron microscope (Raster electron microscopy (RAM) The electron beam quickly scans the sample surface, causing radiation that generates an image on the glowing screen. The RAM is characterized by a high resolution, a large range of zoom (up to 100 000 and higher), a large focus depth (~ 100 μm), a variety of operating modes. The scanning microscope gives a picture of the surfaces and allows you to get a three-dimensional image.

Laser confocal microscopy It makes it possible to get a distinct image and observe objects in focus throughout the field. This method is only suitable for the study of self-losing (fluorescent) objects. When combined with the computer equipment, the spatial reconstruction of the object being studied is possible. In the confocal laser scanning microscope, the images of internal sections are formed by scanning a focused laser beam from different (405, 488, 532, 635 nm) lasers and spatial filtering of radiation. When using a near-field scanning microscopy (SMBP), a high resolution is achieved. The smallest size of the element obtained by the SMBP is 20 nm at a wavelength of light 0.486 nm. In the image of the controlled element, there are no diffraction or interference effects that make it difficult to determine its boundaries. A distinctive feature of the SMBP compared to the atomic-power microscope is the sensitivity to the optical characteristics of the surface of the controlled sample, the wavelength of light, luminescence, etc.

Computer interference microscopy allows you to get a high-contrast image when observing subcellular structures; In many cases, it is used to explore living cells. The principle of operation of an automated interference microscope is based on the interference of the light beams of laser radiation, reflected from the support mirror and the mirror, on which the measured phase object is placed. Theoretically, the extremely achievable resolution can be on average 0.2 nm, it is almost 0.4 μm.

X-ray computed tomography (RTC), positron emission tomography (PAT) Allow objects to observe objects under normal conditions.

Histological technique. Microscopic Methods and Technology

Objective: Get acquainted with the principles of work and use of instruments of special microscopy in research purposes. Secure the skill of microscopation of the histological drug.

the task:

1. Fill in Table 2, noting the main types of microscopy, their varieties, briefly formulate the purpose of using each species.

table 2

Microscopic Methods and Technology

1. Light microscopy.Conventional light microscopes and their varieties are used, in which light sources with different wavelengths are used. In the light microscope, not only individual cells of 4 to 150 μm in size can be seen, but also their intracellular structures - organelles and inclusions. To enhance the contrast of microjects, their staining is used.

a) ultraviolet microscopy.Used shorter ultra-violet rays With a long wave of about 0.2 microns. The image obtained by the eye is converted to visible by registering on a photoplastic or by applying special devices (fluorescent screen, electronic optical converter).

b) Fluorescent (luminescent) microscopy.The essence of the method is that the atoms and molecules of the row of substances, absorbing short-wave rays, go to the excited state. The reverse transition from the excited state into normal occurs with the emission of light, but with a greater wavelength. Ultra-high pressure huts and high-grade housing lamps are used with high brightness in the neighboring ultraviolet and blue-purple rays. Any cell of the living organism has its own fluoroscience (often rather weak).

Distinguish:

Primary fluorescence - serotonin, catecholamines (adrenaline and norepinephrine), contained in nervous, fat and other cells, after fixing tissues in formaldehyde pairs (Fold method).

Secondary fluorescence occurs when processing drugs with special dyes - fluorochromas.

c) phase-contrast microscopy.This methods for obtaining contrast images of transparent and colorless live objects invisible in conventional microscopation methods. For this, unpainted structures are placed in a ring diaphragm placed in a condenser, and a phase plate located in the lens. Such a design of optics makes it possible to transform non-perceived by the phase changes in the light preparation of light into the change of its amplitude, i.e. Brightness of the resulting image.

d) microscopy in a dark field.It reaches the object lented light, which gives the diffraction of structures in the preparation. The microscope has a special condenser, which illuminates the drug strictly slamming. Thus, the field looks dark, and small particles The drug reflects the light, which further falls into the lens. This method is used to study live objects, such as silver grains that look light on a dark field. In the clinic it is used to study crystals in the urine ( uric acid, oxalates), to demonstrate spirochet, etc.

e) interference microscopy.A differential interference microscope is used (with nomaric optics), which is used to study the relief of the cell surface and other biological objects.

In this microscope, the light beam from the illuminator is divided into two streams: one passes through the object and changes the oscillation phase, the second goes, bypassing the object. In the prisms of the lens, both beams are connected and interferred with each other. As a result, an image is built in which the portions of the micro - different thickness And densities differ in the degree of contrast. Conducting a quantitative assessment of changes, the concentration and mass of dry matter determine.

The advantage of such microscopy is the ability to observe cells in the process of movement and mitosis. At the same time, the registration of cell movement can be made using frame microker.

e) dark-based microscope It is used to obtain images of transparent living objects. The sample is considered in it with such a "braid" lighting that the direct light does not have the ability to get into the lens. The image is formed by light diffeped on the object, and as a result, the object looks very light on a dark background (with very large contrast).

2. Polarization microscopy.The polarization microscope is a modification of the lighting microscope in which two polarization filters are installed - the first (polarizer) between the light beam and the lens, and the second (analyzer) between the lens lens and the eye. Both filters can rotate by changing the direction of the beam of light. Structures containing longitudinally oriented molecules (collagen, microtubule, microfilaments), and crystalline structures (in lesidig - balllocites of eggs) when the rotation axis changes appear as glowing. The ability of crystals or paracryistallic formations to split the light wave per ordinary and perpendicular to it is called double beamplan. The fibrils of cross-striated muscles have such ability.

3. Electronic microscopy.Considering the characteristics of the light microscope, you can make sure that the only way to increase the resolution of the optical system will use the lighting source emitting the wave with the smallest length. Such a source can be a hot thread that throws the electron flux in the electric field, the latter can be focused, passing through the magnetic field. This served as the basis for creating an electron microscope, in which a resolution of 0.1 nm is already reached. On the principle of construction, an electronic microscope is very similar to optical: it has a light source (e-gun cathode), a condenser system (condenser magnetic lens), lens (objective magnetic lens), eyepiece (projection magnetic lenses), only instead of retina electrons enter the electron Luminescent screen or photo plastic. The electron microscope uses the electron flow, with a shorter than in a light microscope, wavelengths. Resolved distance of 100,000 times less than in a light microscope. In modern electron microscopes, the resolved distance is about 0.1-0.7 nm.

Currently, transmission and scanning electron microscopes are used, which have a greater depth of field, a wide range of continuous increase changes (from 10-cages to 10 thousand times) and high resolution.

2. Consider the structure of the light microscope. Repeat the rules for working with it.

Working with a microscope. A typical biological microscope device (Fig. 1). The tripod stand is performed in the form of severe casting. It is attached to her hinge a tube holder, carrying all other parts of the microscope.

With the help of a tube in which the lens systems are mounted, they can be moved relative to the sample for focusing. At the lower end of the tube there is a lens.

As a rule, the microscope is equipped with several lenses of different zoom in the revolving head, which allows them to be installed in the working position on the optical axis. In the study of the sample, the operator usually begins with a lens, which has the smallest increase and the most wide field of view, finds its items that interests it, after which it considers them using the lens with a large increase.

The eyepiece is mounted at the end of the retractable holder, with which you can change the length of the tube if necessary. Moving up and down the entire tube with lens and eyepiece, the microscope is on sharpness.

As a sample, a very thin transparent layer or a slice is usually taken, which is placed on a glass plate of a rectangular shape, called the size glass, and from above are covered with a thinner glass plate of smaller sizes, which is called coating glass. To increase the contrast, the sample is often stained with chemicals.

The subject glass is put on the subject table so that the sample is over the central opening of the table. The table is usually supplied with a mechanism for smooth and accurate movement of the sample in the field of view.

The third system of lenses - condenser - concentrates light on the sample. The holder of condensors, which can be somewhat, is under an alignment table. Here is the iris diaphragm for adjusting the aperture. Even below is the lighting mirror installed in a universal hinge. Due to the fact that the mirror discards the light of the lamp to the sample optical microscope system and creates a visible image.

Fig. 1. Microscope for biological research.

A-general view: 1 - base; 2 - Tube holder; 3 - Tubus; 4 - microfering mechanism box; 5 - revolving device; 6 - a substantive table; 7 - macrometer screw; 8 - micrometric screw; 9 - condensor screw; 10 - eyepiece; 11 - lenses; 12 - condenser with iris diaphragm; 13 - mirror; B - Small lenses (a), large (b) and immersion (c) increase.

3. Consider the microspections (Table 3), sketch, sign. Specify the type of dye and increase.

Table 3.

Tissue preparations with different staining

The main method of studying biological microjects is light and electron microscopy, which are widely used in experimental and clinical practice.

Microscopation is the main method of studying micro-lectures used in biology more than 300 years. To study histological preparations, a variety of light microscopes and electron microscopes are used. Since the creation and application of the first microscopes, they are constantly improved. Modern microscopes are complex optical systemsHolding a high resolution. The size of the smallest structure itself, which can be seen using a microscope is determined by the smallest resolved distance (D), which mainly depends on the wavelength of light (λ) and the wavelength of electromagnetic fluctuations of the electron flow, etc. This dependence is approximately determined by the formula d.\u003d λ / 2. Thus, the smaller the wavelength, the less resolved distance, and the smaller the microstructure can be seen in the preparation.

Light microscopy.In order to study histological microjects, conventional light microscopes and their varieties are used, in which light sources with waves of different lengths are used. In conventional light microscopes, a natural or artificial light is served as a light source (Fig. 2.1). The minimum wavelength of the visible part of the spectrum is about 0.4 microns. Consequently, for a conventional light microscope, the smallest resolved distance is approximately 0.2 μm, and the overall increase (the product of the increase in the lens to an increase in eyepiece) can be 1500-2500.

Thus, with the help of a light microscope, not only individual cells with a size of 4 to 150 μm, but also their intracellular structures - organelles, inclusions. To enhance the contrast of microjects, their staining is used.

Ultraviolet microscopy.This is a kind of light microscopy. In the ultraviolet microscope, shorter ultraviolet rays with a wavelength of about 0.2 μm are used. The resolved distance here is 2 times less than in conventional light microscopes, and is approximately 0.1 microns. The image obtained in ultraviolet rays The image is converted to visible by registering on a photoplastic or by applying special devices (luminescent screen, electronically optical converter).

Fluorescent (luminescent) microscopy.Fluorescence phenomena consist in the fact that the atoms and molecules of a series of substances absorbing short

Fig. 2.1.Microscopes for biological research:

but- Luminous biological microscope "Biolam-C": 1 - base; 2 - Tu-buckger; 3 - inclined tube; 4 - eyepiece; 5 - revolver; 6 - lenses; 7 - table; 8 - condenser with iris diaphragm; 9 - condensor screw; 10 - mirror; 11 - micrometric screw; 12 - macrometer screw; b.- Electronic EMV-100 microscope with an automated image processing system: 1 - microscope column (with electron-optical system and sample chamber); 2 - control panel; 3 - camera with luminescent screen; 4 - an image analysis unit; 5 - video signal sensor; in- confocal microscope: 1 - light microscope; 2 - image recorder (photoelectron multiplier);

3 - Scanning device for moving light beam along the axis X, y, z;

4 - power supply and lasers control stand; 5 - computer for image processing

wave rays, go to an excited state. The reverse transition from the excited state into normal occurs with the emission of light, but with a greater wavelength. In the fluorescent microscope, mercury or xse-non-high-pressure dials with high brightness in the range of 0.25-0.4 μm (near ultraviolet rays) are used as sources of light for the excitation of fluorescence, which have a high brightness in the range of 0.25-0.4 μM - pure rays). The length of the light wave of fluorescence is always greater than the wavelength of the exciting light, so they are separated using light filters and study the image of the object only in the light of fluorescence. They distinguish their own, or primary, and induced, or secondary, fluorescence. Any cell of a living organism has its own fluorescence, but it is often extremely weak.

Primary fluorescence is serotonin, catecholamines (adrenaline, norepinerenaline), contained in nervous, fat and other cells, after fixing tissues in formaldehyde pairs at 60-80 ° C (Fold method).

Secondary fluorescence occurs when processing drugs with special dyes - fluorochromas.

There are various fluorochromes that are specifically binding to certain macromolecules (acridine orange, rhodamine, fluorescene, etc.). For example, when processing drugs, acridine orange DNA and its compounds in cells are bright green, and RNA and its derivatives are bright red glow. There are many dyes with which you can reveal proteins, lipids, intracellular calcium ions, magnesium, sodium, etc. Thus, the spectral composition of radiation carries information about the internal structure of the object and its chemical composition. The variant of the method of fluorescent microscopy at which the excitation and the radiation of the fluorescence occur in the ultraviolet region of the spectrum, received the name of the method of ultraviolet fluorescent microscopy.

To increase the contrast of fluorochromed objects applied confocal optionoptical microscope (see Fig. 2.1, B). As lighting, a bunch of monochromatic light of a small diameter, which creates laser source. At each moment of time in the focus of the microscope there is a small plot (volume) of the cell. The light beam moves over the object (scans the object on the axes X, y, z).Each time of moving the light beam, one of the scanning lines, information on the studied structure, which is at this point (volume) on the scanning line (optical cell section), for example, the localization of proteins in the microtubule in the cell is obtained. All information received from each cell scan point is transmitted to a computer, combined with special Program And it is issued on the monitor screen as a contrast image. With this method of microscopy, information is obtained about the form of cells, cytoskel, the kernel structure, chromosome, etc. With the computer, the computer is based on the received information on each scan line creates a volumetric cell image, which makes it possible to consider the cell at different angles of view.

Phase-contrast microscopy.This method is used to obtain contrast images of transparent and colorless living objects, invisible in conventional microscopation methods. The method is based on the fact that light, passing structures with a different refractive index, changes its speed. The design of the microscope optics uses the possibility of transforming the phase changes that have not been perceived by the eye transmitted through the non-colored light into changes in its amplitude, i.e. the brightness of the resulting image. The phase contrast method ensures the contrast of the studied unpainted structures due to the special ring diaphragm placed in the condenser, and the so-called phase plate in the lens. A variety of phase contrast method is a phase-dark-gas contrast method, which gives a negative image compared to a positive phase contrast.

Microscopy in the dark field.In a dark-axis microscope, only light that gives diffraction (envelope by waves) structures in the preparation, reaches a lens. This happens due to the presence of a special condensor in the microscope, which illuminates the drug strictly slamming; Rays from illuminator are directed on the side. Thus, the field looks dark, and small particles in the preparation reflect the light, which further falls into the lens. In the clinic, this method is used to study crystals in the urine (urine acid, oxalate), to demonstrate spirochetes, in particular Treponema Pallidum,causing syphilis, etc.

Interference microscopy.The varieties of the phase-contrast microscope are the interference microscope, which is designed to quantify the mass of the tissue. Differential interference microscope (with nomena optics) is used to study the relief of the surface of cells and other biological objects.

In the interference microscope, the light beam from the illuminator is divided into two streams: one passes through the object and changes through the oscillation phase, the second goes, bypassing the object. In the prisms of the lens, both beams are superimposed on each other. As a result, an image is built in which the micro-line areas of different thickness and density differ in the degree of contrast. Conducting a quantitative assessment of changes, the concentration and mass of dry matter determine.

Phase-contrast and interference microscopes allow you to study live cells.They use interference that occurs when combining two sets of waves and creating an image of microstructures. The advantage of phase-contrast, interference and dark-colored microscopy is the ability to observe cells in the process of motion and mitosis. At the same time, the registration of cell movement can be carried out using a zeitrafer (sample) microvidection.

Polarization microscopy.The polarization microscope is a modification of the lighting microscope in which two polarization filters are installed: the first (polarizer) is between the light beam and the object, and the second (analyzer) is between the lens lens and the eye. Through the first filter, the light passes only in one direction, the second filter has the main axis,

which is perpendicular to the first filter, and it does not miss the light. The effect of a dark field is obtained. Structures containing longitudinally oriented molecules (collagen, microtubule, microfilaments), and crystalline structures, have the property of rotating the axis of light rays, emanating from the polarizer. When changing the axis of rotation, these structures appear as glowing on a dark background. The ability of crystals or paracryistallic formations to split the light wave per ordinary and perpendicular to it is called double beamplan. The fibrils of transverse muscles possess such ability.

Electronic microscopy.A big step forward in the development of microscopy technique was the creation and use of an electron microscope (see Fig. 2.1). The electron microscope uses the flow of electrons with the waves shorter than in the light microscope. At voltage of 50,000 in the wavelength of electromagnetic oscillations arising from the movement of the electron flux in vacuo, is 0.0056 nm. It is theoretically calculated that the resolved distance in these conditions can be about 0.002 nm, or 0.000002 μm, i.e., 100,000 times less than in the light microscope. Practically in modern electron microscopes, the resolved distance is about 0.1-0.7 nm.

Gistology uses transmission (translucent) electronic microscopes (TEM), scanning (raster) electronic microscopes (SEM) and their modifications. With the help of TEM, you can get only a plane image of the microject studied. To obtain a spatial representation of structures, SEM is used that can create a three-dimensional image. The raster electron microscope works on the principle of scanning by the electron microzzle the object under study, i.e., consistently "swells" is sharply focused electron beams of separate surface points. Such a study of the object is called scanning(reading), and the drawing by which the microzond is moving - raster.The resulting image is displayed on a television screen, the electronic beam of which is moving synchronously with the microzzle.

The main advantages of raster electron microscopy are the high depth of field, a wide range of continuous increase change (from tens to tens of thousands of times) and high resolution. Modern options The instruments for studying the surface of the object is an atomic force microscope and a scanning tunnel microscope.

Electronic microscopy using the freezing method- rockingit is used to study the details of the structure of membranes and intercellular compounds. For the manufacture of chips, the cells are frozen at low temperatures (-160 ° C). In the study of the membrane, the chip plane passes through the middle of the bilayer of lipids. Further, the inner surfaces of the obtained halves of membranes are sprayed with metals (platinum, palladium, uranium), they are studied using TEM and micrographs.

Method of cryoelectronic microscopy.A quick frozen thin layer (about 100 nm) of the tissue sample is placed on a microscopic grille and are examined in a microscope vacuum at -160 ° C.

Method of electron microscopy "Freezing - etching"used to study the outer surface of the cell membranes. After the rapid freezing of the cells at a very low temperature, the block split the knife blade. The resulting ice crystals are removed by subliminate water in vacuo. The cells are then calculated by sparing a thin film of heavy metal (for example, platinum). The method allows you to identify the three-dimensional organization of structures.

Thus, freezing methods - rocking and freezing - etching make it possible to study non-fixed cells without the formation of artifacts caused by fixation.

Contrasting methods of heavy metals salts make it possible to investigate separate macromolecules in the electron microscope - DNA, large proteins (for example, myosin). With negative contrast, the aggregates of macromolecules (ribosomes, viruses) or protein filaments (actin yarn) are studied.

Electronic microscopy of ultra-thin sections, obtained by cryo-tombro-tomy.In this case, the method of fabrics without fixation and fill in solid media Quickly cooled in liquid nitro at a temperature of -196 ° C. This ensures the braking of metabolic processes of cells and the transition of water from the liquid phase into solid. Next, blocks are cut on ultramicrotom at low temperatures. This method of preparation of sections is commonly used to determine the activity of enzymes, as well as for immunochemical reactions. Antibodies associated with particles of colloidal gold are used to detect antigens, the localization of which is easy to reveal on the preparations.

Methods of ultrahonshopic microscopy.Electronic microscopes with accelerating voltage up to 3,000,000 V. The advantage of these microscopes is that they allow to investigate the objects of a large thickness (1-10 μm), since at high electron energy they are less absorbed by the object. Stereoscopic shooting allows you to obtain information about the three-dimensional organization of intracellular structures with high resolution (about 0.5 nm).

Microscopic methods Research

ways to study various objects using a microscope. In biology and medicine, these methods allow you to study the structure of microscopic objects, the dimensions of which lie outside the human resolution. Foundation M.M.I. Makes up light and electronic. In practical I. scientific activity Doctors of various specialties - virologists, microbiologists, cytologists, morphologists, hematologists, etc. In addition to conventional light microscopy, phase-contrast, interference, luminescent, polarization, stereoscopic, ultraviolet, infrared microscopy are used. The basis of these methods contains various properties of light. When electron microscopy, the image of the object objects occurs due to the directional flow of electrons.

For light microscopy and the other M. M.I. based on it. In addition to the resolution of the microscope and it also has the direction of the light beam, as well as the features of the object being studied, which can be transparent and opaque. Depending the properties of the object, the physical properties of light change - its and brightness associated with the length and amplitude of the wave, the plane and the direction of the wave propagation. On the use of these properties of light and built various M. M.I. For light microscopy, biological objects are usually painted in order to identify those or other properties ( fig. one ). At the same time, the tissues must be fixed, because Recalls certain structures of only killed cells. In a living cage, the dye is isolated in a cytoplasm in the form of a vacuole and does not blame its structures. However, live biological objects can be studied in the light microscope using the method of vital microscopy. In this case, it is used dark-based, which is embedded in.

A phase-contrast microscopy is also used to study living and unpainted biological objects. It is based on diffraction of the beam of light depending on the characteristics of the radiation object. This changes the length and phase of the light wave. A special phase-contrast microscope contains a translucent phase plate. Live microscopic objects or fixed, but not painted and cells due to their transparency practically do not change the amplitude and the color of the light beam passing through them. Causeing only the phase shift of his wave. However, passing through the object being studied, the rays of light deviate from the translucent phase plate. As a result, the wavelength difference arises between the rays that passed through the object and the rays of the light background. If this difference is at least 1/4 of the wavelength, the visual effect appears, in which the dark object is clearly visible on a light background or vice versa, depending on the characteristics of the phase plate.

Interference microscopy solves the same tasks as phase-contrast. But if the latter allows you to observe only the contours of the objects of the study, then with the help of interference microscopy, you can explore the details of the transparent object and quantify them. This is achieved due to the split of the beam of light in the microscope: one of the rays passes through a particle of the observed object, and the other by it. In the eyepiece of the microscope, both rays are connected and interferred with each other. The emerging phase difference can be measured by determining T. O. Mass of various cellular structures. Sequential measurement of the phase difference with known refractive indices makes it possible to determine the thickness of living objects and non-fixed tissues, the concentration of water and dry matter in them, protein content, etc. On the basis of these interference microscopy, it is indisputable to judge the permeability of membranes, enzyme activity, cellular metabolism of research objects.

Polarization microscopy allows you to study the objects of the study in the light formed by two rays polarized in mutually perpendicular planes, i.e. In polarized light. To do this, use the polaroids or prisms of Nicolas, which are placed in the microscope between the light source and the drug. changes during the passage (or reflection) of the rays of light through various structural components cells and tissues whose properties are heterogeneous. In so-called isotropic structures, the speed of propagation of polarized light does not depend on the polarization plane, in anisotropic structures, the speed of its propagation 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, a positive occurs double bempraneWhen reverse relationships - negative double beamplanation. Many biological objects have a strict molecular orientation, are anisotropic and have a positive double refraction of light. Miofibrils, cilia possesses such properties fiscal epithelium, neurofibrils, collagen fibers and others. Comparison of the nature of the refractive rays of the polarized light and the value of the anisotropy of the object allows you to judge the molecular organization of its structure ( fig. 2. ). Polarization microscopy is one of the histological research methods (histological research methods) , Method of microbiological diagnostics (microbiological diagnostics) , It is used in cytological studies (cytological examination) and others. At the same time, both painted and unspoken and non-fixed, so-called tissue sections can be explored in polarized light.

Wide distribution has luminescent microscopy. It is based on the property of some substances glow - luminescence in UV rays or in the blue-purple part of the spectrum. Many biological substances, such as simple, coenses, some and medicinal products, possess our own (primary) luminescence. Other substances begin to shine only when special dyes are added to them - fluorochromes (secondary). Fluorochromas can be distributed in the cell diffuse or selectively color individual cellular structures or certain chemical compounds biological object. This is based on the use of luminescent microscopy under cytological and histochemical studies (see histochemical research methods) . With the help of immunofluorescence in the luminescent microscope, viral and their concentration in cells are identified, identified, determine the antigens and, hormones, various products of metabolism, etc. ( fig. 3. ). In this regard, luminescent microscopy is used in laboratory diagnostics such infections, such as epidemic, viral, flu, etc., are used in the express diagnosis of respiratory viral infections, examining the prints from the mucous membrane of the patient's nose, and differential diagnosis various infections. In pathorphology, with the help of fluorescent microscopy, they recognize malignant in histological and cytological preparations, determine the areas of heart ischemia when early timing Myocardial infarction, detect tissue biopsy so, etc.

Ultraviolet microscopy is based on the ability of some substances that are part of the living cells, microorganisms or fixed, but not painted, transparent in the visible light of tissues, absorb UV radiation with a certain wavelength (400-250 nM). This property has, such as proteins, aromatic acids (, tryptophan, methylalane), purine and pyramidine bases, etc., using ultraviolet microscopy, clarify the localization and number of these substances, and in the case of the study of live objects - their changes in the process of life.

Infrared microscopy allows you to explore opaque for visible light and UV radiation objects by absorbing them by the structures of light with a wavelength of 750-1200 nM. For infrared microscopy, no preliminary chemical treatment drugs. This M.M.I. Most often used in zoology, anthropology, other branches of biology. In medicine, infrared microscopy is used mainly in neuromorphology and ophthalmology.

For the study of bulk objects use stereoscopic microscopy. The design of stereoscopic microscopes allows you to see the object of research with the right and left eye at different angles. Explore opaque objects with a relatively small magnification (up to 120 times). Stereoscopic microscopy finds use in microsurgery (microsurgery) , in pathorphology, with a special study of biopsy, operational and sectional material, in forensic laboratory studies.

An electron microscopy is used to study on the subcelet and macromolecular levels of the structure of cells, tissues of microorganisms and viruses. This M.M.I. Allowed to switch to a qualitatively new level of studies of matter. He found wide application In morphology, microbiology, virology, biochemistry, oncology, genetics, immunology, a sharp increase in the resolution of an electron microscope is ensured by the flow of electrons passing in vacuo through electromagnetic fields created by electromagnetic lenses. Electrons can pass through the structures of the object under study (transmission electron microscopy) or reflecting OTN (scanning electron microscopy), deviating at different angles, resulting in an image in the fluorescent screen of the microscope. With transmission (transmission) electron microscopy, a plane image of structures is obtained ( fig. four ), when scanning - volume ( fig. five ). The combination of electron microscopy with other methods, for example, with radioautography, histochemical, immunological methods of research (immunological research methods) , Allows electronically radio automatic, electron-histochemical, electron-immunological studies.

Electron microscopy requires special training of research objects, in particular, chemical or physical fixation of tissues and microorganisms. The biopsy material and the sectional material after fixing are dehydrated, poured into epoxy resins, cut with glass or diamond knives on special ultraomes, allowing to obtain ultra-thin sections of tissues with a thickness of 30-50 nM. They are contrasted and then studied in an electron microscope. In the scanning (raster) electron microscope, the surface of various objects is studied, sputting on them in a vacuum chamber electron-dense substances, and investigate the so-called replicas repeating the contours of the sample. See also Microscope .

Fig. 5. Electroniogram of leukocyte and phagocycable bacteria obtained by scanning electron microscopy; × 20,000.

1. Small medical encyclopedia. - M.: Medical Encyclopedia. 1991-96 2. First health care. - M.: Large Russian Encyclopedia. 1994 3. encyclopedic Dictionary medical terms. - M.: Soviet Encyclopedia. - 1982-1984.

• Microscopic technique

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Light microscopy

When using this method, the researcher operates with the following concepts:

Increase– physical property Lens lens and eyepiece. An increase in the microscope is assessed as a product of an increase in lens and an increase in eyepiece.

The minimum size of the observed object (D) and microscope resolution- values \u200b\u200bdepending on the characteristics of the lens lens, wavelength and on the refractive index of the medium separating the object being studied from the lens lens or condenser. Increase microscope resolution using liquid media (immersion media), because The coefficient of their refractive is larger than the refractive index of air. In microscopy, oil, glycerin and aqueous immersion environments are used. Theoretically possible permission limitthe light microscope is 0.2 μm (the minimum distance on which two objects are distinguishable).

Special types of microscopy

Tempal. Use a special condenser separating contrasting structures of an unpainted material. Dumping microscopy allows you to observe live objects. The observed object looks like lit on a dark field. At the same time, the rays from the illuminator fall on the object side, and only the scattered rays come to the microscope lenses.

Phase-contrast Microscopy allows you to study live and unpainted objects. When the light passes through the painted objects, the amplitude of the light wave changes, and when light passes through the unpainted - the light wave phase, which is used to obtain a high-contrast image in phase-contrast and interference microscopy.

Polarization Microscopy is the formation of an image of unpainted anisotropic structures (for example, collagen fibers and myofibrils).

Interference Microscopy combines the principles of phase-contrast and polarization microscopy and is used to obtain a contrast image of unpainted objects.

Luminescent Microscopy is used to observe fluorescent (luminescent) objects. In a luminescent microscope, light from a powerful source passes through two filters. One filter delays the light in front of the sample and skips the light of the wavelength exciting the sample fluorescence. Another filter transmits the light of the wavelength emitted by the fluorescent object. Thus, fluorescent objects absorb the light of one wavelength and emitting in another region of the spectrum.

Fluorescent dyes (fluorescin, rhodamine, etc.) are selectively associated with specific macromolecules.

Electronic microscopy.

The theoretical resolution of the translucent EM is 0.002 nm. The real resolution of modern microscopes is approaching 0.1 nm. For biological objects, the EM permission in practice is 2 nm.

Translucent um It consists of a column through which electrons emitted by cathode thread pass in vacuo. The electron beam focused by ring magnets passes through the prepared sample. The nature of the dispersion of electrons depends on the density of the sample. The electrons passing through the sample focus are observed on the fluorescent screen and recorded using a photoflax.

Scanning EM Apply to obtain a three-dimensional image of the surface of the object under study.

Socular method (freezing-rocking) are used to explore inner structure cell membranes. Cells are frozen at temperatures liquid nitrogen In the presence of a cryoprotector and is used for the manufacture of chip. Square planes pass through the hydrophobic middle of the dual layer of lipids. Nude interior surface Membranes are shaded by platinum, the resulting replicas are studied in the scanning electron microscope.