Plan:

1. What does cytology study?

2. The idea that organisms are made of cells.

3. Research methods used in cytology.

4. Cell fractionation.

5. Autoradiography.

6. Determination of the duration of some stages of the cell cycle using autoradiography.

Cytology is the science of cells. It emerged from other biological sciences almost 100 years ago. For the first time, generalized information about the structure of cells was collected in a book by J.-B. Carnoy's 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, allowing one to judge the properties and functions common to all cells. Cytology also examines the structural features of specialized cells. In other words, modern cytology is the physiology of the cell. Cytology is closely associated with scientific and methodological achievements of biochemistry, biophysics, molecular biology and genetics. This served as the basis for an in-depth study of the cell from the standpoint of these sciences and the emergence of a certain synthetic science about the cell - cell biology, or cell biology. Currently, 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 studies and describes the only unit of all life on Earth – the cell.

A long and careful study of the cell as such led to the formulation of an important theoretical generalization that has general biological significance, namely the emergence of the cell theory. In the 17th century Robert Hooke, a physicist and biologist, distinguished by great ingenuity, created a microscope. Examining a thin section of cork under his microscope, Hooke discovered that it was built from tiny empty cells separated by thin walls, which, as we now know, consist of cellulose. He called these small cells cells. Later, when other biologists began to examine plant tissues under a microscope, it turned out that the small cells discovered by Hooke in a dead, withered plug were also present in living plant tissues, but they were not empty, but each contained a small gelatinous body. After animal tissues were subjected to microscopic examination, it was found that they also consisted of small gelatinous bodies, but that these bodies were 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 became firmly established 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 gained great importance in the last few decades. Among them, electron microscopy occupies a special place, 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 special 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 gives them the opportunity to carry out certain functions necessary for the metabolism of the cell; these structures are called organelles (“little 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 it possible to directly study individual metabolic reactions occurring in organelles.

The method by which organelles are isolated from cells is called fractionation. This method turned out to be very fruitful, giving biochemists the opportunity to isolate various cell organelles in a relatively pure form. It also allows one to determine the chemical composition of organelles and the enzymes they contain and, based on 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 mitochondria and many other cellular organelles remain intact, membrane structures such as the endoplasmic reticulum and the plasma membrane disintegrate into fragments. However, the resulting membrane fragments often close on themselves, resulting in round vesicles 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, which depends on the size, density and shape of the organelles. The resulting precipitate can be collected and examined. Larger, denser structures such as nuclei are the fastest to settle, while smaller, less dense structures such as endoplasmic reticulum vesicles require higher rates and longer times to settle. Therefore, at low centrifugation speeds, the nuclei are sedimented, while other cellular organelles remain in suspension. At higher speeds, mitochondria and lysosomes precipitate, and with prolonged 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, they are then subjected to biochemical analysis to determine the chemical composition and enzymatic activity of the isolated organelles.

Murmansk State Technical University

Department of Biology

Report on the topic:

"Research methods in cytology"

Completed:

1st year student

Faculty of Technology

Departments Biology

Serebryakova Lada Vyacheslavovna

Checked:

Murmansk2001

Plan:

1.What does cytology study?

2.The idea that organisms consist of cells.

3. Research methods used in cytology.

4. Cell fractionation.

5. Autoradiography.

6. Determination of the duration of some stages of the cell cycle using autoradiography.

Cytology is the science of cells. It emerged from other biological sciences almost 100 years ago. For the first time, generalized information about the structure of cells was collected in a book by J.-B. Carnoy's 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, allowing one to judge the properties and functions common to all cells. Cytology also examines the structural features of specialized cells. In other words, modern cytology is the physiology of the cell. Cytology is closely related to 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 from the standpoint of these sciences and the emergence of a certain synthetic science of the cell - cell biology, or cell biology. Currently, 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 studies and describes the only unit of all life on Earth – the cell.

A long and careful study of the cell as such led to the formulation of an important theoretical generalization of general biological significance, namely the emergence of cell theory. In the 17th century Robert Hooke, a physicist and biologist with great ingenuity, created a microscope. Examining a thin section of cork under his microscope, Hooke discovered that it was made of tiny empty cells separated by thin walls, which, as we now know, consist of cellulose. He called these small cells cells. Later, when other biologists began to examine plant tissues under a microscope, it turned out that the small cells discovered by Hooke in a dead, withered plug were also present in living plant tissues, but in them they were not empty, but each contained a small gelatinous body. After animal tissues were subjected to microscopic examination, 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 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 established in these small jelly-like bodies.

The modern understanding of cells 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 gained great importance in the last few decades. Among them, electron microscopy occupies a special place, 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 special 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 allows them to carry out certain functions necessary for the metabolism of the cell; These structures are called organelles (“little 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 it possible to directly study individual metabolic reactions occurring in organelles.

The method by which organelles are isolated from cells is called fractionation. This method turned out to be very fruitful, giving biochemists the opportunity to isolate various cell organelles in a relatively pure form. In addition, it allows one to determine the chemical composition of organelles and the enzymes they contain and, based on 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, which ensures the safety of the organelles and prevents their aggregation. Very often, a sucrose solution is used for this. Although mitochondria and many other cellular organelles remain intact, membrane structures such as the endoplasmic reticulum and plasma membrane disintegrate into fragments. However, the resulting membrane fragments often close on themselves, resulting in round vesicles 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 on the bottom of centrifuge tubes at different centrifugation speeds, which depends on the size, density and shape of the organelles. The resulting precipitate can be collected and examined. Larger, denser structures such as nuclei are the fastest to settle, while smaller, less dense structures such as endoplasmic reticulum vesicles require higher rates for the longest time to settle. Therefore, at low centrifugation speeds, the nuclei are sedimented, while other cellular organelles remain in suspension. At higher speeds, mitochondria and lysosomes precipitate, and with prolonged 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 to some extent contaminated with other organelles. If, nevertheless, it is possible to achieve sufficient purity of the fractions, they are then subjected to biochemical analysis to determine the chemical composition and enzymatic activity of the isolated organelles.

Relatively recently, another method of cell fractionation was created - density gradient centrifugation; In this case, centrifugation is carried out in a test tube in which sucrose solutions of ever-increasing concentration, and therefore increasing density, are first layered on top of each other. During centrifugation, the organelles contained in the homogenate are located in a centrifuge tube at the levels at which sucrose solutions are located, corresponding to them in density. This method gives biochemists the opportunity to separate organelles of the same size, but of different densities (Fig. 1.).

Autoradiography is a relatively new method that has immensely expanded the capabilities of both light and electron microscopy. This is a highly modern method, due to the development of nuclear physics, which made it possible to obtain radioactive isotopes of various elements. Autoradiography requires, in particular, isotopes of those elements that are used by the cell or can bind to substances used by the cell, and that can be administered to animals or added to cultures in quantities that do not disrupt normal cellular metabolism. Because a radioactive isotope (or the substance labeled with it) participates in biochemical reactions in the same way as its non-radioactive counterpart, and at the same time emits radiation, the path of isotopes in the body can be traced using various methods of detecting radioactivity. One way to detect radioactivity is based on its ability to act on photographic film like light; but the radioactive radiation penetrates the black paper used to protect the film from light and has the same effect on the film as light.

In order to detect radiation emitted by radioactive isotopes on preparations intended for study using light or electron microscopes, the preparations are coated in a dark room with a special photographic emulsion, and then left in the dark for some time. Then the preparations are developed (also in the dark) and fixed. Areas of the drug containing radioactive isotopes affect the underlying emulsion, in which dark “grains” appear under the influence of emitted radiation. Thus, radioautographs are obtained (in Greek. radio– radiate, autos– himself and grapho- write).

At first, histologists had only a few radioactive isotopes; for example, many early autoradiography studies used radioactive phosphorus. Later, many more of these isotopes were used; The radioactive isotope of hydrogen, tritium, has found particularly widespread use.

Autoradiography was and still is very widely used to study where and how certain biochemical reactions occur in the body.

Chemical compounds labeled with radioactive isotopes that are used to study biological processes are called precursors. Precursors are usually substances similar to those that the body obtains from food; they serve as building blocks for tissue construction and are incorporated into complex components of cells and tissues in the same way that unlabeled building blocks are incorporated into them. The tissue component into which the labeled precursor is incorporated and which emits radiation is called the product.

Cells grown in culture, although belonging to the same type, will be at different stages of the cell cycle at any given time unless special measures are taken to synchronize their cycles. However, by introducing tritium-thymidine into cells and subsequently making autoradiographs, it is possible to determine the duration of the various stages of the cycle. The time of onset of one stage - mitosis - can be determined without labeled thymidine. To do this, a sample of cells from the culture is kept under observation in a phase-contrast microscope, which makes it possible to directly monitor the progress of mitosis and determine its timing. The duration of mitosis is usually 1 hour, although in some types of cells it takes up to 1.5 hours.

Definition of durationG2-period.

To determine the duration of the G 2 period, a method known as pulse tags: Labeled thymidine is added to the cell culture, and after a short time the culture medium is replaced with fresh one in order to prevent further uptake of labeled thymidine by the cells. In this case, the label is included only in those cells that, during a short stay in a medium with tritium-thymidine, were in the S-period of the cell cycle. The proportion of such cells is small and only a small part of the cells will receive the label. In addition, all cells that include the label will be in interphase - from cells that have barely entered the S-period to those that have almost completed it during exposure to tritium-thymidine. In the sample taken immediately after removal of the labeled thymidine, the label is contained only in interphase nuclei belonging to cells that were in the S-period during the period of exposure to the label; those cells that were in a state of mitosis during this period remain unlabeled.

If you then continue to take samples from the culture at certain intervals and prepare an autoradiograph for each successive sample, then a moment will come when the label begins to appear in mitoticd-chromosomes. Labels will be included in all those cells that were in the S-period during the presence of tritium-thymidine in the medium, and among these cells there will be those that have just entered the S-period and those that have almost completed it. It is quite obvious that these latter will be the first among the labeled cells to undergo mitosis and, therefore, the label will be detected in their mitotic chromosomes. Thus, the interval between 1) the time when labeled thymidine was removed from the culture, and 2) the time of appearance of labeled mitotic chromosomes will correspond to the duration of the G 2 period of the cell cycle.

Definition of durationS-period.

Since the cells that are at the very end of the S-period at the time the label is introduced into the medium will be the first to enter mitosis, therefore, in those cells in which the S-period begins immediately before the label is removed, labeled mitotic chromosomes will appear last. Therefore, if we could determine the interval between the time of entry into mitosis of the cells marked first and the cells marked last, we would establish the duration of the S period. However, although the time when labeled mitotic chromosomes first appear is easy to determine, the time at which the last labeled cells enter mitosis cannot be determined (this is hampered by the very large number of labeled dividing cells in the latter samples). Therefore, the duration of the S-period must be determined in a different way.

When examining autoradiographs of successive samples of cells taken at equal intervals of time, it is discovered that the proportion of cells carrying the label in their mitotic chromosomes gradually increases until literally all dividing cells are labeled. However, as the cells complete mitosis one by one, they become labeled interphase cells. The first to complete mitosis are those of the labeled cells that entered it first; and, accordingly, of the cells with labeled mitotic chromosomes, the last to complete mitosis are those that entered it later than all. Since the duration of mitosis is always the same, then, therefore, if we could determine the interval between: 1) the time of the end of mitosis in the cells that turned on the mark first, and 2) the time of the end of mitosis in the cells that turned on the mark last, we would establish the duration of the S-period. The duration of S- The period can be easily established by determining the interval between: 1) the point in time when 50% of the mitotic cells in the culture are unlabeled, and 2) the point in time after which the culture no longer contains 50% of the labeled cells.

Determination of generation time (total duration of the entire cell cycle).

Continuing to take cell samples from the culture, you can find that the marked mitotic figures at some point completely disappear and then appear again. Such dividing cells are daughter cells derived from those mother cells that turned on the label while being in the S-period at the time of exposure to tritium-thymidine. These mother cells entered the S period, divided, and then went through a second interphase and a second division, that is, they went through one full cycle and part of the next. The time required to complete a complete cell cycle is called time generation. It corresponds to the interval between two successive peaks of label inclusion and usually corresponds to the interval between those points of successive ascending curves in which 50% of the mitotic figures contain the label.

Literature.

A. Ham, D. Cormack “Histology”, volume 1 Moscow “MIR” 1982;

M.G. Abramov “Clinical cytology” Moscow “MEDICINE” 1974;

Y.S.Chentsov “General cytology”

Over the past 4045 years, cytology has transformed from descriptive and morphological into an experimental science that sets itself the task of studying the physiology of a cell, its basic vital functions and the properties of its biology. In other words, this is the physiology of the cell. Carnoy Biology of the Cell published in 1884. Let us highlight some important milestones in the history of the study of cell biology.

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Lecture No. 1

INTRODUCTION TO CYTOLOGY

Subject and objectives of the cytology course.

The place of cytology in the system of biological disciplines

Cytology (from Greek. Kytos cell, cell) science of the cell. Modern cytology studies the structure of cells, their functioning as elementary living systems; explores the functions of individual cellular components, the processes of cell reproduction, their adaptation to environmental conditions, and many other processes that make it possible to judge the properties and functions common to all cells.

Cytology also examines the characteristics of specialized cells, the stages of formation of their special functions and the development of specific cellular structures.

Over the past 40-45 years, cytology has transformed from descriptive and morphological into an experimental science, setting itself the task of studying the physiology of the cell, its basic vital functions and properties, and its biology. In other words, this is the physiology of the cell.

The possibility of such a switch in the interests of researchers arose due to the fact that cytology is closely related to the scientific and methodological achievements of biochemistry, biophysics, molecular biology and genetics.

In general, cytology is closely related to almost all biological disciplines, since everything living on Earth (almost everything!) has a cellular structure, and cytology is precisely the study of cells in all their diversity.

Cytology is closely related to zoology and botany, since it studies the structural features of plant and animal cells; with embryology in the study of the structure of germ cells; with histology cell structure of individual tissues; with anatomy and physiology, since on the basis of cytological knowledge the structure of certain organs and their functioning is studied.

The cell has a rich chemical composition, complex biochemical processes take place in it: photosynthesis, protein biosynthesis, respiration, and also important physical phenomena occur, in particular, the occurrence of excitation, a nerve impulse, therefore cytology is closely related to biochemistry and biophysics.

To understand the complex mechanisms of heredity, it is necessary to study and understand their material carriers - genes, DNA, which are integral components of cellular structures. From this arises a close connection between cytology and genetics and molecular biology.

Cytological research data is widely used in medicine, agriculture, veterinary medicine, and in various industries (food, pharmaceutical, perfumery, etc.). Cytology also occupies an important place in the teaching of biology at school (general biology course in high school).

Brief historical sketch of the development of cytology

In general, cytology is a fairly young science. It emerged from other biological sciences a little over a hundred years ago. For the first time, generalized information about the structure of cells was collected in the book by Zh.B. Carnoy’s “Biology of the Cell,” published in 1884. The appearance of this book was preceded by a long and stormy period of searches, discoveries, and discussions, which led to the formulation of the so-called cell theory, which has enormous general biological significance.

Let us highlight some important milestones in the history of the study of cell biology.

The end of the 16th and the beginning of the 17th century. According to various sources, the inventors of the microscope are Zacharias Jansen (1590, Holland), Galileo Galilei (1610, Italy), Cornelius Drebbel (1619-1620, Holland). The first microscopes were very bulky and expensive and were used by noble people for their own entertainment. But gradually they improved and began to turn from a toy into a scientific research tool.

1665 Robert Hooke (England), using a microscope designed by the English physicist H. Huygens, studied the structure of cork and for the first time used the term “cell” to describe the structural units that make up this tissue. He believed that cells are empty, and living matter is cell walls.

1675-1682 M. Malpighi and N. Grew (Italy) confirmed the cellular structure of plants

1674 Antonio van Leeuwenhoek (Holland) discovered single-celled organisms, including bacteria (1676). He was the first to see and describe animal cells - red blood cells, sperm.

1827 Dolland dramatically improved the quality of lenses. After this, interest in microscopy quickly grew and spread.

1825 Jan Purkinė (Czech Republic) is the first to describe the cell nucleus in the egg of birds. He calls it the “germinal vesicle” and assigns to it the function of “the productive force of the egg.”

1827 Russian scientist Karl Baer discovered the mammalian egg and established that all multicellular organisms begin their development from a single cell. This discovery showed that the cell is the unit not only of structure, but also of development of all living organisms.

1831 Robert Brown (English botanist) first described the nucleus in plant cells. He came up with the name “nucleus” “nucleus” and for the first time stated that it was a common component of any cell, having some essential significance for its life.

1836 Gabriel Valentin, a student of Purkin, discovers the nucleus of animal cells cells of the epithelium of the conjunctiva, the connective membrane of the eye. Inside this “nucleus” he finds and describes the nucleolus.

From that moment on, the nucleus began to be sought out and found in all tissues of plants and animals.

1839 Theodor Schwann (German physiologist and cytologist) published the book “Microscopic studies on the correspondence in the structure and growth of animals and plants,” in which he summarized the existing knowledge about the cell, including the results of research by the German botanist Matthias Jakob Schleiden on the role of the nucleus in plant cells. The main idea of ​​the book (stunning in its simplicity) life is concentrated in cells caused a revolution in biology. In other words, T. Schwann and M. Schleiden formulated the cell theory. Its main provisions then were as follows:

1) both plant and animal organisms consist of cells;

2) cells of plant and animal organisms develop similarly and are close to each other in structure and functional purpose;

3) each cell is capable of independent life.

Cell theory is one of the outstanding generalizations of biology XIX century, which provided the basis for understanding life and revealing the evolutionary connections between organisms.

1840 Jan Purkynė proposed the name “protoplasm” for the cellular contents, making sure that it (and not the cell walls) constituted living matter. Later the term "cytoplasm" was introduced.

1858 Rudolf Virchow (German pathologist and social activist) showed that all cells are formed from other cells through cell division. This position was later also included in the cell theory.

1866 Ernst Haeckel (German biologist, founder of the phylogenetic direction of Darwinism) established that the storage and transmission of hereditary characteristics is carried out by the nucleus.

1866-1888 Cell division was studied in detail and chromosomes were described.

1880-1883 Plastids, in particular chloroplasts, were discovered.

1876 ​​Cell center opened.

1989 Golgi apparatus discovered.

1894 Mitochondria discovered.

1887-1900 The microscope has been improved, as have the methods of fixation, staining of specimens, and preparation of sections. Cytology began to acquire an experimental character. Embryological research is being conducted to determine how cells interact with each other during the growth of a multicellular organism.

1900 Mendel's laws, forgotten since 1865, were rediscovered, and this gave impetus to the development of cytogenetics, which studies the role of the nucleus in the transmission of hereditary characteristics.

The light microscope by this time had almost reached the theoretical limit of resolution; The development of cytology naturally slowed down.

1930s The electron microscope was introduced.

From 1946 to the present day, the electron microscope has become widespread in biology, making it possible to study the structure of the cell in much more detail. This “fine” structure began to be called ultrastructure.

The role of domestic scientists in the development of the doctrine of the cell.

Caspar Friedrich Wolf (1733-1794) member of the St. Petersburg Academy of Sciences, opposed metaphysical ideas about development as the growth of a ready-made organism embedded in the reproductive cell (the theory of preformationism).

P.F. Goryaninov is a Russian biologist who described various forms of cells and, even before Schwann and Schleiden, expressed views close to them.

Second half of the 19th century V. beginning of the twentieth century: Russian cytologist I.D. Chistyakov was the first to describe mitosis in moss spores; I.N. Gorozhankin studied the cytological basis of fertilization in plants; S.T. Navashin discovered double fertilization in plants in 1898.

Basic provisions of modern cell theory

1. The cell, as an elementary living system capable of self-renewal, self-regulation and self-reproduction, underlies the structure and development of all living organisms.

2. The cells of all organisms are built according to a single principle, similar (homologous) in chemical composition, basic manifestations of life activity and metabolism.

3. Cell reproduction occurs through cell division, and each new cell is formed as a result of the division of the mother cell.

4. In multicellular organisms, cells are specialized in the functions they perform and form tissues. Organs and organ systems that are closely interconnected are made up of tissues.

With the development of science, only one position of the cell theory turned out to be not absolutely true - the first. Not all living organisms have a cellular organization. This became clear with the discovery of viruses. This is a non-cellular form of life, but the existence and reproduction of viruses is only possible using the enzymatic systems of cells. Therefore, a virus is not an elementary unit of living matter.

The cellular form of organization of living things, having once emerged, became the basis for all further development of the organic world. The evolution of bacteria, protozoa, blue-green algae and other organisms occurred entirely due to the structural, functional and biochemical transformations of the cell. During this evolution, an amazing variety of cell forms was achieved, but the general plan of the cell structure did not undergo fundamental changes.

The emergence of multicellularity dramatically expanded the possibilities for the progressive evolution of organic forms. The leading changes here were in higher order systems (tissues, organs, individuals, populations, etc.). At the same time, the tissue cells acquired features that were useful for the individual and the species as a whole, regardless of how this feature affected the viability and ability to reproduce the tissue cells themselves. As a result, the cell became a subordinate part of the whole organism. For example, the functioning of a number of cells is associated with their death (secretory cells), loss of the ability to reproduce (nerve cells), and loss of the nucleus (mammalian red blood cells).

Methods of modern cytology

Cytology arose as a branch of microanatomy, and therefore the main method that cytologists use is the method of light microscopy. Currently, this method has found a number of additions and modifications, which has significantly expanded the range of tasks and issues solved by cytology. A revolutionary moment in the development of modern cytology and biology in general was the use of electron microscopy, which opened up unusually broad prospects. With the introduction of electron microscopy, in some cases it is already difficult to draw the line between cytology proper and biochemistry; they are combined at the level of macromolecular study of objects (for example, microtubules, membranes, microfilaments, etc.). Nevertheless, the main methodological technique in cytology remains visual observation of the object. In addition, cytology uses numerous techniques of preparative and analytical biochemistry and methods of biophysics.

Let's get acquainted with some methods of cytological research, which, for ease of study, will be divided into several groups.

I . Optical methods.

1. Light microscopy.Objects of study: preparations that can be viewed in transmitted light. They should be sufficiently transparent, thin and contrasting. Biological objects do not always have these qualities. To study them in a biological microscope, it is necessary to first prepare the appropriate preparations by fixation, dehydration, making thin sections, and staining. The cellular structures in such fixed preparations do not always correspond to the true structures of a living cell. Their study should be accompanied by the study of a living object in dark-field and phase-contrast microscopes, where the contrast is increased due to additional devices to the optical system.

The maximum resolution that a biological microscope can provide under oil immersion is 1700 Ǻ (0.17 μm) in monochromatic light and 2500 Ǻ (0.25 μm) in white light. A further increase in resolution can only be achieved by reducing the wavelength of light.

2. Dark-field microscopy. The method is based on the principle of light scattering at the boundary between phases with different refractive indices. This is achieved in a dark-field microscope or in a conventional biological microscope using a special dark-field condenser, which transmits only very oblique edge rays of the light source. Because the edge rays are highly inclined, they do not enter the lens, and the field of view of the microscope appears dark, while an object illuminated by scattered light appears light. Cell preparations usually contain structures of different optical densities. Against a general dark background, these structures are clearly visible due to their different glow, and they glow because they scatter the rays of light falling on them (Tyndall effect).

Living objects can be studied in a dark field. The resolution of such a microscope is high (less than 0.2 microns).

3. Phase contrast microscopy. The method is based on the fact that individual areas of a transparent drug differ from the environment in refractive index. Therefore, light passing through them travels at different speeds, i.e. experiences a phase shift, which is reflected in a change in brightness. Particles with a refractive index greater than the refractive index of the medium produce dark images on a light background, while particles with an index less than that of the medium produce images lighter than the surrounding background.

Phase contrast microscopy reveals many details and features of living cells and tissue sections. This method is of great importance for studying tissues cultured in vitro.

4. Interference microscopy. This method is close to the method of phase contrast microscopy and makes it possible to obtain contrast images of unstained transparent living cells, as well as calculate the dry weight of the cells. An interference microscope is designed in such a way that a beam of parallel light rays from the illuminator is divided into two streams. One of them passes through the object and acquires changes in the oscillation phase, the other goes bypassing the object. In the lens prisms, both flows are reconnected and interfere with each other. As a result of interference, an image will be built in which areas of the cell with different thicknesses or different densities will differ from each other in the degree of contrast. In this device, by measuring phase shifts, it is possible to determine the concentration and mass of dry matter in an object.

II . Vital (intravital) study of cells.

1. Preparation of live cell preparations.A light microscope allows you to see living cells. For short-term observation, cells are simply placed in a liquid medium on a glass slide; If long-term observation of cells is required, special cameras are used. In any of these cases, cells are studied in specially selected media (water, saline, Ringer's solution, etc.).

2. Cell culture method. Cultivation of cells and tissues outside the body ( in vitro ) is subject to compliance with certain conditions; a suitable nutrient medium is selected, a strictly defined temperature is maintained (about 20 0 for cells of cold-blooded animals and about 37 0 for warm-blooded animals), it is mandatory to maintain sterility and regularly reseed the culture on a fresh nutrient medium. Nowadays, the method of culturing cells outside the body is widely used not only for cytological, but also for genetic, virological and biochemical studies.

3. Microsurgery methods. These methods involve surgical action on the cell. Microoperations on individual small cells began to be carried out from the beginning of the twentieth century, when a device calledmicromanipulator.With its help, cells are cut, individual parts are removed from them, substances are injected (microinjection), etc. The micromanipulator is combined with a conventional microscope, through which the progress of the operation is monitored. Microsurgical instruments are glass hooks, needles, capillaries, which have microscopic dimensions. In addition to mechanical effects on cells, microbeams of ultraviolet light or laser microbeams have recently been widely used in microsurgery. This makes it possible to almost instantly inactivate individual areas of a living cell.

4. Intravital staining methods. When studying living cells, they try to stain them using so-called vital dyes. These are dyes of an acidic (trypan blue, lithium carmine) or basic (neutral red, methylene blue) nature, used at very high dilutions (1:200,000), therefore, the influence of the dye on the vital activity of the cell is minimal. When staining living cells, the dye collects in the cytoplasm in the form of granules, and in damaged or dead cells, diffuse staining of the cytoplasm and nucleus occurs. The time for staining preparations varies greatly, but for most vital dyes it is from 15 to 60 minutes.

III . Cytophysical methods

1. X-ray absorption method. The method is based on the fact that different substances at a certain wavelength absorb X-rays differently. By passing X-rays through a tissue specimen, its chemical composition can be determined from its absorption spectrum.

2. Fluorescence microscopy. The method is based on the property of some substances to fluoresce in ultraviolet rays. For these purposes, an ultraviolet microscope is used, in the condenser of which a light filter is installed that separates blue and ultraviolet rays from the general light beam. Another filter placed in front of the observer's eyes absorbs these rays, allowing fluorescence rays emitted by the drug to pass through. The light source is mercury lamps and incandescent lamps, which produce strong ultraviolet radiation in the overall light beam.

Fluorescence microscopy makes it possible to study a living cell. A number of structures and substances contained in cells have their own (primary) fluorescence (chlorophyll, vitamins A, B 1 and B 2 , some hormones and bacterial pigments). Objects that do not have their own fluorescence can be tinted with special fluorescent dyes fluorochromes . Then they are visible in ultraviolet light (secondary fluorescence). Using this method, you can see the shape of the object, the distribution of fluorescent substances in the object, and the content of these substances).

3. Radiography method. The method is based on the fact that radioactive isotopes, when introduced into the body, enter into general cellular metabolism and are included in the molecules of the corresponding substances. The locations of their localization are determined by the radiation given by isotopes and detected by the illumination of a photographic plate when it is applied to the preparation. The drug is manufactured some time after the introduction of the isotope, taking into account the time of passage of certain stages of metabolism. This method is widely used to determine the localization of sites of biopolymer synthesis, to determine the pathways of substance transfer in a cell, and to monitor the migration or properties of individual cells.

IV . Methods for studying ultrastructure

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

A polarizing microscope differs from a conventional biological microscope in that a polarizer is placed in front of the condenser, and a compensator and analyzer are placed behind the specimen and lens, allowing a detailed study of birefringence in the object under consideration. The polarizer and analyzer are prisms made of Iceland spar (Nicolas prisms). A polarizing microscope makes it possible to determine the orientation of particles in cells and other structures, to clearly see structures with birefringence, and with appropriate processing of preparations, observations can be made on the molecular organization of a particular part of the cell.

2. X-ray diffraction analysis method. The method is based on the property of X-rays to undergo diffraction when passing through crystals. They undergo the same diffraction if biological objects, such as tendon, cellulose, and others, are placed instead of crystals. A series of rings, concentrically located spots and stripes appear on the screen or photographic plate. The diffraction angle is determined by the distance between groups of atoms and molecules in an object. The greater the distance between structural units, the smaller the diffraction angle, and vice versa. On the screen, this corresponds to the distance between the dark areas and the center. Oriented particles give circles, sickles, and points on the diagram; unoriented particles in amorphous substances give the image of concentric rings.

The X-ray diffraction method is used to study the structure of molecules of proteins, nucleic acids and other substances that make up the cytoplasm and nucleus of cells. It makes it possible to determine the spatial arrangement of molecules, accurately measure the distance between them and study the intramolecular structure.

3. Electron microscopy. Considering the characteristics of a light microscope, one can be convinced that the only way to increase the resolution of an optical system is to use an illumination source that emits wavelengths with the shortest wavelength. Such a source can be a hot filament, which in an electric field emits a stream of electrons, the latter can be focused by passing it through a magnetic field. This served as the basis for the creation of the electron microscope in 1933. The main difference between an electron microscope and a light microscope is that it uses a fast flow of electrons instead of light, and electromagnetic fields replace glass lenses. The image is produced by electrons that have passed through the object and not been rejected by it. In modern electron microscopes, a resolution of 1Ǻ (0.1 nm) has been achieved.

Non-living objects preparations are viewed under an electron microscope. It is not yet possible to study living objects, because objects are placed in a vacuum, which is fatal to living organisms. In a vacuum, electrons hit an object without scattering.

Objects studied under an electron microscope must have a very small thickness, no more than 400-500 Ǻ (0.04-0.05 μm), otherwise they turn out to be impenetrable to electrons. For these purposes they useultramicrotomes, the operating principle of which is based on the thermal expansion of the rod that feeds the knife to the object or, conversely, the object to the knife. Specially sharpened small diamonds are used as knives.

Biological objects, especially viruses, phages, nucleic acids, thin membranes, have a weak ability to scatter electrons, i.e. low contrast. Their contrast is increased by sputtering the object with heavy metals (gold, platinum, chromium), carbon sputtering, by treating preparations with osmic or tungstic acids and some salts of heavy metals.

4. Special methods of electron microscopy of biological objects. Currently, electron microscopy methods are being developed and improved.

Freezing method etchingconsists in the fact that the object is first quickly frozen with liquid nitrogen, and then at the same temperature is transferred to a special vacuum installation. There, the frozen object is mechanically chipped with a cooled knife. This exposes the internal zones of frozen cells. In a vacuum, part of the water that has passed into a glassy form is sublimated (“etching”), and the surface of the chip is successively covered with a thin layer of evaporated carbon and then metal. In this way, an impression film is obtained that repeats the intravital structure of the material, which is studied in an electron microscope.

High-voltage microscopy methodselectron microscopes with an accelerating voltage of 1-3 million V have been designed. The advantage of this class of devices is that at high energy electrons, which are less absorbed by the object, samples of greater thickness (1-10 microns) can be examined. This method is also promising in another respect: if the ultra-high energy of electrons reduces their impact on the object, then in principle this can be used in studying the ultrastructure of living objects. Work is currently underway in this direction.

Scanning (raster) electron microscopy methodallows you to study a three-dimensional picture of the cell surface. In this method, a fixed and specially dried object is covered with a thin layer of evaporated metal (most often gold), a thin beam of electrons runs along the surface of the object, is reflected from it and hits a receiving device, which transmits the signal to a cathode ray tube. Thanks to the enormous depth of focus of a scanning microscope, which is significantly greater than that of a transmission microscope, an almost three-dimensional image of the surface under study is obtained.

V . Cyto- and histochemical methods.

Using such methods, it is possible to determine the content and localization of substances in a cell using chemical reagents that, together with the identified substance, produce a new substance of a specific color. The methods are similar to the methods for determining substances in analytical chemistry, but the reaction occurs directly on the tissue preparation, and precisely in the place where the desired substance is localized.

The amount of the final product of a cytochemical reaction can be determined usingcytophotometry method.It is based on determining the amount of chemical substances based on their absorption of light of a certain wavelength. It was found that the intensity of absorption of rays is proportional to the concentration of the substance for the same thickness of the object. Therefore, by assessing the degree of light absorption by a given substance, it is possible to find out its quantity. For this type of research, instruments are used: microscopes-cytophotometers; They have a sensitive photometer behind the lens that records the intensity of the light flux passing through the lens. Knowing the area or volume of the measured structure and the absorption value, it is possible to determine both the concentration of a given substance and its absolute content.

Quantitative fluorometry techniques have been developed that make it possible to determine the content of substances with which fluorochromes bind by the degree of luminescence. Thus, to identify specific proteins, they useimmunofluorescence methodimmunochemical reactions using fluorescent antibodies. This method has very high specificity and sensitivity. It can be used to identify not only proteins, but also individual nucleotide sequences in DNA or to determine the localization of RNADNA hybrid molecules.

VI . Cell fractionation.

In cytology, various methods of biochemistry, both analytical and preparative, are widely used. In the latter case, it is possible to obtain various cellular components in the form of separate fractions and study their chemistry, ultrastructure and properties. Thus, at present, almost any cellular organelles and structures are obtained in the form of pure fractions: nuclei, nucleoli, chromatin, nuclear membranes, plasma membrane, ER vacuoles, ribosomes, Golgi apparatus, mitochondria, their membranes, plastids, microtubules, lysosomes, etc. d.

Obtaining cell fractions begins with the general destruction of the cell, with its homogenization. Fractions can then be isolated from the homogenates. One of the main methods for isolating cellular structures is differential (separation) centrifugation. The principle of its application is that the time for particles to settle in a 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. The resulting fractions, before being analyzed by biochemical methods, must be checked for purity using an electron microscope.

A cell is the elementary unit of living things.

Prokaryotes and eukaryotes

The cell is a self-replicating system. It contains cytoplasm and genetic material in the form of DNA. DNA regulates the life of the cell and reproduces itself, due to which new cells are formed.

Cell sizes . Bacteria diameter 0.2 microns. More often the cells are 10-100 microns, less often 1-10 mm. There are very large ones: eggs of ostriches, penguins, geese - 10-20 cm, nerve cells and milky vessels of plants - up to 1 m or more.

Cell shape : round (liver cells), oval (amphibian red blood cells), multifaceted (some plant cells), stellate (neurons, melanophores), disc-shaped (human red blood cells), spindle-shaped (smooth muscle cells), etc.

But, despite the variety of shapes and sizes, the organization of cells of all living organisms is subject to common structural principles: a protoplast, consisting of cytoplasm and nucleus, and a plasma membrane. Cytoplasm, in turn, includes hyaloplasm, organelles (general organelles and special-purpose organelles) and inclusions.

Depending on the structural features of their constituent parts, all cells are divided intoprokaryotic And eukaryotic.

Prokaryotic cells are characteristic of bacteria and blue-green algae (cyanobacteria). They do not have a true nucleus, nucleoli and chromosomes, they only have nucleoid , devoid of a shell and consisting of a single circular DNA molecule associated with a small amount of protein. Prokaryotes lack membrane organelles: mitochondria, EPS, chloroplasts, lysosomes and the Golgi complex. There are only smaller ribosomes than eukaryotes.

On top of the plasma membrane, prokaryotes have a rigid cell wall and, often, a mucous capsule. The plasma membrane forms invaginations mesosomes , on the membranes of which redox enzymes are located, and in photosynthetic prokaryotes the corresponding pigments (bacteriochlorophyll in bacteria, chlorophyll and phycocyanin in cyanobacteria). Thus, these membranes perform the functions of mitochondria, chloroplasts and other organelles.

Eukaryotes include unicellular animals (protists), fungi, plants, and animals. In addition to the core clearly delimited by a double membrane, they have many other membrane structures. Based on the number of membranes, organelles of eukaryotic cells can be divided into three main groups: single-membrane (ER, Golgi complex, lysosomes), double-membrane (mitochondria, plastids, nucleus), non-membrane (ribosomes, cell center). In addition, the entire cytoplasm is divided by internal membranes into reaction spaces compartments (compartments). In these compartments, various chemical reactions occur simultaneously and independently of each other.

Comparative characteristics of various types

eukaryotic cells (from Lemez, Lisov, 1997)

Similarities and differences between animal and plant cells

Plant and animal cells are similar in the following ways:

1). General plan of the cell structure presence of a cytoplasmic membrane, cytoplasm, nucleus.

2). A unified plan for the structure of the cytoplasmic membrane, built according to the fluid-mosaic principle.

3). Common organelles ribosomes, mitochondria, EPS, Golgi complex, lysosomes.

4). The commonality of life processes metabolism, reproduction, growth, irritability, etc.

At the same time, plant and animal cells differ:

1). In form: plants are more uniform, animals are very diverse.

2). By size: plant larger, animal small.

3). According to their location in tissues: plants are tightly adjacent to each other, animals are loosely located.

4). Plant cells have an additional cellulose wall.

5). Plant cells have large vacuoles. In animals, if they exist, they are small and appear during the aging process.

6). Plant cells have turgor and are elastic. Animals soft.

7). Plant cells contain plastids.

8). Plant cells are capable of autotrophic nutrition, while animal cells are heterotrophs.

9). Plants do not have centrioles (except for some mosses and ferns), animals always have them.

10). Plant cells have unlimited growth.

eleven). Plant cells accumulate starch as a reserve nutrient, while animal cells accumulate glycogen.

12). In animal cells, a glycocalyx is located on top of the cytoplasmic membrane, but in plant cells it is not.

13). ATP synthesis in animal cells occurs in mitochondria, in plant cells in mitochondria and plastids.

Murmansk State Technical University

Department of Biology

Report on the topic:

"Research methods in cytology"

Completed:

1st year student

Faculty of Technology

Departments Biology

Serebryakova Lada Vyacheslavovna

Checked:

Murmansk 2001

Plan:

1. What does cytology study?

2. The idea that organisms are made of cells.

3. Research methods used in cytology.

4. Cell fractionation.

5. Autoradiography.

6. Determination of the duration of some stages of the cell cycle using autoradiography.

Cytology is the science of cells. It emerged from other biological sciences almost 100 years ago. For the first time, generalized information about the structure of cells was collected in a book by J.-B. Carnoy's 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, allowing one to judge the properties and functions common to all cells. Cytology also examines the structural features of specialized cells. In other words, modern cytology is the physiology of the cell. Cytology is closely associated with scientific and methodological achievements of biochemistry, biophysics, molecular biology and genetics. This served as the basis for an in-depth study of the cell from the standpoint of these sciences and the emergence of a certain synthetic science about the cell - cell biology, or cell biology. Currently, 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 studies and describes the only unit of all life on Earth – the cell.

A long and careful study of the cell as such led to the formulation of an important theoretical generalization that has general biological significance, namely the emergence of the cell theory. In the 17th century Robert Hooke, a physicist and biologist, distinguished by great ingenuity, created a microscope. Examining a thin section of cork under his microscope, Hooke discovered that it was built from tiny empty cells separated by thin walls, which, as we now know, consist of cellulose. He called these small cells cells. Later, when other biologists began to examine plant tissues under a microscope, it turned out that the small cells discovered by Hooke in a dead, withered plug were also present in living plant tissues, but they were not empty, but each contained a small gelatinous body. After animal tissues were subjected to microscopic examination, it was found that they also consisted of small gelatinous bodies, but that these bodies were 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 became firmly established 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 gained great importance in the last few decades. Among them, electron microscopy occupies a special place, 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 special 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 gives them the opportunity to carry out certain functions necessary for the metabolism of the cell; these structures are called organelles (“little 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 it possible to directly study individual metabolic reactions occurring in organelles.

The method by which organelles are isolated from cells is called fractionation. This method turned out to be very fruitful, giving biochemists the opportunity to isolate various cell organelles in a relatively pure form. It also allows one to determine the chemical composition of organelles and the enzymes they contain and, based on 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 mitochondria and many other cellular organelles remain intact, membrane structures such as the endoplasmic reticulum and the plasma membrane disintegrate into fragments. However, the resulting membrane fragments often close on themselves, resulting in round vesicles 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, which depends on the size, density and shape of the organelles. The resulting precipitate can be collected and examined. Larger, denser structures such as nuclei are the fastest to settle, while smaller, less dense structures such as endoplasmic reticulum vesicles require higher rates and longer times to settle. Therefore, at low centrifugation speeds, the nuclei are sedimented, while other cellular organelles remain in suspension. At higher speeds, mitochondria and lysosomes precipitate, and with prolonged 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, they are then subjected to biochemical analysis to determine the chemical composition and enzymatic activity of the isolated organelles.

More recently, another method of cell fractionation was created - density gradient centrifugation; In this case, centrifugation is carried out in a test tube in which sucrose solutions of increasing concentrations and, consequently, increasing density are first layered on top of each other. During centrifugation, the organelles contained in the homogenate are located in a centrifuge tube at the same levels as sucrose solutions corresponding to them in density. This method gives biochemists the ability to separate organelles of the same size but different densities (Fig. 1.).

Autoradiography is a relatively new method that has immensely expanded the capabilities of both light and electron microscopy. This is a highly modern method, owing its origin to the development of nuclear physics, which made it possible to obtain radioactive isotopes of various elements. Autoradiography requires, in particular, isotopes of those elements that are used by the cell or can bind to substances used by the cell, and that can be administered to animals or added to cultures in quantities that do not disrupt normal cellular metabolism. Because a radioactive isotope (or the substance labeled with it) participates in biochemical reactions in the same way as its non-radioactive counterpart and at the same time emits radiation, the path of isotopes in the body can be traced using various methods of detecting radioactivity. One way to detect radioactivity is based on its ability to act like light on photographic film; but the radioactive radiation penetrates the black paper used to protect the film from light and has the same effect on the film as light.

So that the radiation emitted by radioactive isotopes can be detected on preparations intended for study using light or electron microscopes, the preparations are coated in a dark room with a special photographic emulsion, and then left for some time in the dark. Then the preparations are developed (also in the dark) and fixed. Areas of the drug containing radioactive isotopes affect the underlying emulsion, in which dark “grains” appear under the influence of the emitted radiation. Thus, radioautographs are obtained (from the Greek. radio– radiate, autos– himself and grapho- write).

At first, histologists had only a few radioactive isotopes; for example, many early autoradiography studies used radioactive phosphorus. Later, much more of these isotopes began to be used; The radioactive isotope of hydrogen, tritium, has found particularly widespread use.

Autoradiography was and still is very widely used to study where and how certain biochemical reactions occur in the body.

Chemical compounds labeled with radioactive isotopes that are used to study biological processes are called precursors. Precursors are usually substances similar to those the body obtains from food; they serve as building blocks for tissue construction and are incorporated into complex components of cells and tissues in the same way that unlabeled building blocks are incorporated into them. The tissue component into which the labeled precursor is incorporated and which emits radiation is called the product.

Cells grown in culture, although belonging to the same type, will be at different stages of the cell cycle at any given time unless special measures are taken to synchronize their cycles. However, by introducing tritium-thymidine into cells and then making autoradiographs, the duration of the various stages of the cycle can be determined. The time of onset of one stage - mitosis - can be determined without labeled thymidine. To do this, a sample of cells from the culture is kept under observation in a phase-contrast microscope, which makes it possible to directly monitor the progress of mitosis and determine its timing. The duration of mitosis is usually 1 hour, although in some types of cells it takes up to 1.5 hours.

G 2-period .

To determine the duration of the G 2 period, a method known as pulse tag: Labeled thymidine is added to the cell culture, and after a short time the culture medium is replaced with fresh one in order to prevent further uptake of labeled thymidine by the cells. In this case, the label is included only in those cells that, during a short stay in a medium with tritium-thymidine, were in the S-period of the cell cycle. The proportion of such cells is small and only a small part of the cells will receive the label. In addition, all cells that include the label will be in interphase - from cells that have barely entered the S-period to those that have almost completed it during exposure to tritium-thymidine. In a sample taken immediately after removal of labeled thymidine, the label is contained only in interphase nuclei belonging to cells that were in the S-period during the period of exposure to the label; the same cells that were in a state of mitosis during this period remain unlabeled.

If you then continue to take samples from the culture at certain intervals and make an autoradiograph for each successive sample, then a moment will come when the label begins to appear in mitotic d -chromosomes. The labels will be included in all those cells that were in the S-period during the presence of tritium-thymidine in the medium, and among these cells there will be both those that have just entered the S-period and those that have almost completed it. It is quite obvious that these latter will be the first among the labeled cells to undergo mitosis and, therefore, the label will be detected in their mitotic chromosomes. Thus, the interval between 1) the time when labeled thymidine was removed from the culture and 2) the time of appearance of labeled mitotic chromosomes will correspond to the duration of the G 2 period of the cell cycle.

Determining Duration S -period .

Since the cells that are at the very end of the S-period at the time the label is introduced into the medium will be the first to enter mitosis, therefore, in those cells in which the S-period begins immediately before the label is removed, labeled mitotic chromosomes will appear last. Therefore, if we could determine the interval between the time of entry into mitosis of the cells marked first and the cells marked last, we would establish the duration of the S period. However, although the time when labeled mitotic chromosomes first appear is easy to determine, the time at which the last labeled cells enter mitosis cannot be determined (this is hampered by the very large number of labeled dividing cells in the latter samples). Therefore, the duration of the S-period has to be determined in a different way.

When examining autoradiographs of successive samples of cells taken at regular intervals, it is discovered that the proportion of cells carrying the label in their mitotic chromosomes gradually increases until literally all dividing cells are labeled. However, as the cells complete mitosis one by one, they become labeled interphase cells. The first to complete mitosis are those of the labeled cells that entered it first; and accordingly, of the cells with labeled mitotic chromosomes, the last to complete mitosis are those that entered it later than all. Since the duration of mitosis is always the same, then, therefore, if we could determine the interval between: 1) the time of the end of mitosis in the cells that turned on the mark first, and 2) the time of the end of mitosis in the cells that turned on the mark last, we would establish the duration of the S-period . The duration of the S-period can be easily determined by determining the interval between: 1) the point in time when 50% of the mitotic cells in the culture carry the label, and 2) the point in time after which the culture no longer contains 50% of the labeled cells.

Determination of generation time (total duration of the entire cell cycle).

Continuing to take cell samples from the culture, you can find that the marked mitotic figures completely disappear at some point, and then appear again. Such dividing cells are daughter cells derived from those mother cells that turned on the label while being in the S period at the time of exposure to tritium-thymidine. These mother cells entered the S period, divided, and then went through a second interphase and a second division, that is, they went through one full cycle and part of the next. The time required to complete a complete cell cycle is called time generation. It corresponds to the interval between two successive peaks of label incorporation and usually corresponds to the segment between those points of successive ascending curves at which 50% of the mitotic figures contain the label.

Literature.

A. Ham, D. Cormack “Histology”, volume 1 Moscow “MIR” 1982;

M.G. Abramov “Clinical Cytology” Moscow “MEDICINE” 1974;

Y.S.Chentsov “General cytology”

Galileo-Galilei (1564 -1642) (Italian philosopher, mathematician, physicist and astronomer, who had a significant influence on the science of his time; inventor of the microscope) One of the first microscopes (1876)

Light microscopy Robert Hooke (1635 -1703) 1665 – monograph “Micrography”, which describes his microscopic and telescopic observations

DEVICE AND PRINCIPLE OF OPERATION OF A MICROSCOPE Modern light microscope 1. Mechanical part 1. 1. Body 1. 2. Mechanical (specimen) stage 1. 3. Binocular attachment 1. 4. Focusing mechanism 2. Lighting system 2. 1. Light source 2. 2. Collector 2. 3. Condenser 3. Optical part 3. 1. Objectives 3. 2. Eyepieces

Path of rays in a standard microscope light source condenser sample lens eyepiece eye Path of rays in a modern microscope light source sample collector condenser eyepiece lens p image of the sample. DEVICE AND PRINCIPLE OF OPERATION OF THE MICROSCOPE

Lens opening angle: MICROSCOPE RESOLUTION Rayleigh formula: Field resolution of a microscope is the minimum distance between two points of the image it forms while they are still visible separately. where is the wavelength of the light used, n is the refractive index of the medium, and is the opening angle of the lens. light source sample collector condenser lens eyepiece p sample image Abbe's formula: where NA is the numerical aperture of the lens, equal to n sin (/2). NAd 61, 0 2/sin 2 n d

2 114 n. NA ndz Microscope depth resolution – depth of focus. Young's formula:

Diffraction of a laser beam with a wavelength of 650 nm passing through a hole with a diameter of 0.2 mm MICROSCOPE AS A DIFFRACTION CONVERTER dxxuixuxfu. F)]2 sin()2)2 sin()2))