In the course of a cytological study, the structure of cells is studied to detect malignant, benign tumors and lesions of a non-tumor nature. The main purpose of the study is to confirm or refute the fact of malignancy of the cells taken for analysis.

Methods of cytological research are based on the study under a microscope of the structure of cells, the cellular composition of fluids and tissues.

There are such methods of cytological studies:

• light microscopy;
• electron microscopy;
• centrifugation method. It is used when it is necessary to separate cell membranes from the general structure;
• labeled atom method. They are used to study biochemical processes in cells: for this, a labeled radioactive isotope is introduced into them;
• lifetime study. This research method makes it possible to study the dynamic processes occurring in the cell.

The conclusion of the cytological study is based on the features of changes in the cytoplasm, the cell nucleus, the nuclear-cytoplasmic ratio, the formation of complexes and cell structures.

A cytological analysis is used during a preventive examination, to clarify the diagnosis, during surgery, for the timely detection of relapses, and control over the course of treatment.

Cytological examination of smears

As materials for analysis use:

• fluids: urine, prostate secretion, sputum, swabs obtained during endoscopy of various organs, discharge from the nipples, prints and scrapings from ulcerative and eroded surfaces, wounds and fistulas, fluid from serous and articular cavities;
• punctates: biological materials obtained during diagnostic puncture performed with a thin needle;
• smears from the cavity and cervix.

Most of these cytological studies of smears are carried out if necessary, to establish and clarify the diagnosis. But a cytological examination of a smear from the cervix (Pap smear) is recommended: once a year - for women over 19 who are sexually active; twice a year - women who take hormonal contraceptives have had genital herpes; more than twice a year - women who suffer from infertility, uterine bleeding, obesity, who often change sexual partners, take estrogens, who have warts on the genitals, have genital herpes.

Cytological examination of the cervix

For a cytological examination of the cervix, a smear is taken from the outer and inner parts of the cervix and from the vaults of the vagina using a special wooden spatula. Then it is transferred to glass and fixed.

A cytological examination of the cervix is ​​\u200b\u200bcarried out to detect cancerous cell changes, and in conclusion, the doctor indicates one of the five stages of the state of the cells:

• stage 1. Cells with deviations are not found;
• stage 2. There are minor changes in the structure of cells caused by inflammation of the internal genital organs. This condition of the cells does not cause fear, but the woman is recommended to undergo additional examination and treatment;
• stage 3. A small number of cells with structural deviations were found. In this case, it is recommended to take a smear again or conduct a histological examination of the altered tissue;
• stage 4. Individual cells with malignant changes are found. The final diagnosis is not made, an additional examination is prescribed;
• stage 5. A large number of cancer cells were found in the smear.

The reliability of such a cytological study is high, but it can only provide information about the area from which the cells were taken for analysis. In order to assess the condition of the fallopian tubes, ovaries, uterus, you should undergo a comprehensive examination.

The main feature of the morpho-functional method for studying cells is the desire to understand the structural basis of the biochemical processes that determine a given function, that is, to associate these processes with specific cellular structures.

The end goal of this method is identical to that pursued by molecular biology and cellular structural biochemistry. However, the methods used by these sciences to solve a common problem are fundamentally different. If in molecular biology and structural biochemistry an indispensable condition is the destruction of the cell and the isolation of the structure under study in the form of a more or less pure fraction, then in cytological studies, on the contrary, the preservation of the integrity of the cell is a prerequisite. In this case, it is necessary to strive to reduce external interference to a minimum, and try to investigate the structural and biochemical organization of certain components precisely within the boundaries of an integral cellular system.

Morphofunctional studies have developed rapidly over the past decades. At that time, a large number of fundamentally new methods for the qualitative and quantitative analysis of cellular structures were developed. This approach is closely related to new branches of biological sciences and, in particular, to molecular biology, which determines the very significant contribution of such studies to the progress of our knowledge about the general patterns of cell organization.

electron microscopy

One of the most common, which has become a classic method used in structural and biochemical studies, is the method of electron microscopy in its various modifications. These modifications are due to both different approaches to the analysis of the structures under study and the peculiarities of cell preparation for ultrastructural studies. High resolutions of conventional transmission (transmission) microscopes make it possible to analyze not only all organelles of the nuclear and cytoplasmic apparatuses, but also some structures located at the supramolecular level of organization, for example, supporting and contractile microfibrils, microtubules, and some multienzyme complexes. Currently, to study cells at the systemic and subsystemic levels of their organization, the method of high-voltage electron microscopy is increasingly being successfully used. Due to the much higher energy of the penetrating electron beam compared to a transmission electron microscope, this method makes it possible to study “thick” sections or even whole spread cells under a microscope, which makes it possible, for example, to analyze the complex system of submembrane fibrils of the cell surface apparatus as a whole.

In the study of the function of the surface apparatus of the cell, the relationship of individual subsystems of the surface apparatus of the nucleus and a number of other issues of general cytology, the method of scanning electron microscopy, which makes it possible to study the surface of an object in volume, becomes essential.

Freeze-chipping method

A special and fundamentally important place in cytological studies of the morphobiochemical direction is occupied by the freezing-cleaving method. It is the most sparing method of preparing biological objects for ultrastructural analysis, i.e., it causes minimal changes in cellular structures compared to their native state. The essence of the method is as follows. The object is placed in an atmosphere of liquid nitrogen, which immediately stops all metabolic processes. Then chips are made from the frozen object. From the surface of the chips, replicas are obtained by applying a metal film on them. These films are further examined under an electron microscope. The advantage of the freeze-cleavage method is that the cleavage plane usually passes through the hydrophobic phase of the membrane, and this makes it possible to study on the cleavages the quantity, size, and nature of the arrangement of integral membrane proteins, i.e., directly the internal morphobiochemical organization of membranes. The method gave very valuable results in the study of various kinds of membrane structures and special formations, for example, certain types of cell contacts.

Cytochemical method

For the main task of the structural-biochemical aspect of cytological studies - the elucidation of the functional significance of structures through the analysis of their biochemical organization - cytochemical methods play an exceptionally important role. At present, they are being continuously improved both in terms of accurate qualitative identification of chemical compounds in the structures under study, and in terms of their quantitative assessment. With the help of special instruments that allow quantitative cytospectrophotometry, it is possible to determine the content of a given substance, such as RNA and DNA, not only in the cell as a whole, but also at the level of nuclear or cytoplasmic structures. Thanks to interference microscopy, it is possible to assess the total amount of protein in the cell and its changes during its life.

There is a method of cytochemical identification of enzymes, which makes it possible to judge not only the localization and amount of a particular compound in cellular structures, but also the processes of synthesis and intracellular transport of these compounds.

The cytochemistry of enzymes is based on the principle of substrate-enzyme interaction with the use of marker compounds that precipitate in this case. Determining the localization, and in some cases the activity of enzymatic systems, we can judge the localization of certain biochemical processes in cellular structures.

Autoradiography

The method of autoradiography, as well as the cytochemistry of enzymes, opens up the possibility of studying intracellular synthesis and transport, but at the same time it has even wider possibilities. The method of the author-diography is based on the use of radioactive precursors of the synthesis of macromolecules labeled with artificial isotopes (3 H, 14 C, 35 S, etc.). It allows not only to localize the sites of synthesis of certain macromolecules, but also to trace specific routes of intracellular transport of these compounds, to give a relative quantitative assessment of the intensity of synthesis and the rate of movement of macromolecules in cellular structures. In this way, in particular, the movement of RNA from the nucleus into the cytoplasm of cells was shown for the first time, the localization of synthesis and intracellular secretion transport in secretory cells were traced in detail, and many other facts important for general cytology were revealed. At its core, this method is one of the most typical methods characteristic of the structural-biochemical direction of research, since it allows you to directly study the processes of metabolism in intracellular structures in an integral, undestroyed (as in biochemical studies) cell. The essence of this method is based on the detection of molecules labeled with an artificial isotope using a photographic emulsion, which covers sections of cells and tissues fixed at different times after the introduction of a labeled precursor.

Immunocytochemical method

At present, a very accurate qualitative analysis of individual proteins of cellular structures within an integral cellular system is also possible. Such an analysis is performed using immunocytochemical methods. The essence of these methods lies in the fact that a specific protein serves as an antigen, to which specific antibodies are produced in the body of any mammals. The latter are combined with a fluorescent dye or other marker. Then, the studied cell is treated with serum with labeled antibodies. In this case, specific marked antibodies bind strictly selectively to the structures containing the studied proteins. Using this method, in particular, the localization of the main and auxiliary contractile proteins of the actin-myosin system in the submembrane fibrillar apparatus of cells was revealed, and the modification of their distribution during the formation of the mitotic apparatus and during cytotomy was shown. The same method was successfully used to prove the validity of the fluid-mosaic model of membrane organization.

Complex methods of cell research

Recently, especially great progress in the study of the structural and biochemical organization of cells has been achieved with the complex use of ultrastructural analysis methods, cytochemistry and autoradiography methods. These successes are mainly due to the development of special methods of cytochemistry and autoradiography at the ultrastructural level, which make it possible to directly analyze metabolic processes at the named level of cell organization, “structure” biochemical processes, and find out the specific significance of certain cellular structures. in separate links of complex processes of intracellular metabolism. In this regard, extensive material has been accumulated on the role of various types of the membrane phase of the cytoplasm in synthetic anabolic processes and processes of intracellular catabolism.

Major successes have been achieved, in particular, in the study of the organization and functioning of the lysosomal apparatus of cells. Important new facts were obtained in the study of the nuclear apparatus of cells. With the help of cytochemical methods, it is possible to identify ribonucleoproteins (RNPs) and deoxyribonucleoproteins (DNPs) at the ultrastructural level and thereby make significant progress in studying the organization of transcription, maturation, and intranuclear transport of various types of RNPs in eukaryotic cells, and the use of the electron autoradiography made it possible to detail the role of individual cellular structures in these processes. For example, it was possible to study in detail the function of the nucleolus and concretely structure the processes of formation of ribosomal RNA in it.

Such a synthesis of molecular-biological and structural-biochemical aspects and methods is also very typical for the development of many other important questions about the fine organization of individual cell components. At the same time, the close relationship between molecular biological and morphobiochemical cytological analysis is manifested not only in the synthesis of the final results, but also in their interaction in the process of the study itself. Such interaction is carried out either by carrying out complex work using both biochemical and cytological methods by specialists, biochemists and cytologists, or by using special complex methods that are on the border of biochemical and cytological analysis of cellular structures.

An example of the first kind is the combination of methods for the biochemical isolation of cell components with their fine ultrastructural analysis. In this way, photographs of working genes were obtained for the first time with the identification of DNA, RNA polymerases and transcribed RNA molecules on them. The improvement of this method now makes it possible in some cases to take into account the intensity of transcription by directly counting the number of RNA polymerase complexes. Using an electron microscope, you can directly study patterns of DNA replication on biochemically isolated, circular or linear DNA molecules. Methods of ultrastructural analysis are also widely used in the immunocytochemical study of the localization of individual proteins, in ribosome subparticles, in the study of various levels of DNP organization, and in many other cases.

A typical example of specially developed complex methods is the hybridization of DNA and RNA on sections. Its essence is as follows. DNA, which is part of the DNP of a whole cell, is denatured, and then processed by RNA fractions labeled with radioactive isotopes. As a result, DNA autoradiographically reveals regions that are complementary to given RNA fractions, i.e., the transcription sites of the latter, in other words, it becomes possible to accurately determine the localization of certain genes.

Within the framework of the experimental method, the functional organization of the cell as a whole or its individual components is studied by changing its state with the help of external influence. Observing then changes in the vital activity of the cell or its components, one can draw conclusions about certain properties of the studied mechanisms. This kind of method is now very widespread in some sections of cytology, and in some of its areas, the cytophysiological aspect of the analysis of cellular structures still occupies a dominant position.

This is precisely the state of the problem of the transport function of the surface apparatus of the cell. On the one hand, significant progress has been made in the study of this issue: based on the results of cytophysiological analysis, it was possible to identify varieties of transmembrane transport of substances, to characterize various properties of transport systems. On the other hand, the final solution of the question of the mechanisms of trans-membrane transport is possible only if the specific organization of the lipid-protein system of membranes is clarified and the exact knowledge of the properties and role of the remaining components of membrane transport systems, i.e., at the level of structural and biochemical analysis of the plasma membrane and the entire surface apparatus of the cell.

The limited possibilities of the cytophysiological study of transmembrane transport are clearly manifested by the example of the state of the issue of the organization of ion channels, which play a major role in many important processes, such as, for example, the propagation of a nerve impulse. With the help of a whole arsenal of various cytophysiological methods, it was shown that in the plasma membrane there are special channels for Na, K, Cl ions, which differ in their properties. However, specific knowledge of their structural organization is still limited by indirect data on their protein nature. Thus, the solution of the question of the organization of ion channels in particular and membrane transport systems in general seems to fall into the hands of scientists who are proficient in structural biochemical methods, because in this case, numerous and very valuable facts obtained in cytophysiological studies are is only the first phenomenological stage in the analysis of these general cellular mechanisms. Nevertheless, in certain aspects of the study of the cell, the cytophysiological approach can give a lot.

At present, the variety of methods of cytophysiological studies is determined both by the ever-increasing arsenal of agents that appear in cytologists, and by the use of subtle methods for analyzing the changes that occur as a result of the action of these agents on the cell. If earlier, to analyze changes in cells under the action of external agents, such methods familiar to physiologists as registration of electrical potentials, assessment of cellular respiration by oxygen uptake, quantitative assessment of dye sorption, registration of qualitative changes in cell staining, etc. were used. , now for such purposes, methods characteristic of the structural and functional direction are increasingly used: electron microscopic study of ultrastructural changes, autoradiographic analysis of synthetic processes, etc.

Among the agents used in experimental studies, two main groups can be distinguished. The first group consists of substances whose "point of application" inside the cell is more or less known - these are substances that block individual links of intracellular metabolism (for example, actinomycin D, which inhibits transcription, or puromycin, which blocks protein synthesis, 2,4-dinitrophenol, which uncouples respiration and oxidative phosphorylation), substances that selectively destroy certain cellular structures (for example, colchicine, which destroys microtubules, or cytochalasin B, which acts on microfibrils). The second group consists of agents of the so-called complex action that change cell metabolism in general - temperature, osmotic pressure, pH, etc. The use of such agents as, for example, 2,4-dinitrophenol, made it possible to clarify a number of questions concerning the conjugation of respiration and phosphorylation in the respiratory chain of mitochondria; the use of inhibitors of RNA and protein synthesis made it possible to study some links of protein synthesis in ribosomes and transcription processes; using colchicine and cytochalasin, the role of microtubules and microfilaments in the processes of intracellular transport has been elucidated.

Agents of the second group (complex action) have the advantage that they are, as it were, more natural for cells, because cells in natural conditions encounter similar changes in the external environment. At the same time, they affect almost all aspects of cellular metabolism, making it difficult to analyze the changes that occur in this case. Nevertheless, the study of the effect of such agents on the cell is of independent importance and is absolutely necessary for studying the mechanisms of cell adaptation to changing environmental factors, resolving the issue of the ratio of specific and nonspecific processes in the response of cells to external influences and other similar tasks. , which play an important role in developing the problem of cellular integration.

In the study of the functional organization of cells, the analysis of the mechanisms of interaction of individual cell systems is of great importance. In many cases, this problem can be solved by creating special experimental models. The most typical examples of this kind are nuclear transplants in different objects (protozoa, amphibian eggs); hybridization of somatic cells; transplantation of cell parts in protozoa; research using a number of other microsurgical techniques, conducted on protozoological objects and in vitro cultured mammalian cells.

With the help of such models, the most important general cytological issues were studied. For example, the results of experiments on transplanting the nuclei of differentiated amphibian cells into an ovum devoid of its own nucleus were one of the most convincing arguments in favor of the theory of differential gene activity. The essence of the latter is to state the structural identity of the genomes of differentiated cells of a multicellular organism. This implies a fundamentally important position that the process of differentiation occurs not through irreversible changes in the hereditary apparatus of cells, but through regulation of the activity of a set of genes that is the same for all cells of a given organism.

Very interesting facts were found on an experimental model for studying the process of dedifferentiation of a hybrid cell — a chicken erythrocyte and a mammalian cancer cell. The peculiarity of this heterokaryon lies in the fact that when a chicken erythrocyte merges with a cancer cell, hemoglobin hemolysis occurs and a normal, almost completely inactivated erythrocyte nucleus is found in the cytoplasm of the cancer cell. Thus, transplantation of the differentiated nucleus into unusual conditions of the active cytoplasm is carried out here. Careful observations of changes in the structural organization of these nuclei showed that under new conditions there is a significant increase in their volume. Proteins coming from the cytoplasm play a significant role in the swelling of the nuclei. These external changes in the nuclear apparatus of the erythrocyte reflect the deep processes of restructuring of its internal organization, resulting in the resumption of transcription of "chicken" messenger RNA. However, the implementation of the information contained in it in the form of the synthesis of "chicken" proteins does not occur until the nucleolus is formed in the nuclear apparatus of chicken erythrocytes and the synthesis of ribosomal RNA begins. Thus, a thorough analysis of experimental models showed the presence of complex cytoplasmic control over the activity of the nuclear apparatus.

With the help of experimental models, it was possible to solve a number of other important general cytological issues. For example, the question of the mechanisms of movement of anaphase chromosomes was successfully studied on the native mitotic apparatus isolated from the crushing sea urchin blastomeres and working outside the cell. Mostly on experimental models, it was possible to establish a widespread general pattern of cell organization, namely, the absence of a rigid cause-and-effect principle in the relationship of complex intracellular processes. It turned out that such multi-component processes as cell reproduction, the processes of synthesis and intracellular transport of high-polymer compounds, etc., consist of separate, relatively autonomous stages that are not connected by a rigid causal relationship. The elucidation of this pattern, on the one hand, creates the prerequisites for understanding the mechanisms of the amazing plasticity of cellular organization. On the other hand, the same regularity is the basis for studying the mechanisms of integration of such processes in an integral cellular system under normal conditions.

Currently, the number and variety of experimental models designed to solve certain specific general cytological problems is increasing. This significantly contributes to the progress of our knowledge in a relatively poorly studied area of ​​cytology - the mechanisms of interaction and integration of the work of subcellular systems.

It should be emphasized that the specifics of research carried out within the framework of the experimental approach to the analysis of the patterns of cell organization is the ever greater deepening of the criteria and signs by which the analysis of the integrating mechanisms and specific functions of individual cellular structures is carried out. At the same time, it becomes clear that the successful solution of the tasks facing such research is possible only with the widespread introduction of the methods of the structural-functional approach into practice.

The essence of the comparative cytological method of research in general cytology is the elucidation of the general patterns of cell organization using the whole variety of their varieties provided to the scientist by living nature. The comparative method has two aspects. On the one hand, it is traditionally used to identify related relationships between individual types of cells (especially for unicellular organisms). On the basis of the phylogenetic systematics of prokaryotic and lower and higher eukaryotic cells created in this way and carried out using fine cytological criteria, it becomes possible to trace the formation of both individual particular cell systems and the general mechanisms of regulation and integration of the cell as an integral system. As an example this kind of application of comparative cytological analysis in cell research can provide interesting data on the fine organization of the nuclear apparatus in prokaryotic, lower and higher eukaryotic cells

The principal features of the organization of the nuclear apparatus of eukaryotic cells are the presence of a complex surface apparatus of the nucleus, a significantly larger amount of DNA concentrated in chromosomes compared to prokaryotic cells, and, finally, a kind of packaging of DNA using the main proteins - histones. analysis of the nuclear apparatus of lower eukaryotes made it possible to identify among them cells that, in terms of the structure of the nucleus, occupy an intermediate position between pro- and eukaryotic cells. Armored flagellates have a typical surface apparatus of the nucleus, but their chromosomes, as in the case of prokaryotes, are formed by ring-shaped DNA molecules, which are organized into compact structures without the participation of histones, characteristic of all eukaryotes

Recently, in connection with the discovery of fundamental features in the organization of the genome of pro- and eukaryotic cells, the comparison of the processes of transcription and maturation of RNA in these organisms, as well as in mesokaryotes and cells of lower eukaryotes, has become important. As a result of such comparisons, there may be significant changes have been made to our traditional ideas about kinship relationships in the main groups of organisms and, in particular, the relationship between pro- and eukaryotic cells.

The second example of the traditional application of the evolutionary approach to cytological problems can be attempts to propose a hypothesis of the complication of the mechanisms of the equally hereditary distribution of chromosomes between daughter cells in the process of evolution, developed on the basis of a comparative analysis of numerous variants of chromosome divergence in protozoa and in lower plants. In these cases, the membranes of the nuclear envelope take an active part in the processes of chromosome segregation, which allows for a certain homology with prokaryotic cells, in which the cell membrane plays a leading role in the uniform distribution of sister chromosomes between daughter cells.

Finally, the widely accepted symbiotic hypothesis of the origin of mitochondria and chloroplasts can serve as a third example of the traditional evolutionary approach to general cytological problems. Its essence lies in the assumption that these important organelles of energy metabolism arose from prokaryotic organisms that invaded eukaryotic cells at a relatively early stage of eukaryotic evolution.

Despite the importance of this kind of general biological constructions for the development of general cytology, the traditional historical approach to the development of general cytological problems is now still of rather limited use. One of the main reasons for this situation is the presence of specific and still insufficiently studied features of the evolutionary process at the cellular and subcellular levels of organization, which makes it extremely difficult to determine the relationship between individual groups of unicellular organisms, and, consequently, the construction of reasonable evolutionary nyh hypotheses in the field of general cytology.

At present, another aspect of using the comparative cytological method is more widespread, not pursuing the goal of direct clarification of the historical conditionality of a particular cellular structure or process. In modern general cytology, this aspect of the application of the comparative method has undergone several modifications.

At the first stage, during the introduction of fundamentally new morphobiochemical methods into the practice of cytological analysis, the choice of the object of study was determined by the following considerations. First, the convenience of this or that object for the application of the method used mattered. Secondly, the degree of expression of this trait in the studied cell played an important role. So, to study the general patterns of organization of eukaryotic cells, mammalian liver cells with their harmoniously developed system of membrane organelles were a favorite object. mammals.

For complex cytological and molecular biological studies of the organization of prokaryotic cells, Escherichia coli was widely used; The models for studying the organization of lower eukaryotes were yeasts and molds. At the same time, it turned out that the regularities established on these objects are of universal significance, since in many cases they are fundamentally similar in all eukaryotic or all prokaryotic cells. Moreover, a number of patterns of subcellular organization, especially at the molecular and supramolecular levels, turned out to be universal for cells of both pro- and eukaryotic types (organization of membranes, the principle of the structure of ribosomes, etc.), despite the fact that that these types of cells are fundamentally different from each other in some respects. This circumstance gave rise to the idea that it is possible to develop the main general cytological problems on a limited range of objects that are methodologically convenient, and then extend the established patterns to other cells due to their fundamentally similar organization.

However, in recent years such a simplified use of the comparative approach has begun to be criticized as modern cytological methods are introduced into the special biological sciences of the cell - particular cytology, protozoology, botany of lower plants. The morphobiochemical analysis applied in these fields of science made it possible to establish facts that testify to the enormous diversity of the specific implementation of one or another common feature of cell organization, the diversity is much greater than follows from the results obtained earlier on "model" objects. This diversity is especially great at the highest subsystem and systemic levels of cell organization. It is also typical for such complex and multicomponent processes as the processes of intracellular metabolism and transport or the equal hereditary distribution of genetic material during cell division.

The generalization of a large comparative cytological material, obtained at the level of modern methodological capabilities, forced us to abandon the above-mentioned simplified idea of ​​the role of the comparative method. In this regard, in general cytology (especially with regard to eukaryotic cells), the dominant position is acquired by the idea of ​​the need to use a comparative method for the analysis of individual cell systems or processes similar in functional activity in all the variety of their manifestations in specific cells. RES is caused not by "typical", "average" cells, but, on the contrary, by cells that sharply deviate from the average type of organization, cells in which certain signs are hypertrophied.

The largest number of such "evading" variants is found among the cells of higher multicellular organisms, where far-reaching specialization of cells as part of individual tissue systems is developed. Cases of "deviation" from the middle type are also widespread among the higher protozoa, which have undergone evolution, while maintaining the unicellular level of organization. It was during the study of this kind of atypical cells that it was possible to reveal a large number of new interesting facts that significantly deepen our understanding of both the general laws of cellular organization and its evolutionary plasticity, which determines the observed diversity of cellular systems. At the same time, as noted above, in the case of special sciences, the specific manifestation of common features characteristic of all cells in various objects is of greatest interest.

In contrast to the special sciences, with a general cytological approach, this question is posed in a slightly different plane, because the researcher seeks to find out how widespread the specific manifestations of this trait are in different cells, what combination of general mechanisms and what exactly it is due to. So, for example, protozoologists managed to discover a very interesting dynamics of macronucleus formation after conjugation in gastrociliary ciliates. In the emerging macronucleus, a significant increase in the amount of DNA occurs, and then a sharp reduction in the hereditary material is observed (up to 93%). Such a process of reduction of genetic material also takes place in the somatic cells of a number of groups of multicellular animals (some insects, nematodes). The remaining DNA, small in total quantity, but containing all the information necessary for the functioning of the macronucleus, is replicated many times. As a result, a definitive macronucleus is created, which differs from the micronucleus not only in the amount of DNA, but also in its qualitative composition. The vast majority of non-functioning genes are absent here, while functioning loci are represented by a significant number of copies.

These facts are of great general cytological interest precisely because, as a rule, the phenomena observed here are not just paradoxical features characteristic only of higher unicellular organisms. Thus, the processes of polytenization, selective replication of individual sections of chromosome DNA, and, finally, selective reduction of significant sections of the genome - all these phenomena also take place in specialized cells of multicellular organisms. They are carried out, probably, on the basis of common elementary mechanisms. And the specificity of the complex process of changes in the nuclear apparatus during the formation of the macronucleus in gastrociliated ciliates is mainly due to a peculiar combination of general, universal elementary mechanisms for eukaryotic cells. Ideas of this kind are now widely used in general cytology. They are extremely stimulating directed comparative cytological studies devoted to the elucidation of important general cytological problems. material from the site

An example of a targeted comparative cytological study is the study of the mechanism of the equal hereditary distribution of chromosomes during mitosis in eukaryotes by analyzing the mitosis of different types of diatoms: on these objects, in contrast to typical mitoses of metazoan cells, it is possible to clearly morphologically trace complex changes in microtubule organizing centers, the formation and mutual divergence of microtubular semi-spindles, divergence of chromosomes to the poles of the cell with the help of the formation in the metaphase of a peculiar structure - a collar.

In the light of recent data on the leading role of the tubulin-dynein mechanochemical system in the anaphase movement of chromosomes in metazoan cells, it is very likely that this system is also present in diatoms, i.e. here, too, there is only a peculiar combination of elementary mechanisms common to all cells and causing mechanochemical processes during mitosis.

Obviously, for the analysis of these mechanisms, the elucidation of which is one of the most urgent problems of general cytology, it would be promising to have such an object where they are clearly differentiated and morphologically expressed.

The number of such examples is constantly growing. This is due, on the one hand, to the ever-expanding introduction of complex modern methods into the practice of particular cytological, protozoological and botanical studies, on the other hand, the accumulation in the comparative cytological studies themselves of facts that are becoming increasingly important for the general cytology and are in the center of its attention. And all this, in turn, leads to the fact that comparative cytological analysis begins to occupy an exceptionally important place in cytology.

A brief description of the main directions and aspects of modern general cytological research shows that at this stage in the development of cytology, there is both a fairly clear distinction between individual areas and their synthesis. There is a distinction both in methodological terms and in terms of the logic of solving specific problems set within each of the directions and approaches. In the morphofunctional aspect of cytological studies, a discrete approach to the analysis of cellular structures dominates. One of the most important features of the experimental approach to the study of the patterns of cellular organization is its focus on the analysis of the general integrating mechanisms of organization of cellular systems and the whole cell. At the same time, as already emphasized above, the solution of the problems facing such studies is impossible without the widespread use of methods inherent in the morphofunctional approach. Experimental analysis gives a phenomenological characterization of the properties of certain cellular mechanisms and intracellular processes, thereby creating the necessary basis for the application of a rich arsenal of structural and biochemical methods.

Thus, at the present stage of development of general cytology, there are prerequisites for a very close combination of these two aspects of cytological studies. This is natural, since in the end both approaches pursue the same goal - to elucidate the functional organization of cellular structures and the mechanisms of regulation of processes in an integral cellular system.

The comparative cytological approach to the analysis of general cytological problems occupies a special position in modern general cytology. Comparative cytological analysis is carried out on the basis of data obtained on the basis of morphofunctional and experimental approaches, i.e., methodically, all the main aspects of cytological studies are closely related to each other.

The specificity of the comparative cytological approach, the specificity that determines its special position, is the purposeful use of various objects of wildlife to study the general patterns of organization of individual cellular structures, intracellular processes and integrating mechanisms in all the variety of their manifestations in various cell types.

So, as can be seen from the above, the main directions of cytological research largely determine the specifics of the current stage in the development of general cytology and determine its close relationship with related biological sciences. One of the most characteristic features of this stage is the close interconnection of all the most important areas of cytological research in a methodological sense. Moreover, such methodical integration often goes beyond the scope of general cytology.

In cytological work, purely biochemical and molecular biological methods are widely used, and vice versa, in biochemical and molecular biological studies, cytological morphological methods are widely used. The methodical integration of related sciences and the unity of their ultimate goals led to the formation of a new synthetic science of the cell - cell biology. It combines cytology, structural biochemistry, molecular biology, molecular genetics and private biological sciences about the cellular level of organization. Such a union of related sciences is undoubtedly a progressive phenomenon. However, despite such a synthesis, each of the sciences retains both its methodological specifics and specifics in the formulation and methods of developing problems of cell organization. At present, the dominant position in this synthetic science belongs to research in the molecular biological and molecular genetic areas. This situation is due to the rapid progress of our knowledge of the lower levels of cell organization, but it is only a temporary phenomenon.

In fact, the leading place in the new synthetic science of the cell should be occupied by general cytology - the science of the general patterns of the cellular level of organization of living matter. Of the modern biological sciences dealing with this level of organization of living matter, general cytology, expanding the purposeful comparative cytological approach based on structural biochemical methods, is most prepared for a deep general biological generalization of a huge amount of factual material on discrete analysis of individual -ny cell structures in numerous varieties of cells. The leading position should be taken by general cytology in the analysis of general cellular integrating mechanisms. An important prerequisite for this is the rapid development of new experimental models. Their in-depth analysis by modern methods and the widespread introduction of experimental models in targeted comparative cytological studies should ensure progress in solving one of the main problems of cell organization - the problem of cell integration.

With the accumulation of factual material on the elementary universal mechanisms of cell integration and the scope of their modifications, it is general cytology that is faced with the task of conducting a deep analysis of the historical conditionality of the organization of particular cellular systems and cellular organization as a whole, as well as the specifics of the evolutionary process on the cellular and subcellular levels of organization of living matter. The solution of this problem is facilitated by the tendency now clearly emerging in general cytological studies to combine discrete analysis of individual components of a cell with the study of its kg. to a complete system.

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Plan:

1. What does cytology study.

2. The idea that organisms are made up of cells.

3. Research methods used in cytology.

4. Fractionation of cells.

5. Radio autography.

6. Determining the duration of some stages of the cell cycle by autoradiography.

Cytology is the science of the cell. It stood out from the environment of other biological sciences almost 100 years ago. For the first time, generalized information about the structure of cells was collected in the book by J.-B. Carnoy's The Biology of the Cell, published in 1884. Modern cytology studies the structure of cells, their functioning as elementary living systems: the functions of individual cellular components, the processes of cell reproduction, their repair, adaptation to environmental conditions, and many other processes are studied, which make it possible to judge the properties and functions common to all cells. Cytology also considers the structural features of specialized cells. In other words, modern cytology is cell physiology. Cytology is closely associated with the scientific and methodological achievements of biochemistry, biophysics, molecular biology and genetics. This served as the basis for an in-depth study of the cell already from the standpoint of these sciences and the emergence of a certain synthetic science of the cell - cell biology, or cell biology. At present, the terms cytology and cell biology coincide, since their subject of study is the cell with its own patterns of organization and functioning. The discipline "Cell Biology" refers to the fundamental sections of biology, because it explores and describes the only unit of all life on Earth - the cell.

A long and close study of the cell as such led to the formulation of an important theoretical generalization of general biological significance, namely, the emergence of the cell theory. In the 17th century Robert Hooke, a physicist and biologist of great ingenuity, created the microscope. Examining a thin section of cork under his microscope, Hooke discovered that it was made up of tiny empty cells separated by thin walls, which, as we now know, are composed of cellulose. He called these little cells cells. Later, when other biologists began to examine plant tissues under a microscope, it turned out that the small cells found by Hooke in a dead dried cork are also found in living plant tissues, but they are not empty, but each contain a small gelatinous body. After microscopic examination of animal tissues, it was found that they also consist of small gelatinous bodies, but that these bodies are only rarely separated from each other by walls. As a result of all these studies, in 1939, Schleiden and Schwann independently formulated the cell theory, which states that cells are the elementary units from which all plants and all animals are ultimately built. For some time, the double meaning of the word cell still caused some misunderstandings, but then it was firmly entrenched in these small jelly-like bodies.

The modern understanding of the cell is closely related to technical advances and improvements in research methods. In addition to conventional light microscopy, which has not lost its role, polarization, ultraviolet, fluorescence, and phase-contrast microscopy have acquired great importance in the past few decades. Among them, a special place is occupied by electron microscopy, the resolution of which made it possible to penetrate and study the submicroscopic and molecular structure of the cell. Modern research methods have made it possible to reveal a detailed picture of cellular organization.

Each cell consists of a nucleus and cytoplasm, separated from each other and from the external environment by membranes. The components of the cytoplasm are: membrane, hyaloplasm, endoplasmic reticulum and ribosomes, Golgi apparatus, lysosomes, mitochondria, inclusions, cell center, specialized organelles.

A part of an organism that performs a specific function is called an organ. Any organ - lung, liver, kidney, for example - each has its own special structure, thanks to which it plays a certain role in the body. In the same way, there are special structures in the cytoplasm, the peculiar structure of which enables them to carry out certain functions necessary for cell metabolism; these structures are called organelles ("small organs").

Elucidation of the nature, function and distribution of cytoplasmic organelles became possible only after the development of methods of modern cell biology. The most useful in this regard were: 1) electron microscopy; 2) cell fractionation, with the help of which biochemists can isolate relatively pure fractions of cells containing certain organelles, and thus study individual metabolic reactions of interest to them; 3) autoradiography, which made possible the direct study of individual metabolic reactions occurring in organelles.

The method by which organelles are isolated from cells is called fractionation. This method proved to be very fruitful, giving biochemists the ability to isolate various cell organelles in a relatively pure form. It also makes it possible to determine the chemical composition of organelles and the enzymes contained in them and, on the basis of the data obtained, to draw conclusions about their functions in the cell. As a first step, the cells are destroyed by homogenization in some suitable medium that preserves the organelles and prevents their aggregation. Very often, a sucrose solution is used for this. Although the mitochondria and many other cell organelles remain intact, membrane tangles such as the endoplasmic reticulum and the plasma membrane are fragmented. However, the resulting membrane fragments often close on themselves, resulting in rounded bubbles of various sizes.

At the next stage, the cell homogenate is subjected to a series of centrifugations, the speed and duration of which increases each time; this process is called differential centrifugation. Different cell organelles are deposited at the bottom of centrifuge tubes at different centrifugation speeds, depending on the size, density and shape of the organelles. The resulting precipitate can be sampled and examined. Larger, denser structures such as nuclei precipitate the fastest, while smaller, less dense structures, such as endoplasmic reticulum vesicles, require higher rates and longer times. Therefore, at low centrifugation speeds, nuclei are precipitated, while other cell organelles remain in suspension. At higher speeds, mitochondria and lysosomes are precipitated, and with long centrifugation and very high speeds, even small particles such as ribosomes precipitate. Precipitates can be examined using an electron microscope to determine the purity of the resulting fractions. All fractions are contaminated to some extent with other organelles. If, nevertheless, it is possible to achieve sufficient purity of the fractions, then they are then subjected to biochemical analysis in order to determine the chemical composition and enzymatic activity of the isolated organelles.

For the progress of histology, cytology and embryology, the introduction of the achievements of physics and chemistry, new methods of related sciences - biochemistry, molecular biology, genetic engineering is of great importance.

Modern research methods make it possible to study tissues not only as a whole, but also to isolate individual cell types from them to study their vital activity for a long time, to isolate individual cell organelles and their macromolecules (for example, DNA), and to study their functional features.

Such opportunities have opened up in connection with the creation of new instruments and technologies - various types of microscopes, computer technology, X-ray diffraction analysis, the use of nuclear magnetic resonance (NMR), radioactive isotopes and autoradiography, electrophoresis and chromatography, fractionation of cell contents using ultracentrifugation, separation and cultivation of cells, obtaining hybrids; the use of biotechnological methods - obtaining hybridomas and monoclonal antibodies, recombinant DNA, etc.

Thus, biological objects can be studied at the tissue, cellular, subcellular and molecular levels. Despite the introduction into the natural sciences of various biochemical, biophysical, physical and technological methods necessary to solve many issues related to the vital activity of cells and tissues, histology basically remains a morphological science with its own set of methods. The latter make it possible to characterize the processes occurring in cells and tissues, their structural features.

The main stages of cytological and histological analysis are the choice of the object of study, its preparation for examination under a microscope, the use of microscopy methods, and the qualitative and quantitative analysis of images.

The objects of study are living and fixed cells and tissues, their images obtained in light and electron microscopes or on a television display screen. There are a number of methods that allow the analysis of these objects.

Methods of microscopy of histological preparations

The main methods for studying biological microobjects are light and electron microscopy, which are widely used in experimental and clinical practice.

Microscopy is the main method of studying micro-objects used in biology for over 300 years. Since the creation and use of the first microscopes, they have been constantly improved. Modern microscopes are a variety of complex optical systems with high resolution. The size of the smallest structure that can be seen under a microscope is determined by the smallest resolvable distance (d o ), which mainly depends on the wavelength of the light. (\) and wavelengths of electromagnetic oscillations of the electron flow, etc. This dependence is approximately determined by the formula d 0 = 1 / 2 \. Thus, the smaller the wavelength, the smaller the resolvable distance and the smaller the microstructures that can be seen in the preparation. Various types of light microscopes and electron microscopes are used to study histological preparations.

Rice. 1. Microscopes for biological research.

A - light biological microscope "Biolam-S": 1 - base; 2 - tube-holder; 3 - inclined tube; 4 - eyepiece, 5 - revolver; 6 - lenses; 7 - table; 8 - condenser with iris diaphragm; 9 - condenser screw; 10 - mirror; 11 - micrometer screw; 12 - macrometric screw. B - electron microscope EMV-100AK with an automated image processing system: 1 - microscope column (with an electron-optical system and a camera for samples); 2 - control panel; 3 - camera with luminescent screen; 4 - image analysis block; 5 - video signal sensor.

Light microscopy. To study histological micro-objects, ordinary light microscopes and their varieties are used, which use light sources with different wavelengths. In conventional light microscopes, the source of illumination is natural or artificial light (Fig. 1, A). The minimum wavelength of the visible part of the spectrum is approximately 0.4 µm. Therefore, for a conventional light microscope, the smallest the resolution distance is approximately 0.2 µm ( d o = "/, - 0.4 μm = 0.2 μm), and the total magnification (the product of the lens magnification and the eyepiece magnification) can be 1500-2500.

Thus, in a light microscope, one can see not only individual cells ranging in size from 4 to 150 microns, but also their intracellular structures - organelles, inclusions. To enhance the contrast of micro-objects, their staining is used.

ultraviolet microscopy. This is a type of light microscopy. The ultraviolet microscope uses shorter ultraviolet rays with a wavelength of about 0.2 µm. The resolved distance here is 2 times less than in conventional light microscopes, and is approximately 0.1 μm (d o = V 2 - 0.2 μm = 0.1 μm). The image obtained in ultraviolet rays, invisible to the eye, is converted into a visible one by registering on a photographic plate or by using special devices (luminescent screen, electron-optical converter).

Fluorescent (luminescent) microscopy. The phenomenon of fluorescence lies in the fact that the atoms and molecules of a number of substances, absorbing short-wavelength rays, go into an excited state. The reverse transition from the excited state to the normal state occurs with the emission of light, but with a longer wavelength. In a fluorescent microscope, mercury or ultrahigh-pressure xenon lamps are used as light sources for excitation of fluorescence, which have high brightness in the spectral region of 0.25-0.4 μm (near ultraviolet rays) and 0.4-0.5 μm (blue-violet rays). The wavelength of the fluorescence light wave is always greater than the wavelength of the exciting light, so they are separated using light filters and the image of the object is studied only in the light of fluorescence. Distinguish between own, or primary, and induced, or secondary, fluorescence. Any cell of a living organism has its own fluorescence, but it is often extremely weak.

Serotonin, catecholamines (adrenaline, noradrenaline) contained in nerve, mast and other cells have primary fluorescence after tissue fixation in formaldehyde vapor at 60-80 °C (Falk method).

Secondary fluorescence occurs when preparations are treated with special dyes - fluorochromes.

There are various fluorochromes that specifically bind to certain macromolecules (acridine orange, rhodamine, fluorescein, etc.). For example, when processing preparations, fluorochrome acridine orange is most often used. In this case, DNA and its compounds in cells are bright green, and RNA and its derivatives - a bright red glow. Thus, the spectral composition of radiation carries information about the internal structure of the object and its chemical composition. A variant of the method of fluorescence microscopy, in which both excitation and emission of fluorescence occur in the ultraviolet region of the spectrum, is called the method ultraviolet fluorescence microscopy.

Phase contrast microscopy. This method is used to obtain contrast images of transparent and colorless living objects, invisible with conventional microscopy methods. As already mentioned, in a conventional light microscope, the necessary contrast of structures is achieved by staining. The phase contrast method provides the contrast of the studied unstained structures due to a special annular diaphragm placed in the condenser and the so-called phase plate located in the objective. This design of the microscope optics makes it possible to convert the phase changes of the light passing through the unstained specimen, which are not perceived by the eye, into a change in its amplitude, i.e. brightness of the resulting image. Increasing the contrast allows you to see all structures that differ in refractive index. A variation of the phase contrast method is the method phase-dark-field contrast, giving a negative versus positive phase contrast image.

Dark field microscopy. In a dark-field microscope, only the light that diffracts the structures in the preparation reaches the objective. This happens due to the presence of a special condenser in the microscope, which illuminates the preparation with strictly oblique light; rays from the illuminator are directed from the side. Thus, the field looks dark, and the small particles in the preparation reflect the light, which then enters the lens. The resolution of this microscope cannot be better than that of a brightfield microscope because the same wavelength is used. But there is more contrast here. It is used to study living objects, autoradiographic objects such as silver grains that appear bright in a dark field. In the clinic, it is used to study crystals in the urine (uric acid, oxalates), to demonstrate spirochetes, in particular treponema pallidum, which causes syphilis, etc.

interference microscopy. Varieties of the phase contrast microscope are the interference microscope, which is designed to quantify tissue mass, and the differential interference microscope (with Nomarsky optics), which is specifically used to study the surface relief of cells and other biological objects.

In an interference microscope, the beam of light from the illuminator is divided into two streams: one passes through the object and changes the phase of the oscillation, the second goes bypassing the object. In the prisms of the objective, both beams are connected and interfere with each other. As a result, an image is constructed in which sections of a micro-object of different thickness and density differ in contrast. After quantifying the changes, determine the concentration and mass of dry matter.

Phase-contrast and interference microscopes make it possible to study living cells. They use the interference effect that occurs when two sets of waves are combined to create an image of microstructures. The advantage of phase-contrast, interference and dark-field microscopy is the ability to observe cells in the process of movement and mitosis. In this case, cell movement can be recorded using time-lapse (frame-by-frame) microfilming.

polarizing microscopy. A polarizing microscope is a modification of a light microscope in which two polarizing filters are installed - the first (polarizer) between the light beam and the object, and the second (analyzer) between the objective lens and the eye. Light passes through the first filter in only one direction, the second filter has a main axis that is perpendicular to the first filter, and it does not transmit light. This creates a dark field effect. Both filters can be rotated to change the direction of the light beam. If the analyzer is rotated 90° with respect to the polarizer, no light will pass through them. Structures containing longitudinally oriented molecules (collagen, microtubules, microfilaments) and crystalline structures (in Leydig cells 1) appear as luminous when the rotation axis changes. The ability of crystals or paracrystalline formations to split a light wave into an ordinary wave and a wave perpendicular to it is called birefringence. This ability is possessed by fibrils of striated muscles.

Electron microscopy. A big step forward in the development of microscopy technology was the creation and use of an electron microscope (see Fig. 1, B). The electron microscope uses a stream of electrons with shorter wavelengths than the light microscope. At a voltage of 50,000 V, the wavelength of electromagnetic oscillations arising from the movement of a stream of electrons in a vacuum is 0.0056 nm. It is theoretically calculated that the resolvable distance under these conditions can be about 0.002 nm, or 0.000002 µm, i.e. 100,000 times less; than in a light microscope. In practice, in modern electron microscopes, the resolvable distance is about 0.1-0.7 nm.

Currently, transmission (transmission) electron microscopes (TEM) and scanning (scanning) electron microscopes (SEM) are widely used. With the help of TEM, only a planar image of the microobject under study can be obtained. To obtain a spatial representation of the structures, SEMs are used that can create a three-dimensional image. A scanning electron microscope works on the principle of scanning an object under study with an electron microprobe, i.e., it sequentially "feels" individual points of the surface with a sharply focused electron beam. To study the selected area, the microprobe moves along its surface under the action of deflecting coils (the principle of television scanning). Such an examination of an object is called scanning (reading), and the pattern along which the microprobe moves is called a raster. The resulting image is displayed on a television screen, the electron beam of which moves synchronously with the microprobe.

The main advantages of scanning electron microscopy are a large depth of field, a wide range of continuous changes in magnification (from tens to tens of thousands of times) and high resolution.

Freezing electron microscopy- chipping used to study the details of the structure of membranes and intercellular connections. Cells are frozen at a low temperature (-160°C) to make chips. When examining the membrane, the cleavage plane passes through the middle of the lipid bilayer. Further, metals (platinum, palladium, uranium) are deposited on the inner surfaces of the obtained halves of the membranes, they are studied using TEM and microphotography.

Method of cryoelectron microscopy. A rapidly frozen thin layer (about 100 nm) of the tissue sample is placed on a microscopic grid and examined under a microscope vacuum at -160°C.

The method of electron microscopy "freezing- etching" used to study the outer surface of cell membranes. After rapidly freezing the cells at a very low temperature, the block is split open with a knife blade. The resulting ice crystals are removed by sublimation of water in a vacuum. Then areas of the cells are shaded by sputtering a thin film of a heavy metal (for example, platinum). The method makes it possible to reveal the three-dimensional organization of structures.

Thus, the methods of freezing-cleavage and freezing-etching make it possible to study non-fixed cells without the formation of fixation-induced artifacts in them.

Methods of contrasting with salts of heavy metals make it possible to study individual macromolecules - DNA, large proteins (for example, myosin) in an electron microscope. With negative contrasting, aggregates of macromolecules (ribosomes, viruses) or protein filaments (actin filaments) are studied.

Electron microscopy of ultrathin sections obtained by cryoultra-microtomy. With this method, tissue pieces without fixation and pouring into solid media are quickly cooled in liquid nitrogen at a temperature of -196 °C. This provides inhibition of the metabolic processes of cells and the transition of water from the liquid phase to the solid. Next, the blocks are cut on an ultramicrotome at low temperature. This method of sectioning is usually used to determine the activity of enzymes, as well as for carrying out immunochemical reactions. To detect antigens, antibodies associated with particles of colloidal gold are used, the localization of which is easy to identify on preparations.

Methods of ultrahigh-voltage microscopy. Electron microscopes with an accelerating voltage of up to 3,000,000 V are used. The advantage of these microscopes is that they allow you to study objects of great thickness (1-10 microns), since at high electron energy they are less absorbed by the object. Stereoscopic imaging allows obtaining information about the three-dimensional organization of intracellular structures with high resolution (about 0.5 nm).

X-ray diffraction analysis. To study the structure of macromolecules at the atomic level, methods are used using X-rays having a wavelength of about 0.1 nm (hydrogen atom diameter). The molecules that form a crystal lattice are studied using diffraction patterns, which are recorded on a photographic plate in the form of many spots of varying intensity. The intensity of the spots depends on the ability of various objects in the array to scatter radiation. The position of the spots in the diffraction pattern depends on the position of the object in the system, and their intensity indicates its internal atomic structure.

Methods for studying fixed cells and tissues

Study of fixed cells and tissues. The main object of research are histological preparations, made from fixed structures. The drug may be a smear (for example, a smear of blood, bone marrow, saliva, cerebrospinal fluid, etc.), an imprint (for example, of the spleen, thymus, liver), a film of tissue (for example, connective or peritoneal, pleura, pia mater) , thin cut. Most often, a section of a tissue or organ is used for study. Histological preparations can be studied without special processing. For example, a prepared blood smear, print, film, or section of an organ can be immediately viewed under a microscope. But due to the fact that the structures have a "weak contrast", they are poorly detected in a conventional light microscope and the use of special microscopes (phase contrast, etc.) is required. Therefore, specially processed preparations are more often used.

The process of manufacturing a histological preparation for light and electron microscopy includes the following main steps: 1) taking the material and fixing it, 2) compacting the material, 3) preparing sections, 4) staining or contrasting sections. For light microscopy, one more step is necessary - the conclusion of sections in a balm or other transparent media (5). Fixation ensures the prevention of decomposition processes, which helps to preserve the integrity of the structures. This is achieved by the fact that a small sample taken from an organ is either immersed in a fixative (alcohol, formalin, solutions of heavy metal salts, osmic acid, special fixative mixtures) or subjected to heat treatment. Under the action of the fixative, complex physico-chemical changes occur in tissues and organs. The most significant of them is the process of irreversible coagulation of proteins, as a result of which vital activity ceases, and the structures become dead, fixed. Fixation leads to a compaction and reduction in the volume of the pieces, as well as to an improvement in the subsequent staining of cells and tissues.

sealing pieces, necessary for the preparation of sections, is made by impregnating the previously dehydrated material with paraffin, celloidin, organic resins. Faster compaction is achieved by using the method of freezing the pieces, for example in liquid carbonic acid.

Section preparation produced on special devices - microtomes(for light microscopy) and ultramicrotomes(for electron microscopy).

Section staining(in light microscopy) or spraying them with metal salts(in electron microscopy) is used to increase the image contrast of individual structures when viewed under a microscope. Methods for staining histological structures are very diverse and are selected depending on the objectives of the study. Histological stains are divided into acidic, basic and neutral. Examples include the best known basic dye, azure II, which stains the nuclei purple, and the acidic dye, eosin, which stains the cytoplasm pink-orange. The selective affinity of structures for certain dyes is due to their chemical composition and physical properties. Structures that stain well with acid dyes are called oxyphilic(acidophilic, eosinophilic), and staining basic - basophilic. Structures that accept both acidic and basic dyes are neutrophilic(heterophilic). Colored preparations are usually dehydrated in alcohols of increasing strength and cleared in xylene, benzene, toluene, or some oils. For long-term preservation, a dehydrated histological section is enclosed between a slide and cover slip in Canadian balsam or other substances. The finished histological preparation can be used for microscopic examination for many years. For electron microscopy, sections obtained on an ultramicrotome are placed on special grids, contrasted with salts of manganese, cobalt, etc., after which they are viewed under a microscope and photographed. The obtained microphotographs serve as the object of study along with histological preparations.

Methods for studying living cells and tissues

The study of living cells and tissues allows you to get the most complete information about their life - to trace the movement, the processes of division, destruction, growth, differentiation and interaction of cells, the duration of their life cycle, reactive changes in response to the action of various factors.

In vivo studies of cells in the body (invivo). One of the vital research methods is the observation of structures in a living organism. With the help of special translucent microscopes-illuminators, for example, it is possible to study the dynamics of blood circulation in microvessels. After anesthesia in the animal, the object of study (for example, the mesentery of the intestine) is taken out and examined under a microscope, while the tissues must be constantly moistened with isotonic sodium chloride solution. However, the duration of such observation is limited. The best results are obtained by implanting transparent chambers into the body of an animal.

The most convenient organ for the implantation of such cameras and subsequent observation is the ear of an animal (for example, a rabbit). A section of the ear with a transparent chamber is placed on the microscope stage and under these conditions the dynamics of changes in cells and tissues is studied over a long period of time. In this way, the processes of leukocyte eviction from blood vessels, various stages of the formation of connective tissue, capillaries, nerves, and other processes can be studied. The eye of experimental animals can be used as a natural transparent camera. Cells, tissues, or organ samples are placed in the fluid of the anterior chamber of the eye at the angle formed by the cornea and iris and can be observed through the transparent cornea. In this way, a fertilized egg was transplanted and the early stages of embryonic development were traced. Monkeys were transplanted small pieces of the uterus and studied changes in the lining of the uterus in different phases of the menstrual cycle.

The method of transplantation of blood and bone marrow cells from healthy donor animals to recipient animals subjected to lethal radiation has found wide application. Recipient animals after transplantation remained alive due to the engraftment of donor cells forming colonies of hematopoietic cells in the spleen. The study of the number of colonies and their cellular composition makes it possible to identify the number of parental hematopoietic cells and the various stages of their differentiation. Using the method of colony formation, the sources of development for all blood cells were established.

Vital and supravital staining. During vital (lifetime) staining of cells and tissues, the dye is introduced into the animal's body, while it selectively stains certain cells, their organelles or intercellular substance. For example, using trypan blue or lithium carmine, phagocytes are detected, and using alizarin, a newly formed bone matrix.

Supravital staining refers to the staining of living cells isolated from the body. In this way, young forms of erythrocytes are detected - blood reticulocytes (brilliant cresyl blue dye), mitochondria in cells (Janus green dye), lysosomes (neutral red dye).

Studies of living cells and tissues in culture (invitro). This method is one of the most common. Cells isolated from the human or animal body, small samples of tissues or organs are placed in glass or plastic vessels containing a special nutrient medium - blood plasma, embryonic extract, as well as artificial media. There are suspension cultures (cells suspended in the medium), tissue, organ and monolayer cultures (explanted cells form a continuous layer on the glass). The sterility of the medium and the temperature corresponding to body temperature are ensured. Under these conditions, cells for a long time retain the main indicators of vital activity - the ability to grow, reproduce, differentiate, and move. Such cultures can exist for many days, months, and even years if the culture medium is renewed and viable cells are transplanted into other vessels. Some types of cells, due to changes in their genome, can persist and multiply in culture, forming continuous cell lines. A. A. Maksimov, A. V. Rumyantsev, N. G. Khlopin, A. D. Timofeevsky, and F. M. Lazarenko made a great contribution to the development of methods for cultivating cells and tissues. At present, cell lines of fibroblasts, myocytes, epitheliocytes, macrophages, etc. have been obtained, which have existed for many years.

The use of the cultivation method made it possible to reveal a number of patterns of differentiation, malignant transformation of cells, cellular interactions, interactions of cells with viruses and microbes. The ability of cartilage cells to form an intercellular substance in culture and the ability of adrenal cells to produce hormones were shown. The cultivation of embryonic tissues and organs made it possible to trace the development of bone, skin, and other organs. A technique for cultivating nerve cells has been developed.

The tissue culture method is of particular importance for conducting experimental observations on human cells and tissues. Cells taken from the human body during puncture or biopsy can be used in tissue culture to determine sex, hereditary diseases, malignant degeneration, and to identify the effects of a number of toxic substances.

In recent years, cell cultures have been widely used for cell hybridization.

Methods have been developed for separating tissues into cells, isolating individual cell types and culturing them.

First, the tissue is converted into a cell suspension by destroying intercellular contacts and the intercellular matrix with the help of proteolytic enzymes (trypsin, collagenase) and compounds that bind Ca 2+ (using EDTA - ethylenediaminetetraacetic acid). Further, the resulting suspension is separated into fractions of cells of various types by centrifugation, which allows separating heavier cells from lighter ones, large from small ones, or by sticking cells to glass or plastic, the ability of which is different for different types of cells. To ensure specific adhesion of cells to the glass surface, antibodies are used that specifically bind to cells of the same type. Adhering cells are then separated by breaking down the matrix with enzymes, thus obtaining a suspension of homogeneous cells. A more subtle method of cell separation is labeling with antibodies associated with fluorescent dyes. Labeled cells are separated from unlabeled cells using a sorter (electronic fluorescence-activated cell analyzer). The cell analyzer sorts in 1 with about 5000 cells. Isolated cells can be studied under culture conditions.

The method of cell cultivation makes it possible to study their vital activity, reproduction, differentiation, interaction with other cells, the influence of hormones, growth factors, etc.

Cultures are usually prepared from a cell suspension prepared by the tissue dissociation method described above. Most cells are unable to grow in suspension, they need a solid surface, which is the surface of a plastic culture dish, sometimes with extracellular matrix components, such as collagen. Primary crops are called cultures prepared immediately after the first stage of cell fractionation, secondary- cell cultures transplanted from primary cultures into a new medium. Cells can be transplanted sequentially over weeks and months, while the cells retain their characteristic signs of differentiation (for example, epithelial cells form layers). The starting material for cell cultures is usually fetal and neonatal tissues.

Mixtures of salts, amino acids, vitamins, horse serum, chicken embryo extract, embryonic serum, etc. are used as nutrient media. Special media have been developed for cultivating various cell types. They contain one or more protein growth factors necessary for cells to live and reproduce. For example, nerve growth factor (NGF) is required for the growth of nerve cells.

Most cells in culture have a certain number of divisions (50-100), and then they die. Sometimes mutant cells appear in culture, which multiply endlessly and form a cell line (fibroblasts, epitheliocytes, myoblasts, etc.). Mutant cells are different from cancer cells, which are also capable of continuous division but can grow without being attached to a solid surface. Cancer cells in culture dishes form a denser population than normal cell populations. A similar property can be induced experimentally in normal cells by transforming them with tumor-like viruses or chemical compounds, which results in the formation of neoplastically transformed cell lines. Cell lines of non-transformed and transformed cells can be stored for a long time at low temperatures (-70 °C). The genetic homogeneity of cells is enhanced by cloning, when a large colony of homogeneous cells is obtained from one cell during its successive division. A clone is a population of cells derived from a single progenitor cell.

cell hybrids. When two cells of different types merge, a heterokaryon is formed - a cell with two nuclei. To obtain a heterokaryon, a cell suspension is treated with polyethylene glycol or inactivated viruses to damage the cell plasmolemms, after which the cells are capable of fusion. For example, the inactive nucleus of a chicken erythrocyte becomes active (RNA synthesis, DNA replication) when cells merge and are transferred to the cytoplasm of another cell growing in tissue culture. The heterokaryon is capable of mitosis, resulting in the formation of hybrid cell. The shells of the nuclei of the heterokaryon are destroyed, and their chromosomes are combined in one large nucleus.

Cloning of hybrid cells leads to the formation of hybrid cell lines that are used to study the genome. For example, in a mouse-human hybrid cell line, the role of human chromosome 11 in insulin synthesis has been established.

Hybridomas. Hybridoma cell lines are used to obtain monoclonal antibodies. Antibodies are produced by plasma cells, which are formed from B-lymphocytes during immunization. A specific type of antibody is obtained by immunizing mice with specific antigens. If such immunized lymphocytes are cloned, a large amount of homogeneous antibodies can be obtained. However, the lifetime of B-lymphocytes in culture is limited. Therefore, they merge with "immortal" tumor cells (B-lymphomas). As a result, hybrids are formed. (hybrid cell, with a genome from two different cells; ohm - ending in tumor names). Such hybridomas are able to multiply for a long time in culture and synthesize antibodies of a certain type. Each hybridoma clone is a source of monoclonal antibodies. All antibody molecules of a given species have the same antigen-binding specificity. It is possible to generate monoclonal antibodies against any protein contained in a cell and use them to localize proteins in a cell, as well as to isolate a protein from a mixture (protein purification), which allows one to study the structure and function of proteins. Monoclonal antibodies are also used in gene cloning technology.

Antibodies can be used to study the function of various molecules by introducing them through the plasmalemma directly into the cytoplasm of cells with a thin glass pipette. For example, the introduction of antibodies to myosin into the cytoplasm of a fertilized sea urchin egg stops the division of the cytoplasm.

Recombinant DNA technology. Classical genetic methods make it possible to study the function of genes by analyzing the phenotypes of mutant organisms and their offspring. Recombinant DNA technology complements these methods, allowing detailed chemical analysis of genetic material and obtaining large quantities of cellular proteins.

Hybridization methods are widely used in modern biology to study the structure of genes and their expression.

Methods for studying the chemical composition and metabolism of cells and tissues

To study the chemical composition of biological structures - the localization of substances, their concentration and dynamics in metabolic processes, special research methods are used.

Cyto- and histochemical methods. These methods make it possible to detect the localization of various chemicals in the structures of cells, tissues and organs - DNA, RNA, proteins, carbohydrates, lipids, amino acids, minerals, vitamins, enzyme activity. These methods are based on the specificity of the reaction between a chemical reagent and a substrate that is part of cellular and tissue structures, and on the staining of chemical reaction products. Enzymatic control is often used to increase the specificity of the reaction. For example, to detect ribonucleic acid (RNA) in cells, gallocyanin is often used - a dye with basic properties, and the presence RNA confirmed by control treatment with ribonuclease, which cleaves RNA. Gallocyanin stains RNA in blue-violet. If the section is pre-treated with ribonuclease and then stained with gallocyanin, then the absence of staining confirms the presence of ribonucleic acid in the structure. Numerous cyto- and histochemical methods are described in specific manuals.

In recent years, the combination of histochemical methods with the method of electron microscopy has led to the development of a new promising area - electron histochemistry. This method makes it possible to study the localization of various chemicals not only at the cellular, but also at the subcellular and molecular levels.

To study cell macromolecules, very sensitive methods are used using radioactive isotopes and antibodies, which make it possible to detect even a small content of molecules (less than 1000).

radioactive isotopes during the decay of the nucleus, they emit charged particles (electrons) or radiation (for example, gamma rays), which can be registered in special devices. Radioactive isotopes are used in radioautography. For example, with the help of radioisotopes of 3 H-thymidine, nuclear DNA is examined, with the help of 3 H-uridine - RNA.

radioautographic method. This method makes it possible to most fully study the metabolism in different structures. The method is based on the use of radioactive elements (for example, phosphorus - 32 P, carbon - 14 C, sulfur - 35 S, hydrogen - 3 H) or compounds labeled by them. Radioactive substances in histological sections are detected using a photographic emulsion, which is applied to the preparation and then developed. In areas of the drug, where the photographic emulsion comes into contact with a radioactive substance, a photoreaction occurs, as a result of which illuminated areas (tracks) are formed. This method can be used to determine, for example, the rate of incorporation of labeled amino acids into proteins, the formation of nucleic acids, iodine metabolism in thyroid cells, etc.

Methods of immunofluorescent analysis. The use of antibodies. Antibodies are protective proteins produced by plasma cells (derivatives of B-lymphocytes) in response to the action of foreign substances (antigens). The number of different forms of antibodies reaches a million. Each antibody has sites for "recognition" of the molecules that caused the synthesis of this antibody. Due to the high specificity of antibodies for antigens, they can be used to detect any cell proteins. To identify the localization of proteins, antibodies are stained with fluorescent dyes, and then the cells are examined using fluorescence microscopy. Antibodies can also be used to study antigens at the ultrastructural level using an electron microscope. For this, antibodies are labeled with electron-dense particles (colloidal gold microspheres). To enhance the specificity of the reaction, monoclonal antibodies are used, formed by a cell line - clones obtained by the hybridoma method from one cell. The hybridoma method makes it possible to obtain monoclonal antibodies with the same specificity and in unlimited quantities.

Methods of immunofluorescent analysis are widely and effectively used in modern histology. These methods are used to study the processes of cell differentiation, to identify specific chemical compounds and structures in them. They are based on antigen-antibody reactions. Each cell of the body has a specific antigenic composition, which is mainly determined by proteins. The reaction products can be stained and detected in a fluorescent microscope, for example, the detection of actin and tubulin in a cell using the immunofluorescent analysis method (see chapter IV).

Modern research methods make it possible to analyze the chemical composition of various structural components of cells, both fixed and living. The study of individual intracellular structures became possible after the development of cell contents fractionation technologies.

Fractionation of cellular contents

Cell structures and macromolecules can be fractionated by various methods - ultracentrifugation, chromatography, electrophoresis. These methods are described in more detail in textbooks of biochemistry.

Ultracentrifugation. Using this method, cells can be divided into organelles and macromolecules. First, cells are destroyed by osmotic shock, ultrasound, or mechanical action. In this case, the membranes (plasmolemma, endoplasmic reticulum) break up into fragments, from which the smallest bubbles are formed, and the nuclei and organelles (mitochondria, Golgi apparatus, lysosomes and peroxisomes) remain intact and are in the forming suspension.

A high-speed centrifuge (80,000-150,000 rpm) is used to separate the above cell components. First, larger parts (nuclei, cytoskeleton) settle (sediment) at the bottom of the tube. With a further increase in the centrifugation speed of the supernatant fractions, smaller particles sequentially settle down - first mitochondria, lysosomes and peroxisomes, then microsomes and the smallest vesicles, and finally ribosomes and large macromolecules. During centrifugation, different fractions settle at different rates, forming separate bands in the test tube, which can be isolated and examined. Fractionated cell extracts (cell-free systems) are widely used to study intracellular processes, for example, to study protein biosynthesis, decipher the genetic code, etc.

Chromatography is widely used for protein fractionation.

Electrophoresis makes it possible to separate protein molecules with different charges by placing their aqueous solutions (or in a solid porous matrix) in an electric field.

Chromatography and electrophoresis methods are used to analyze peptides obtained by splitting a protein molecule and to obtain the so-called peptide maps of proteins. These methods are described in detail in biochemistry textbooks.

The study of the chemical composition of living cells. To study the distribution of substances and their metabolism in living cells, nuclear magnetic resonance and microelectrode techniques are used.

Nuclear magnetic resonance (NMR) makes it possible to study small molecules of low molecular weight substances. A tissue sample contains atoms in different molecules and in different environments, so it will absorb energy at different resonant frequencies. The absorption diagram at resonant frequencies for a given sample will be its spectrum NMR. In biology, the NMR signal from protons (hydrogen nuclei) is widely used to study proteins, nucleic acids, etc. To study macromolecules inside a living cell, 3 H, 13 C, 35 K, 31 P isotopes are often used to obtain an NMR signal and monitor its change. during the life of the cell. So, 3| P is used to study muscle contraction - changes in the content of ATP and inorganic phosphate in tissues. The 13 C isotope makes it possible to study many processes in which glucose is involved using NMR. The use of NMR is limited by its low sensitivity: 1 g of living tissue must contain at least 0.2 mm of the test substance. The advantage of the method is its harmlessness to living cells.

Microelectrode technology. Microelectrodes are glass tubes filled with an electrically conductive solution (usually a KC1 solution in water), the end diameter of which is measured in fractions of a micron. The tip of such a tube can be introduced into the cytoplasm of the cell through the plasmalemma and determine the concentration of H + , Na + , K + , C1", Ca 2+ , Mg 2+ ions, the potential difference on the plasma membrane, and also inject molecules into the cell. For determination of the concentration of a particular ion, ion-selective electrodes are used, which are filled with an ion-exchange resin that is permeable only to this ion.In recent years, microelectrode technology has been used to study the transport of ions through special ion channels (specialized protein channels) in the plasma membrane.In this case, a microelectrode with a thicker the tip, which is tightly pressed against the corresponding part of the plasmalemma.This method allows you to study the function of a single protein molecule.Change in the concentration of ions inside the cell can be determined using luminescent indicators.For example, to study the intracellular Ca 2+ concentration, the luminescent protein aquarin (isolated from jellyfish) is used, which radiates light t in the presence of Ca 2+ ions and reacts to changes in the concentration of the latter in the range of 0.5-10 μM. Fluorescent indicators have also been synthesized that bind strongly to Ca 2+ . The creation of various new types of intracellular indicators and modern methods of image analysis makes it possible to accurately and quickly determine the intracellular concentration of many low molecular weight substances.

Quantitative Methods

At present, along with qualitative methods, quantitative histochemical methods have been developed and used for determining the content of various substances in cells and tissues. A feature of quantitative-histochemical (as opposed to biochemical) research methods is the possibility of studying the concentration and content of chemical components in specific cell and tissue structures.

Cytospectrophotometry- method of quantitative study of intracellular substances by their absorption spectra.

Cytospectrofluorometry- a method for quantitative study of intracellular substances by their fluorescence spectra or by fluorescence intensity at one pre-selected wavelength (cytofluorometry).

Modern microscopes - cytofluorometers make it possible to detect small amounts of a substance in various structures (up to 10-14 -10-16 g) and to estimate the localization of the substances under study in microstructures.

Methods for image analysis of cellular and tissue structures

The obtained images of microobjects in a microscope, on a television screen, on electron microphotographs can be subjected to special analysis - the identification of morphometric, densitometric parameters and their statistical processing.

Morphometric methods make it possible to determine using special grids (E. Veibel, A. A. Glagolev, S. B. Stefanova) the number of any structures, their areas, diameters, etc. In particular, in cells, the areas of nuclei, cytoplasm, their diameters, nuclear-cytoplasmic ratios, etc. There are manual morphometry and automated morphometry, in which all parameters are measured and recorded in the device automatically.

In recent years, more and more widespread automationintegrated image processing systems (ASOIS), allowing the most effective implementation of the above quantitative methods for studying cells and tissues. At the same time, the analytical capabilities of quantitative microscopy are supplemented by methods of analysis and recognition of samples based on the processing by means of electronic computers (computers) of information extracted from images of cells and tissues. In essence, we can talk about devices that not only enhance the optical capabilities of the human visual analyzer, but also greatly expand its analytical capabilities. An opinion is expressed that ASOIz makes the same revolution in morphology, which happened about 300 years ago due to the invention of the light microscope, and about 50 years ago - the electron microscope, since they not only immeasurably increase the productivity of the researcher and not only objectify observations, but also allow one to obtain new information about previously undetectable processes, numerically model and predict their development in cells and tissues.

At the same time, participation in a computer experiment requires a new approach from the researcher to its implementation, skills in compiling algorithms for the research process, accuracy of reasoning, and, ultimately, raising the scientific and methodological level of research.

One of the methods that significantly expanded the number of morphological problems to be solved is optical-structural machine analysis (OSMA), proposed in 1965 by K.M. Bogdanov. In 1978, the author of the method was awarded the State Prize of the USSR. With the advent of OSMA, a qualitatively new step has been taken in the development of a unified methodology for the quantitative analysis of microstructures based on statistical characteristics. Recently, OSMA has found effective application in research practice and the national economy.

On fig. 2 shows the Protva-MP automated image processing system created in our country by LOMO. The system is designed for complex studies of cells and tissues using absorption, fluorescent microscopy and autoradiography.

A special scanning optical or electron microscope, which is part of the system, sequentially scans the image of the preparation in two coordinates, converts it into a digital form, and enters it into a computer, which, in turn, digitally processes the image and provides information about the geometric and other characteristics of the analyzed object.

Using a color display, the researcher can "dissect" the image, highlighting only those structural components that interest him. The capacious information storage devices included in the computer on magnetic disks or tapes make it possible to store both the images themselves and the results of their processing for subsequent storage and documentation.

We will consider the use of methods for automated analysis of micro-objects using the example of processing an image of a blood leukocyte (Fig. 3) A scanning microscope-photometer allows you to “view” the optical density values ​​line by line with a step specified by the researcher As a result, the optical signal corresponding to the optical density of the object is converted into digital form The resulting digital matrix subject to preparation using a special mathematical apparatus

First, the background is removed and a "clean" object is isolated - the image of the cell (1a), then any detail of interest to the researcher is selected from the image of the cell, for example, the cytoplasm (16) and the nucleus (I order average and integral value of optical density, dispersion, asymmetry, kurtosis, etc. Based on the image of the object, morphometric parameters are obtained: area, perimeter, diameter, nuclear-cytoplasmic ratio, shape factor, etc.

The next stage of image processing is the construction of two-dimensional diagrams of optical density interdependence for the whole cell (see Fig. 3), its cytoplasm (Wb) and nucleus (Nm). and kernels. These diagrams allow you to calculate the histogram parameters of the second order - homogeneity, local contrast, entropy, etc.

Rice. 3. Automated cell image processing (diagram).

Image of a leukocyte (a), its cytoplasm (b) and nucleus (c). I - digital image; II - histograms of optical density; III - two-dimensional histograms of the dependence of optical density values.

The parameters obtained in this way represent a multidimensional "portrait" of the cell and have a specific numerical expression. They can be subjected to various methods of statistical processing, allow extremely accurate classification of micro-objects, reveal features of their structure that are not visually detectable.

Thus, the application of new research methods in histology, cytology and embryology makes it possible to elucidate the general patterns of organization of tissues and cells, the structural foundations of biochemical processes that determine the function of specific structural components of the cell.

The structure, ultrastructure and functioning of cellular organelles are currently being studied using the following main methods: light and electron, dark-field, phase-contrast, polarization, luminescence microscopy, used to study the structure, ultrastructure of fixed cells, and differential centrifugation, which makes it possible to isolate individual organelles and analyze them by cytochemical, biochemical, biophysical, and other methods.

Light microscopy.

The principle of the method is that a beam of light, passing through an object, enters the lens system of the objective, and builds a primary image, which is enlarged with the help of the eyepiece lenses. The main optical part of the microscope, which determines its main capabilities, is the lens.

In modern microscopes, lenses are interchangeable, which allows you to study cells at different magnifications. The main characteristic of a microscope as an optical system is its resolution, i.e. the ability to give a separate image of two objects close to each other.

Images given by the lens can be enlarged many times by using a strong eyepiece or, for example, projections onto a screen (up to 10 5 times). The resolution of a light microscope is limited by the wavelength of light: the shorter the wavelength, the higher the resolution. Typically, light microscopes use light sources in the visible region of the spectrum (400-700 nm), so the maximum resolution of the microscope in this case may not be higher than 200-350 nm (0.2-0.35 microns). If you use violet light (260-280 nm), then you can increase the resolution to 130 - 140 nm (0.13-0.14 microns). This will be the limit of the theoretical resolution of the light microscope, determined by the wave nature of light.

Thus, all that a light microscope can give as an auxiliary device to our eye is to increase its resolution by about 1000 times (the human naked eye has a resolution of about 0.1 mm, which is equal to 100 microns). This is the "useful" magnification of the microscope, above which we will only increase the contours of the image without revealing new details in it. Therefore, when using the visible region of light, 0.2-0.3 µm is the ultimate resolution limit of the light microscope.

Electron microscopy.

For a scanning electron microscope, the material is often frozen to produce an ice-covered surface. In this case, the loss of water of water-soluble substances is excluded, and chemical changes in structures are also smaller. When analyzing data obtained using an electron microscope, it must be remembered that this method examines the static states of the cell at the moment of a rapid stop in the movement of the cytoplasm caused by the action of fixing chemicals.

Dark field microscopy.

Its essence is that, like dust particles in a beam of light (Tyndall effect), tiny particles (less than 0.2 microns) glow in a cell under side illumination, the reflected light of which enters the microscope lens. This method has been successfully used in the study of living cells.

To determine the localization of sites for the synthesis of biopolymers, to determine the transfer of substances in a cell, to monitor the migration or properties of individual cells, they are widely used autoradiography method- registration of substances labeled with isotopes. For example, using this method, using labeled RNA precursors, it was shown that all RNA is synthesized only in the interphase nucleus, and the presence of cytoplasmic RNA is the result of the migration of synthesized molecules from the nucleus.

In cytology, various analytical and preparative methods biochemistry. In the latter case, various cellular components can be obtained in the form of separate fractions and their chemistry, ultrastructure, and properties can be studied. Currently, almost any cell organelles and structures are obtained in the form of pure fractions.

One of the main ways to isolate cellular structures is differential (separating) centrifugation. The principle of its application is that the time for particles to settle in the homogenate depends on their size and density: the larger the particle or the heavier it is, the faster it will settle to the bottom of the test tube. To speed up this settling process, the accelerations created by the centrifuge are used.

By repeated fractional centrifugation of mixed subfractions, pure fractions can be obtained. In cases of finer separation of fractions, sucrose density gradient centrifugation is used, which allows a good separation of components, even slightly differing from each other in specific gravity. The obtained fractions, before they are analyzed by biochemical methods, must be checked for purity using an electron microscope.

Test questions:

1. Levels of organization of living matter

2. Cellular theory of organization of organisms

3. Research methods in cytology

4. Tasks and subject of cytology

5. The device of a light microscope

6. Electron microscope device

7. Safety precautions for cytological work

8. Requirements for the preparation of biological material for cytological examination

9. Fixing agents, mechanism of action

10. Cytochemistry, material requirements and capabilities

11. Quantitative analysis (morphometry), requirements and possibilities

12. Artifacts in cytology, ways to objectify the results

1. Zavarzin A.A., Kharazova A.D. Fundamentals of General Cytology. - L., 1982.

2. Chentsov Yu.S. Fundamentals of Cytology. - M., 1984.

3. Shubnikova E.A. Functional morphology of tissues. - M., Publishing House of Moscow State University, 1981.