Using light microscopy in a plant cell. Methods for studying plant cells

Confocal microscope and images made with its help: anther cracking, xylem vessels, chloroplasts in stigma cells.

Light microscopy

One of the main methods of cytology today remains microscopy, designed to study the structure of the cell; it is widely used in fundamental and applied research. The invention of the microscope is associated with the names of Galileo Galilei (Italian) and the Jansen brothers (Dutch) in 1609-1611. The term "microscope" was coined by Faber (German) in 1625.

At the moment, there are two main types of microscopy - light and electron. The differences between them lie in the principle of considering the object. In the first case, the object is considered in the flow of the visible part of electromagnetic radiation (wavelength = 400-750 nm), in the second case - in the flow of electrons. These two methods have different resolutions. Resolution or resolution limit is the minimum distance between two points at which they are visible separately. The resolution limit of the microscope is set by the wavelength of the radiation flux in which the object is being studied. Therefore, radiation of a given wavelength can be used to study only such structures, the minimum dimensions of which are comparable to the wavelength of the radiation itself. The resolution limit of light microscopy was reached by microscope designers at the end of the 19th century and amounted to 0.2 microns. This means that two objects, if separated by less than 0.2 microns, will appear as one, even if we greatly enlarge the image, for example by projecting it onto a screen. Therefore, with the help of a light microscope it is not possible to examine two centrioles in the cell center; they look like one point (it must be said that in modern commercially produced microscopes, the maximum resolution is not realized). Due to the limited resolution of a light microscope, it can be used to study a limited number of intracellular structures, including: the nucleus, plastids, large vacuoles, and the plant cell wall. The smallest objects clearly visible in a light microscope are bacteria and mitochondria, the size of which is about 500 nm (0.5 microns), smaller objects are not clearly visible, increasing the precision of lens processing cannot overcome this limitation, which is set by the wave nature of light.

Resolution depends not only on the wavelength of the light source, but also on the refractive index of the medium through which the object is observed, as well as on the angle at which the light rays enter the lens. The standard set of microscope lenses consists of: low magnification lenses (x8) with an aperture of A = 0.2 and high magnification lenses (x20) with A = 0.40 and - (x40) with A = 0.65. These lenses are called “dry”, since the object is viewed through the air (refractive index n=1). But most microscopes are also equipped with special immersion objectives, which require a special immersion environment (n=1). Such a medium can be water; the x40 VI lens has an aperture of 0.75. The most common is oil immersion (n=1.51), at x90 the lens aperture value is A=1.25. If immersion is used, the resolution of the light microscope improves. However, high-resolution lenses have disadvantages: shallow depth of field and low contrast.

The most common method of light microscopy is the brightfield method, in which light rays from an illuminator pass through the object and enter the lens. Fixed and stained cells are studied in this way. The discovery of basic cellular structures involves the development and use of a set of dyes that selectively stain cell components and provide contrast for their observation. There is a wide variety of dyes. Some of them are extracted from plants and animals; there are still no synthetic analogues. For example, the widely used hematoxylin is an extract of the tropical logwood tree, and carmine is the pigment of the fat body of some types of aphids. These are all so-called nuclear dyes that color structures containing nucleic acids. The use of a non-nuclear dye, silver nitrate, allowed Camillo Golgi in 1898 to observe and describe what was later called the Golgi apparatus.

Staining a living cell is possible only in rare cases, so other methods are used to study them. Unlike the bright field method, when observing objects using the dark field method, the illuminator rays do not enter the lens and the image is created only by scattered rays coming from the object. In this case, against a dark background you can see luminous particles, which are smaller in size than the resolution of the lens, although the size and shape of the particles are difficult to determine. Transmitted light dark-field microscopy is used to study transparent objects normally invisible in bright field and, especially, to view living cells. Living and dying cells look completely different against a dark background. The protoplast of dying cells glows brighter, there is no explanation for this fact. This method was invented by Zsigmondy (Austria) in 1912. In a light microscope, one can distinguish objects that change the amplitude of the illumination rays, but living cells are transparent to visible light and the rays, passing through the cell, practically do not change the amplitude. The human eye is not able to perceive the phase shift of rays without changing the amplitude. Therefore, the methods of phase contrast (invented by Zernike (Dutch) in 1934) and interference microscopy (invented by Lebedev in 1932) are used specifically to study living cells. In such systems, the passage of light through a living cell is accompanied by a change in the phase of the light wave. Light is delayed when passing through thick areas of the cell, such as the nucleus. A recombination of two sets of waves occurs, which create an image of cellular structures.

To study objects that are birefringent (starch grains, plant fibers, crystals), polarization microscopy is used, the foundations of which were laid by Ebner in 1882. In this method, a special polarizer device is used, which converts multidirectional light waves and they acquire one direction.

In fluorescence microscopy, an object is viewed in the light emitted by itself. The first fluorescent microscope was designed by Keller and Zindentokf in 1908. This method is based on the ability of a number of substances to glow when illuminated with short-wavelength rays (violet or ultraviolet). Fluorescence microscopy is often used to identify specific proteins, antibodies, and Koons was the first to use fluorochromes to bind to antibodies, and this reaction was named after him. In cytoembryological studies, this method is used to study structures containing the carbohydrate callose. For this method, a special optical system with a mercury lamp connected to a light microscope is used.

Recently, the capabilities of light microscopy have increased significantly due to the use of sensitive video systems. The image created by a light microscope is processed in a video camera. It is cleared of “noise”, converted into digital signals and sent to a computer, where it undergoes additional processing to extract hidden information. Computer interference microscopy allows you to achieve high contrast and analyze transparent objects and living cells.

Electron microscopy

Long continuous efforts to improve research methods brought the desired results at the end of the Second World War. It was then, thanks to an amazing coincidence of circumstances, almost at the same time, that scientists were enriched with a number of new powerful tools and research methods. In morphology, such a tool was the electron microscope. Created back in the 30s of the 20th century, it had sufficient resolving power to penetrate the cell, down to nanometer-sized structures. At the same time, the electron beam had weak penetrating power, and this required the preparation of very thin samples of the material and a high vacuum. Such stringent requirements created serious difficulties, but in a surprisingly short time it was possible to develop methods for preparing tissue samples and construct devices for obtaining thin sections from them. The quality of the objects steadily improved and by the early 1960s many previously unknown cellular structures had been described.

So, the resolution of an electron microscope is much higher than that of a light microscope. Theoretically, at a voltage of 100,000 V, its resolution is 0.002 nm, but due to the correction of electronic lenses, it decreases and in reality is 0.1 nm in modern electron microscopes. Significant difficulties in observing biological objects further reduce the normal resolution; it does not exceed 2 nm. However, this is 100 times greater than that of a light microscope, which is why electron microscopy is called ultramicroscopic.

The general design of a transmission electron microscope resembles that of a light microscope. It is significantly larger than the light one and seems to be upside down. The source of radiation in an electron microscope is a cathode filament that emits electrons (electron gun). The electrons are emitted from the top of a cylindrical column about two meters high. To ensure that there are no obstacles to the movement of electrons, this happens in a vacuum, the electrons are accelerated by the anode and penetrate through a tiny hole into the lower part of the column with a narrow electron beam. The electron beam is focused by ring magnets along the column, which act like the glass lenses of a light microscope. The sample is placed in the path of the electron beam. At the moment of its passage through the sample, part of the electrons is scattered in accordance with the density of the substance, the rest of the electrons are focused, forming an image on a photographic plate or on a screen.

The first electron microscope was created by Siemens in 1939. It made it possible to see many amazing structures in the cell. But for this, completely new methods of preparing drugs had to be invented, which began to be used since 1952. Cells are fixed in this case with glutaraldehyde, which covalently binds proteins, and then with osmic acid, which stabilizes the protein and lipid layers. The sample is dehydrated and impregnated with resins, which form a solid block after polymerization. Sections for electron microscopy should be approximately 1:200 the thickness of a single cell. To make such sections, an ultramicrotome (1953) was created, which uses glass or diamond knives. The resulting sections are placed on a special copper mesh. The electron microscope image depends on electron scattering, which is determined by the atomic number of the substance. Biological objects consist mainly of carbon, oxygen and hydrogen, which have a low atomic number. To enhance the contrast, they are impregnated with heavy metals such as osmium, uranium, and lead. Thin sections with transmission electron microscopy do not allow one to judge the three-dimensional structure of the cell; this deficiency can be compensated for by a series of sections from which the cell is reconstructed. It's a long process.

There is also a direct method for studying the three-dimensional structure of biological objects - scanning electron microscopy - it was created in 1965. In this case, electrons scattered or emitted by the surface of the object, which must be fixed, dried and covered with a film of heavy metal, are used to obtain an image. This method is only applicable for studying surfaces and its resolution is low - about 10 nm.

Electron microscope

Transmission, probe and scanning electron microscopes. Electron microscopic image of the surface of an anther and pollen grain

Chemical methods for studying cells

A classic light microscope has low resolution, which does not allow studying the details of the structure of a cell smaller than 0.25 microns in size. The second stage of studying the cell dates back to the time when microscopists worked to improve their instruments. At the same time - the end of the 18th century. - French scientist Antoine de Lavoisier and Englishman Joseph Priestley create a new science - chemistry. Unlike morphology, which progresses from complex to simple, chemistry progresses from simple to complex. Chemistry began with the identification of elements, atoms, and then moved along the path of studying some of their simplest combinations - molecules.

The synthesis of the biological molecule urea, first carried out in 1828 by the German scientist Friedrich Wöhler, helped cross the border between inorganic and organic chemistry and allow penetration into the living world of chemistry. This marked the beginning of the use of a chemical approach to the study of cells. Over the next hundred years, amino acids, sugars, fats, purines, pyrimidines, and other small molecules were discovered, purified, structurally studied, and synthesized. Scientists were able to get an idea of ​​the metabolism of these substances in the body and the ways in which basic biological molecules are formed from them: proteins, polysaccharides and nucleic acids. But again insurmountable obstacles arose on the path of progress: classical chemistry was powerless in the face of the complexities of the structural complexity of these large molecules. For a long time, cells were studied mainly by observing them. But as the experimental method developed in the natural sciences, it began to be used in the study of living organisms. This was facilitated by powerful biomedical research carried out in the second half of the 19th century. At the beginning of the 20th century. American Ross Garrison and Frenchman Alexis Carrel found that animal cells can be cultured in vitro, similar to how they do with single-celled organisms. Thus, they demonstrated the ability of cells to live independently and created a cultivation method, which is now one of the most relevant.

But all these methods, essentially revolutionary, were still indirect; the cell remained a closed black box. The huge gap between the smallest particle visible in a light microscope and the largest molecule accessible to chemical study remained unknown. In this unknown space, important concepts and concepts were hidden, the functions of the described cellular structures, their connection with known biomolecules remained unknown - without all this, the life of the cell remained unsolved.

In turn, biochemistry has also been enriched with a number of fundamentally new devices and methods. Of particular interest was chromatography, based on a very simple phenomenon - the formation of a rim or halo around a stain (what we see when we try to remove a stain with a special solution). This phenomenon is based on differences in the speed of movement of different paints in the flow of spreading liquid. At the beginning of the 20th century, Russian physiologist and biochemist Mikhail Semenovich Tsvet was the first to use this phenomenon. By passing the extract from the leaves through a vertical tube filled with an adsorbent powder, he was able to separate the main leaf pigments - green and orange - and obtain them in the form of individual colored stripes or rings along the tube. He called his method chromatography (Greek khroma - color, graphein - to record). Color died relatively young and the potential of his method remained unexploited until the early 40s. There are now many variations of chromatography - applicable to all substances that can be identified chemically.

Close to chromatography is gel electrophoresis, in which it is not the flow of solvent, but the electromotive force that promotes the movement and separation of electrically charged components. These methods revolutionized the field of chemical analysis. Now analysis can be carried out on trace amounts of a mixture of almost any composition.

The second method that radically changed the chemical study of living cells was the method of isotope labeling. Isotopes are varieties of the same chemical element that differ in atomic mass. Some isotopes exist in nature, many can be produced artificially through nuclear reactions. Isotopes are used to specifically label certain molecules so that such molecules can be distinguished from related ones without disturbing the overall structure. This method is used in the analysis of biosynthetic processes that could not be studied in any other way. For example, with the production of labeled amino acids, it became possible to study their combination into proteins in a living organism or under experimental conditions, even despite the infinitesimal amount of newly formed protein, due to its radioactivity. This method became widespread with the creation of nuclear reactors and the production of a wide range of radioisotopes. Without the tagged atom method, advances in cellular and molecular biology would have been impossible.

Thus, both morphology and biochemistry, enriched by new methods, were constantly improved, the gap between their knowledge became less and less and disappeared completely when it became possible to divide the cell into parts in such a way that each part could be independently studied.

The methods used for such fractionation are based primarily on centrifugation. This method uses differences in the physical properties, in particular the size and density, of certain components of the cell to separate them from each other. This made it possible to study a large part of the cell and combine morphological and biochemical knowledge.

However, one part of the cell—its crucial central part, the nucleus—remained largely inaccessible until another event occurred. It began with an attempt to analyze, using genetics, the characteristics of some simple viruses that infect bacteria and are called bacteriophages or bacteria eaters. This research turned out to be the right approach to solving the problem of genetic organization, which even in the simplest noncellular organisms was unusually complex. For a long time, the new discipline known today as molecular biology was limited to the study of viruses and bacteria, but then it literally burst into the eukaryotic cell, making it possible to study the regulation of cell activity.

To study the molecular basis of cell organization, detailed biochemical analysis is required. It requires a significant number of cells of a certain type, so it is impossible to use pieces of tissue, because they contain cells of different types. At the first stage of work, pieces of fabric are turned into a suspension. This can be done by destroying the intercellular substance and intercellular connections. To do this, the tissue is treated with proteolytic enzymes that destroy proteins (trypsin, collagenase). Calcium plays an important role in connecting cells and their adhesion, so chelating substances that bind calcium are also used. The tissues are then subjected to gentle mechanical destruction and separated into individual cells. The second stage is the separation of the suspension into separate fractions. To do this, they use centrifugation, with the help of which large cells are separated from small ones, and light ones from heavy ones, or antibodies are used, and the ability of cells to attach to glass or plastic with different strengths. The third stage is the introduction of isolated cells into culture. The first experiments were carried out in 1907 by Harrison, who cultivated the spinal cord of amphibians in a plasma clot. Culture media have a rather complex composition. The standard medium was developed in the early 70s; it contains a set of 13 amino acids, 8 vitamins, and mineral salts. In addition, the medium may include glucose, penicillin, streptomycin, horse or calf serum. As Hayflick and Moorhead showed in 1961, most mammalian cells die in culture after a certain number of divisions. Human skin cells divide in culture 50-100 times. However, mutant cells sometimes appear in culture and can multiply indefinitely, forming a cell line. In 1952, a continuous cell line was isolated from cervical cancer known as the HeLa line. Such lines are stored at a temperature of -70 C, after thawing they retain the ability to divide. The method of culturing plant cells was developed by 1964. Using it, it was possible to grow a whole carrot plant in vitro from root cells.

In order to be able to see a small object, it is necessary to enlarge it. Magnification is achieved using a system of lenses located between the examiner's eye and the object. Contrast and resolution are of great importance for microscopic observations, allowing one to clearly distinguish an object from the background and separately see very close details of the image. Depending on the principle of image creation, microscopy is divided into light, electron and laser.

Modern light microscopes are complex and have three lens systems (Fig. 2.1). The condenser system is responsible for properly illuminating the field of view and is located between the light source and the object. With an external light source, the rays are directed into the condenser by a mirror. Many modern microscopes have a built-in light source and do not have a mirror. The image of the objective lens system facing the object and the eyepiece in contact with the researcher’s eye are magnified. Total magnification is defined as the product of the objective magnification and the eyepiece magnification. The resolving power of a microscope depends on the wavelength of the light used, the optical properties of the lenses, and the refractive index of the medium in contact with the outer objective lens.

Rice. 2.1.

The simplest technique to increase the resolution of a microscope is the use of immersion. A drop of liquid whose refractive index exceeds the refractive index of air is placed between the outer lens of the objective and the object. A special immersion lens is used for each liquid. The most common are water (white ring) and oil (black ring) lenses. Modifications of conventional bright-field microscopy are ultraviolet, dark-field, and phase-contrast microscopy.

The use of shorter wavelength ultraviolet rays also improves the resolution of the microscope. However, the use of special light sources and quartz optics lead to a significant increase in the cost of microscopic studies.

In dark-field microscopy, the object is illuminated only by oblique side beams using a special dark-field condenser. With this lighting, the field of view remains dark, and small particles glow with reflected light. Dark-field microscopy allows one to discern the contours of objects that lie beyond the visibility of a conventional microscope, such as prokaryotic flagella. However, with this method of observation it is impossible to consider the internal structure of the object.

When using a phase-contrast device, you can observe living transparent objects that practically do not differ in density from the surrounding background. The color and brightness of the rays passing through such objects almost do not change, but a phase shift occurs that is not registered by the human eye. A phase contrast device, used as an attachment to a conventional microscope, converts phase differences in light waves into changes in their color and brightness. Transparent objects become clearer, and even individual structures and inclusions can be observed in the cells of large microorganisms.

Microscopic studies as a method of cell knowledge

The diameter of a typical animal cell is 10-20 microns, which is five times smaller than the smallest visible particle. Only with the advent of advanced light microscopes at the beginning of the 19th century was it possible to establish the fact that all tissues of animals and plants consist of individual cells. This discovery, summarized in the form of cell theory by Schleiden and Schwann in 1838, marks the beginning of cell biology.

Being extremely small in size, animal cells are also colorless and transparent: hence, the discovery of their basic structures was made possible by the development of a set of dyes at the end of the 19th century. It was the dyes that provided sufficient contrast to observe subcellular structures. A similar situation was observed in the early 40s of our century, when the invention of a powerful electron microscope required new methods for preserving and staining cells. It was only after they were developed that the full complexity of cellular structure began to emerge. Microscopy as a methodology is still based on methods of sample preparation and the capabilities of the microscope itself.

Conventional optical microscopy

In the general case, radiation of a given wavelength can be used to study only those structures whose minimum dimensions are still comparable to the wavelength of the radiation itself. This principle limits the capabilities of any microscope. The resolution limit of a light microscope is set by the wavelength of light, which for visible light ranges from 0.4 µm (violet) to 0.7 µm (dark red). It follows that the smallest objects that can still be observed in a light microscope are bacteria and mitochondria (their width is ~ 0.5 µm). The smaller elements of the cell are distorted by effects caused by the wave nature of light.

To prepare a permanent preparation that can be stained and observed under a microscope, the cells are treated with a fixing agent to immobilize, kill, and preserve them. Modern methods typically use aldehydes, such as formaldehyde or glutaraldehyde, which form covalent bonds with the free amino groups of proteins and thus cross-link adjacent molecules.

Once fixed, the tissue is usually cut into very thin “slices” (sections) using a microtome. Sections with a thickness of 1 to 10 μm are placed on the surface of a glass slide. Paraffin or special resin is used as enclosing media. In liquid form, these media impregnate and surround the fixed tissue: they then harden by cooling or by polymerization, forming a solid block that can be conveniently cut with a microtome.

There is a serious risk that fixation or imprisonment procedures may damage the structure of cells or cellular macromolecules. That is why another method of preparing sections has been proposed - quick freezing. Frozen tissue is cut on a cryostat in a special microtome installed in a cold chamber.

The contents of most cells, which usually consist of 70% water, have virtually no components that can interfere with the passage of light rays. Therefore, in their natural state, most cells, even after fixation and sectioning, are practically invisible in a conventional light microscope. One way to see them is to stain the cells with dyes.

Fluorescence microscopy

Because most macromolecules are present in relatively few copies in cells, one or two dye molecules bound to a macromolecule may go undetected. An alternative approach to the sensitivity problem is to use fluorescence.

Fluorescent dyes absorb light of one wavelength and emit light of another, longer wavelength. If such a substance is irradiated with light whose wavelength matches the wavelength of the light absorbed by the dye, and then a filter is used for analysis that transmits light with a wavelength corresponding to the light emitted by the dye, the fluorescent molecule can be detected by glowing in the dark field. The high intensity of emitted light is a characteristic feature of such molecules.

The use of fluorescent dyes to stain cells requires the use of a special fluorescent microscope. Such a microscope is similar to a conventional light microscope, but here the light from the illuminator, emitted by a powerful source, passes through two sets of filters - one to stop the light in front of the sample and the other to filter the light received from the sample.

Fluorescence microscopy is often used to identify specific proteins or other molecules that become fluorescent after being covalently bound to fluorescent dyes. For example, fluorescent dyes can be bound to antibody molecules, which immediately turns them into highly specific and convenient dye reagents that selectively bind to specific macromolecules on the surface of a living or inside a fixed cell. Two dyes are commonly used for this purpose: fluorescein, which produces intense yellow-green fluorescence after excitation with light blue light, and rhodamine, which produces dark red fluorescence after excitation with yellow-green light.

Phase contrast and interference microscopy

The possibility of specimens being lost or damaged during preparation has always been a concern for microscopists. The only way to solve this problem is to study living cells without fixation or freezing. Microscopes with special optical systems are very useful for this purpose.

When light passes through a living cell, the phase of the light wave changes according to the cell's refractive index: light passing through relatively thin or relatively thick areas of the cell, such as the nucleus, is delayed and its phase is correspondingly shifted relative to the phase of light passing through relatively thin areas cytoplasm. Both phase contrast and interference microscopes use interference effects that occur when two sets of waves recombine to create images of cellular structures. Both types of light microscopy are widely used to observe living cells.

The simplest way to see the details of a cell's structure is to observe the light scattered by the various components of the cell. In a dark-field microscope, the rays from the illuminator are directed from the side, and only scattered rays enter the microscope lenses. Accordingly, the cell looks like an illuminated object on a dark field. One of the main advantages of phase contrast, interference and dark field microscopy is the ability to observe the movement of cells during mitosis and migration

Video cameras and related image processing technologies have greatly enhanced the capabilities of light microscopy. This made it possible to observe cells for long periods of time in low light, eliminating prolonged exposure to bright light (or heat). Since the image is produced by the video camera in the form of electronic signals, it can be suitably converted into numerical signals, sent to a computer, and then further processed to extract hidden information. These and similar image processing methods make it possible to compensate for the optical deficiencies of microscopes and practically reach the resolution limit.

The high contrast achievable with computer interference microscopy makes it possible to observe even very small objects, such as individual microtubules whose diameter is less than one tenth of the wavelength of light (0.025 μm). Individual microtubules can also be seen using fluorescence microscopy. However, in both cases, diffraction effects are inevitable, greatly changing the image. In this case, the diameter of the microtubules is overestimated (0.2 μm), which makes it impossible to distinguish individual microtubules from a bundle of several microtubules.



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

For example:

hematoxylin dye colors some components of the nucleus blue or violet;

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

Sudan III dye stains suberized cell membranes pink;

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

When conducting microscopic examinations, most 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 device uses the bread slicer principle. Slightly thicker sections are made for plant tissues than for animal tissues because plant cells are relatively larger. Thickness of plant tissue sections for - 10 microns - 20 microns. Some tissues are too soft to cut straight away. Therefore, after fixation, they are poured into molten paraffin or a special resin, which saturate the entire fabric. After cooling, a solid block is formed, which is then cut using a microtome. This is explained by the fact that plant cells have strong cell walls that make up the tissue framework. Lignified shells are especially durable.

When using the filling during preparation, the cut runs the risk of damaging the cell structure; to prevent this, use the method of quick freezing. When using this method, you can do without fixing and filling. Frozen tissue is cut using a special microtome - cryotome (Appendix 1, Fig. 2).

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

phase-contrast (Appendix 1, Fig. 3) and interference microscopes (Appendix 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 (superpose) 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.

Lecture 13. Microscopy as a method for studying cells and tissues.

1. Light microscopy.

2. Electron microscopy.

Modern cytology has numerous and varied research methods, without which it would be impossible to accumulate and improve knowledge about the structure and functions of cells. In this chapter we will get acquainted only with the basic, most important research methods.

The modern light microscope is a very advanced device, which is still of paramount importance in the study of cells and their organelles. Using a light microscope, magnification of 2000-2500 times is achieved. The magnification of a microscope depends on its resolution, i.e., the smallest distance between two points that are visible separately.

The smaller the particle visible through a microscope, the greater its resolution. The latter, in turn, is determined by the lens aperture (aperture is the actual opening of the optical system, determined by the size of the lenses or diaphragms) and the wavelength of the light.

The resolution of a microscope is determined using the formula: a = 0.6, where a is the minimum distance between two points; -- wavelength of light; n is the refractive index of the medium located between the preparation and the first, i.e., frontal, objective lens; a is the angle between the optical axis of the lens and the most strongly deviating ray entering the lens, or the angle of diffraction of the rays.

The value indicated in the denominator of the fraction (n sin a) is constant for each lens and is called its numerical aperture. The numerical aperture as well as the magnification are engraved on the lens barrel. The relationship between the numerical aperture and the minimum resolvable distance is as follows: the larger the numerical aperture, the smaller this distance, i.e., the higher the resolution of the microscope.

Increasing the resolution of the microscope, which is absolutely necessary for studying the details of the cell structure, is achieved in two ways:

1) increasing the numerical aperture of the lens;

2) reducing the wavelength of light that illuminates the drug.

In order to increase the numerical aperture, immersion objectives are used. The liquids used are: water (r = 1.33), glycerin (r = 1.45), cedar oil (/1 = 1.51) compared to air n equal to 1.

Since the refractive index of immersion liquids is greater than 1, the numerical aperture of the lens increases and rays that make a larger angle with the optical axis of the lens can enter it than in the case when there is air between the front lens of the lens and the specimen.

The second way to increase the resolution of a microscope is to use ultraviolet rays, the wavelength of which is shorter than the wavelength of visible light rays.

However, the resolution of a microscope can only be increased to a certain limit, limited by the wavelength of light. The smallest particles that are clearly visible in a modern light microscope must have a value greater than 1/3 wavelength of light. This means that when using the visible part of daylight with a wavelength from 0.004 to 0.0007 mm, particles of at least 0 will be visible in the microscope .0002-0.0003 mm. Consequently, with the help of modern microscopes it is possible to examine those details of the cell structure that have a size of at least 0.2-0.3 microns.

Currently, many different models of light microscopes have been created. They provide the opportunity for a multifaceted study of cellular structures and their functions.

Biological microscope. The biological microscope (MBI-1, MBI-2, MBI-3, MBR, etc.) is intended for studying preparations illuminated by transmitted light. It is this type of microscope that is most widely used for studying the structure of cells and other objects.

However, with the help of a biological microscope it is possible to study in detail mainly fixed and stained cell preparations. Most living, unstained cells are colorless and transparent in transmitted light (they do not absorb light), and they cannot be examined in detail.

Phase contrast microscopy. A phase contrast device provides a contrast image of preparations of living cells, almost invisible when observing them in a biological microscope).

The phase contrast method is based on the fact that individual areas of a transparent sample differ from the surrounding environment in refractive index. Therefore, light passing through them travels at different speeds, i.e., it experiences a phase shift, which is expressed in a change in brightness. Phase changes of light waves are converted into light vibrations of different amplitudes, and a contrast image of the preparation is perceived by the eye, in which the distribution of illumination corresponds to the distribution of wide opportunities for studying living cells, their organelles and inclusions in an intact state. This circumstance plays an important role, since fixing and staining cells, as a rule, damages cellular structures.

A phase contrast device for a biological microscope consists of a set of phase lenses that differ from conventional ones in the presence of an annular phase plate, a condenser with a set of annular diaphragms, and an auxiliary microscope that magnifies the image of the annular diaphragm and the phase plate when they are combined.

Interference microscopy. The interference contrast method is close to the phase contrast microscopy method and makes it possible to obtain contrast images of unstained transparent living cells, as well as to calculate the dry weight of cells. A special interference microscope used for these purposes is designed in such a way that a beam of parallel light rays coming from a light source is divided into two parallel branches - upper and lower.

The lower branch passes through the preparation, and the phase of its light vibration changes, while the upper wave remains unchanged. For the drug, i.e. in the lens prisms, both branches reconnect and interfere with each other. As a result of interference, areas of the drug that have different thicknesses or unequal refractive indices are painted in different colors and become contrasting and clearly visible.

Fluorescence microscopy. Like phase contrast, fluorescence (or luminescence) microscopy makes it possible to study a living cell. Fluorescence is the glow of an object, excited by light energy absorbed by it. Fluorescence can be excited by ultraviolet, blue and violet rays.

A number of structures and substances contained in cells have their own (or primary) fluorescence. For example, the green pigment chlorophyll, found in the chloroplasts of plant cells, has a characteristic bright red fluorescence. A rather bright glow is produced by vitamins A and B and some pigments of bacterial cells; this makes it possible to recognize individual types of bacteria.

However, most substances contained in cells do not have their own fluorescence. Such substances begin to glow, revealing a variety of colors, only after pre-treatment with luminescent dyes (secondary fluorescence). These dyes are called fluorochromes. These include fluorescein, acridine orange, berberine sulfate, phloxin, etc. Fluorochromes are usually used in very weak concentrations (for example, 1:10000, 1:100000) and do not damage a living cell. Many of the fluorochromes selectively stain individual cellular structures and substances in a specific light. Thus, acridine orange, under certain conditions, colors deoxyribonucleic acid (DNA) green and ribonucleic acid (RNA) orange. Therefore, secondary fluorescence with acridine orange is now one of the important methods for studying the localization of nucleic acids in the cells of various organisms.

In addition, the use of fluorochromes makes it possible to obtain contrasting, easy-to-observe preparations in which the desired structures can be easily found, bacterial cells can be recognized and counted. The fluorescence microscopy method also makes it possible to study changes in cells and individual intracellular structures under different functional states, and makes it possible to distinguish between living and dead cells.

When blue and violet light rays are used as a source of fluorescence, the equipment consists of a conventional biological microscope, a low-voltage lamp (for a microscope) with a blue light filter that transmits rays that excite fluorescence, and a yellow light filter that removes excess blue rays. The use of ultraviolet rays as a source of fluorescence requires a special fluorescent microscope with quartz optics that transmits ultraviolet rays.

Polarization microscopy. The method of polarization microscopy is based on the ability of various components of cells and tissues to refract polarized light. Some cellular structures, such as spindle filaments, myofibrils, cilia of the ciliated epithelium, etc., are characterized by a certain orientation of molecules and have the property of birefringence. These are so-called anisotropic structures.

Anisotropic structures are studied using a polarizing microscope. It differs from a conventional biological microscope in that a polarizer is placed in front of the condenser, and a compensator and analyzer are placed behind the specimen and lens, allowing a detailed study of birefringence in the object under consideration. In this case, light or colored structures are usually observed in the cells, the appearance of which depends on the position of the drug relative to the plane of polarization and on the magnitude of birefringence.

A polarizing microscope makes it possible to determine the orientation of particles in cells and other structures, to clearly see birefringent structures, and with appropriate processing of preparations, observations can be made on the molecular organization of a particular part of the cell.

Dark field microscopy. The study of drugs in the dark is carried out using a special condenser. A dark-field condenser differs from a conventional bright-field condenser in that it transmits only very oblique edge rays from the light source. Because the edge rays are highly inclined, they do not enter the lens, and the field of view of the microscope appears dark, while an object illuminated by scattered light appears light.

Cell preparations usually contain structures of different optical densities. Against a general dark background, these structures are clearly visible due to their different glow, and they glow because they scatter the rays of light falling on them (Tyndall effect).

In a dark field, a variety of living cells can be observed.

Ultraviolet microscopy. Ultraviolet (UV) rays are not perceived by the human eye, making direct study of cells and their structures within them impossible. For the purpose of studying cell preparations in UV rays, E.M. Brumberg (1939) designed the original MUF-1 ultraviolet microscope, and several models of this microscope are currently available. Method E.M. Broomberg is based on the fact that many substances that make up cells have characteristic absorption spectra of UV rays.

When studying various substances in living or fixed unstained cells and tissues in such a microscope, the preparation is photographed three times (on the same plate) in the rays of three different zones of the UV spectrum.

For photography, the UV wavelengths are selected so that in each zone there is an absorption band of any one substance that does not absorb rays in the other two zones. Therefore, the substances that are visible in the photographs turn out to be different in all photographs.

The resulting images are then placed in a special device called a chromoscope. One picture is viewed in blue, the second in green, and the third in red.

Three color images are obtained, which are combined into one in a chromoscope, and in this final image of the object, the various substances of the cell turn out to be painted in different colors.

But an ultraviolet microscope allows not only photography, but also visual observations of tissues and cells, for which it has a special fluorescent screen.

Using this microscope, it is possible to examine particles of slightly smaller sizes than in a conventional biological microscope, due to the fact that UV rays have a much shorter wavelength than ordinary light rays.

Therefore, the resolution of a UV microscope is 0.11 μm, while the resolution of a biological microscope using conventional lighting is 0.2-0.3 μm.

Using an ultraviolet microscope, a quantitative determination of the absorption of UV rays by nucleic acids and other substances contained in cells is carried out, i.e., the amount of these substances in one cell is determined.

Microphotography. Microphotography of various microscopic preparations is carried out in order to obtain their enlarged image - a microphotography. Micrographs are convenient for studying individual structures of cells and other objects; microphotographs represent documents that very accurately reflect all the details of the structure of a microscopic specimen.

Photographing of microscopic preparations is carried out using special microphoto installations or microphoto attachment cameras. The latter are widely used and are suitable for microphotography with a biological and any other microscope. A microphoto camera is a camera in which the lens has been removed and replaced with a microscope.

The optical system of the microscope serves as the lens of this camera. There are several types of microphoto attachments. Microphoto attachments like MFN-8 are very convenient.

There is also a special biological microscope MBI-6 with a permanent camera. MBI-6 allows for routine visual examination of drugs and their photography in transmitted and reflected light, in light and dark fields of view, with phase contrast and in polarized light.

Micro-filming plays an important role in studying the life processes of cells. To study the details of the most important processes occurring in the cell, such as division, phagocytosis, cytoplasmic flow, etc., a time-lapse device is used.

Using this device, it is possible to produce either accelerated filming, which is usually used in rapidly occurring processes, or slow-motion filming of those changes in the cell that are characterized by a slow flow.

Microcine filming is not only a method that allows one to study in detail various structures and processes in a living cell, but also a method for documenting these processes and all the changes that are associated with them.