What method is used in cytology. Cytology - the science of the cell Modern research methods. Cellular level of life organization

Fundamentals of Cytology

Cell. Cell theory.

Cell- the smallest structure capable of self-reproduction. The term "cell" was introduced by R. Hooke in 1665 (he studied with a microscope a cut of an elder stem - a core and a cork; although Hooke himself saw not cells, but their shells). The improvement of microscopic technology made it possible to reveal the variety of cell shapes, the complexity of the structure of the nucleus, the process of cell division, etc. The microscope was improved by Antony van Leeuwenhoek (his microscopes gave an increase of 270-300 times).

Other cell research methods:

1. differential centrifugation- based on the fact that different cell structures have different densities. With a very fast rotation in the device (ultracentrifuge), the organelles of finely ground cells precipitate from the solution, arranged in layers in accordance with their density. These layers are separated and studied.
2. electron microscopy- has been used since the 30s of the 20th century (when the electron microscope was invented - it gives an increase of up to 10 6 times); using this method, they study the structure of the smallest structures of the cell, incl. individual organelles and membranes.
3. autoradiography- a method that allows you to analyze the localization in cells of substances labeled with radioactive isotopes. This is how the sites of synthesis of substances, the composition of proteins, and the ways of intracellular transport are revealed.
4. phase contrast microscopy- used to study transparent colorless objects (living cells). When passing through such a medium, light waves are displaced by an amount determined by the thickness of the material and the speed of light passing through it. A phase contrast microscope converts these shifts into a black and white image.
5. x-ray diffraction analysis- the study of the cell with the help of x-rays.

In 1838-1839. botanist Matthias Schleiden and physiologist Theodor Schwann created cell theory. Its essence was that the main structural element of all living organisms (plants and animals) is a cell.

1. cell - elementary living system; the basis of the structure, life, reproduction and individual development of organisms.
2. cells of various body tissues and cells of all organisms are similar in structure and chemical composition.
3. new cells arise only by dividing pre-existing cells.
4. the growth and development of any multicellular organism is a consequence of the growth and reproduction of one or more initial cells.

The molecular composition of the cell.

Chemical elements that make up cells and perform any function are called biogenic. According to the content of the elements that make up the cell, they are divided into three groups:

1. macronutrients- make up the bulk of the cell - 99%. Of these, 98% falls on 4 elements: C, O, H and N. This group also includes K, Mg, Ca, P, C1, S, Na, Fe.
2. trace elements- these include mainly ions that are part of enzymes, hormones and other substances. Their concentration is from 0.001 to 0.000001% (B, Cu, Zn. Br, I, Mo, etc.).
3. ultramicroelements- their concentration does not exceed 10 -6%, and the physiological role is not revealed (Au, Ag, U, Ra).

The chemical components of living things are divided into inorganic(water, mineral salts) and organic(proteins, carbohydrates, lipids, nucleic acids, vitamins).

Water. With a few exceptions (bone and tooth enamel), water is the predominant component of cells - an average of 75-85%. In the cell, water is in a free and bound state. The water molecule is dipole- at one end there is a negative charge, at the other - positive, but in general the molecule is electrically neutral. Water has a high heat capacity and a relatively high thermal conductivity for liquids.

The biological significance of water: universal solvent (for polar substances, non-polar substances do not dissolve in water); environment for reactions, a participant in reactions (protein breakdown), participates in maintaining the thermal equilibrium of the cell; source of oxygen and hydrogen during photosynthesis; the main means of transport of substances in the body.

Ions and salts. Salts are part of the bones, shells, shells, etc., i.e. perform supporting and protective functions, and also participate in mineral metabolism. Ions are part of various substances (iron - hemoglobin, chlorine - hydrochloric acid in the stomach, magnesium - chlorophyll) and are involved in regulatory and other processes, as well as in maintaining homeostasis.

Squirrels. According to the content in the cell, they rank first among organic matter. Proteins are irregular polymers made up of amino acids. Proteins are made up of 20 different amino acids. Amino acid:

NH2-CH-COOH | R

The connection of amino acids occurs as follows: the amino group of one acid is combined with the carboxyl group of another, and a water molecule is released. The resulting connection is called peptide(a kind of covalent), and the compound itself - peptide. A compound of many amino acids is called polypeptide. If a protein consists of only amino acids, then it is called simple ( protein), if it includes other substances, then complex ( proteid).

The spatial organization of proteins includes 4 structures:

1. Primary(linear) - polypeptide chain, i.e. a string of amino acids connected by covalent bonds.
2. Secondary- the protein thread is twisted into a spiral. It creates hydrogen bonds.
3. Tertiary- the helix further coils, forming a globule (coil) or fibril (elongated structure). Hydrophobic and electrostatic interactions arise in it, as well as covalent disulfide -S-S- bonds.
4. Quaternary- connection of several protein macromolecules together.

The breakdown of protein structure is called denaturation. It can be irreversible (if the primary structure is damaged) or reversible (if other structures are damaged).

Protein Functions:

1. enzymes are biologically active substances, they catalyze chemical reactions. More than 2000 enzymes are known. Properties of enzymes: specificity of action (each act only on a certain substance - substrate), activity only in a certain environment (each enzyme has its own optimal pH range) and at a certain temperature (as the temperature rises, the likelihood of denaturation increases, so the activity of the enzyme decreases), greater efficiency actions with little content. Any enzyme has active center- this is a special site in the structure of the enzyme, to which the substrate molecule is attached. Currently, based on the structure, enzymes are divided into two main groups: fully protein enzymes and enzymes consisting of two parts: apoenzyme (protein part) and coenzyme (non-protein part; this is an ion or molecule that binds to the protein part, forming a catalytically active complex). Coenzymes are metal ions, vitamins. Without the coenzyme, the apoenzyme does not function.
2. regulatory - hormones.
3. transport - hemoglobin.
4. protective - immunoglobulins (antibodies).
5. movement - actin, myosin.
6. building (structural).
7. energy - extremely rarely, only after carbohydrates and lipids are over.

Carbohydrates- organic substances, which include C, O and H. General formula: C n (H 2 O) n, where n is at least 3. They are divided into 3 classes: monosaccharides, disaccharides (oligosaccharides) and polysaccharides.

Monosaccharides(simple carbohydrates) - consist of one molecule, these are solid crystalline substances, highly soluble in water, having a sweet taste. Ribose And deoxyribose(C 5) - are part of DNA and RNA. Glucose(C 6 H 12 O 6) - is a part of polysaccharides; the main primary source of energy in the cell. Fructose And galactose isomers of glucose.

Oligosaccharides- consist of 2, 3 or 4 monosaccharide residues. Most important disaccharides- they consist of 2 residues; highly soluble in water, sweet in taste. sucrose(C 12 H 22 O 11) - consists of glucose and fructose residues; widely distributed in plants. Lactose (milk sugar)- consists of glucose and galactose. The most important source of energy for young mammals. Maltose- consists of 2 molecules of glucose. It is the main structural element of starch and glycogen.

Polysaccharides- macromolecular substances, consisting of a large number of monosaccharide residues. Poorly soluble in water, have no sweet taste. Starch- It is represented by two forms: amylose (consists of glucose residues connected in an unbranched chain) and amylopectin (consists of glucose residues, linear and branched chains). Glycogen- polysaccharide of animals and mushrooms. The structure resembles starch, but is more branched. Fiber (cellulose)- the main structural polysaccharide of plants, is part of the cell walls. It is a linear polymer.

Functions of carbohydrates:

1. energy - 1 g with complete decay gives 17.6 kJ.
2. Structural.
3. Support (in plants).
4. Supply of nutrients (starch and glycogen).
5. Protective - viscous secrets (mucus) are rich in carbohydrates and protect the walls of hollow organs.

Lipids- combine fats and fat-like substances - lipoids. Fats are esters of fatty acids and glycerol. Fatty acids: palmitic, stearic (saturated), oleic (unsaturated). Vegetable fats are rich in unsaturated acids, so they are fusible, liquid at room temperature. Animal fats contain mainly saturated acids, so they are more refractory, at room temperature - solid. All fats are insoluble in water, but readily soluble in non-polar solvents; conduct heat poorly. Fats are phospholipids(this is the main component of cell membranes) - they include a residue of phosphoric acid. Lipoids include steroids, waxes, etc.

Lipid functions:

1. structural
2. energy - 1 g with complete decay gives 38.9 kJ.
3. Nutrient storage (adipose tissue)
4. Thermoregulation (subcutaneous fat)
5. Suppliers of endogenous water - when 100 g of fat is oxidized, 107 ml of water is released (camel principle)
6. Protection of internal organs from damage
7. Hormones (estrogens, androgens, steroid hormones)
8. Prostaglandins are regulatory substances that maintain vascular and smooth muscle tone and are involved in immune responses.

ATP (adenosine triphosphate). The energy released during the breakdown of organic substances is not used immediately for work in cells, but is first stored in the form of a high-energy compound - ATP. ATP is made up of three residues of phosphoric acid, ribose (a monosaccharide), and adenine (a nitrogenous base residue). When one residue of phosphoric acid is cleaved, ADP is formed, and if two residues are cleaved, then AMP is formed. The cleavage reaction of each residue is accompanied by the release of 419 kJ/mol. This phosphorus-oxygen bond in ATP is called macroergic. ATP has two macroergic bonds. ATP is formed in mitochondria from AMP, which first attaches one, then the second phosphoric acid residue with the absorption of 419 kJ / mol of energy (or from ADP with the addition of one phosphoric acid residue).

Examples of energy-intensive processes: protein biosynthesis.

Nucleic acids- These are high-molecular organic compounds that provide storage and transmission of hereditary information. First described in the 19th century (1869) by the Swiss Friedrich Miescher. There are two types of nucleic acids.

DNA (deoxyribonucleic acid)

The content in the cage is strictly permanent. It is mainly located in the nucleus (where it forms chromosomes consisting of DNA and two types of proteins). DNA is an irregular biopolymer whose monomer is a nucleotide consisting of a nitrogenous base, a phosphoric acid residue, and a deoxyribose monosaccharide. There are 4 types of nucleotides in DNA: A (adenine), T (thymine), G (guanine) and C (cytosine). A and G are purine bases, C and T are pyrimidine bases. At the same time, in DNA the number of purine bases is equal to the number of pyrimidine bases, as well as A \u003d T and C \u003d G (Chargaff's rule).

In 1953, J. Watson and F. Crick discovered that the DNA molecule is a double helix. Each helix consists of a polynucleotide chain; the chains are twisted one around the other and together around a common axis, each turn of the helix contains 10 pairs of nucleotides. The chains are held together by hydrogen bonds that arise between the bases (between A and T - two, between C and G - three bonds). Polynucleotide chains are complementary to each other: opposite to adenine in one chain there is always thymine in the other and vice versa (A-T and T-A); opposite cytosine - guanine (C-G and G-C). This principle of DNA structure is called the principle of complement or complementarity.

Each strand of DNA has a specific orientation. Two strands in a DNA molecule are located in the opposite direction, i.e. antiparallel.

The main function of DNA is the storage and transmission of hereditary information.

RNA (ribonucleic acid)

1. i-RNA (messenger RNA) - contained in the nucleus and cytoplasm. Its function is to transfer information about the structure of a protein from DNA to the site of protein synthesis.
2. t-RNA (transfer RNA) - mainly in the cytoplasm of the cell. Function: transport of amino acid molecules to the site of protein synthesis. This is the smallest RNA.
3. r-RNA (ribosomal RNA) - is involved in the formation of ribosomes. This is the largest RNA.

Cell structure.

The main components of a cell are: the outer cell membrane, the cytoplasm and the nucleus.

Membrane. In the composition of the biological membrane ( plasmalemma) includes lipids that form the basis of the membrane and high molecular weight proteins. Lipid molecules are polar and consist of charge-bearing polar hydrophilic heads and non-polar hydrophobic tails (fatty acids). The membrane contains mainly phospholipids(they have a phosphoric acid residue in their composition). Membrane proteins can be superficial, integral(permeate the membrane through) and semi-integral(immersed in the membrane).

The modern model of the biological membrane is called "Universal Fluid Mosaic Model", according to which globular proteins are immersed in a double lipid layer, while some proteins penetrate it through, others partially. It is believed that integral proteins are amphiphilic, their non-polar regions are immersed in the lipid bilayer, and the polar ones protrude outward, forming a hydrophilic surface.

Submembrane cell system (submembrane complex). It is a specialized peripheral part of the cytoplasm and occupies a border position between the working metabolic apparatus of the cell and the plasma membrane. In the submembrane system of the surface apparatus, two parts can be distinguished: peripheral hyaloplasm, where the enzymatic systems associated with the processes of transmembrane transport and reception are concentrated, and structurally musculoskeletal system. The musculoskeletal system consists of microfibrils, microtubules, and skeletal fibrillar structures.

Supramembrane structures eukaryotic cells can be divided into two broad categories.

1. Supramembrane complex proper, or glycocalyx 10-20 nm thick. It consists of peripheral membrane proteins, carbohydrate parts of glycolipids and glycoproteins. Glycocalyx plays important role in the receptor function, provides the "individualization" of the cell - it contains tissue compatibility receptors.
2. Derivatives of supramembrane structures. These include specific chemical compounds not produced by the cell itself. They are best studied on microvilli of mammalian intestinal epithelial cells. Here they are hydrolytic enzymes adsorbed from the intestinal cavity. Their transition from a suspended to a fixed state creates the basis for a qualitatively different type of digestion, the so-called parietal digestion. The latter, in its essence, occupies an intermediate position between the cavity and intracellular.

Functions of a biological membrane:

1. barrier;
2. receptor;
3. cell interaction;
4. maintaining the shape of the cell;
5. enzymatic activity;
6. transport of substances into and out of the cell.

Membrane transport:

1. for micromolecules. Distinguish between active and passive transport.

   TO passive include osmosis, diffusion, filtration. Diffusion- the transport of a substance towards a lower concentration. Osmosis- the movement of water towards the solution with a higher concentration. With the help of passive transport, water and fat-soluble substances move.

   TO active transport include: the transfer of substances with the participation of carrier enzymes and ion pumps. The carrier enzyme binds the transferred substance and "drags" it into the cell. The mechanism of the ion pump is considered on the example of operation potassium-sodium pump: during its operation, three Na + are transferred from the cell for every two K + into the cell. The pump operates on the principle of opening and closing channels and, by its chemical nature, is a protein-enzyme (breaks down ATP). The protein binds to sodium ions, changes its shape, and a channel is formed inside it for the passage of sodium ions. After passing through these ions, the protein changes shape again and a channel opens through which potassium ions pass. All processes are energy dependent.

   The fundamental difference between active transport and passive transport is that it comes with energy costs, while passive transport does not.

2. for macromolecules. Occurs with the help of active capture by the cell membrane of substances: phagocytosis and pinocytosis. Phagocytosis- capture and absorption of large particles by the cell (for example, the destruction of pathogenic microorganisms by macrophages of the human body). First described by I.I. Mechnikov. pinocytosis- the process of capture and absorption by the cell of liquid droplets with substances dissolved in it. Both processes occur according to a similar principle: on the surface of the cell, the substance is surrounded by a membrane in the form of a vacuole, which moves inward. Both processes are associated with energy consumption.

Cytoplasm. In the cytoplasm, the main substance (hyaloplasm, matrix), organelles (organelles) and inclusions are distinguished.

Base substance fills the space between the plasmalemma, nuclear membrane and other intracellular structures. It forms the internal environment of the cell, which unites all intracellular structures and ensures their interaction with each other. The cytoplasm behaves like a colloid capable of changing from a gel state to a sol and vice versa. Sol- this is a state of matter characterized by low viscosity and devoid of crosslinks between microfilaments. Gel- this is a state of matter characterized by high viscosity and the presence of bonds between microfilaments. The outer layer of the cytoplasm, or ectoplasm, has a higher density and is devoid of granules. Examples of processes taking place in the matrix: glycolysis, decomposition of substances to monomers.

Organelles- structures of the cytoplasm that perform specific functions in the cell.

Organelles are:

1. membrane (one- and two-membrane (mitochondria and plastids)) and non-membrane.
2. organelles of general importance and special. The former include: ER, Golgi apparatus, mitochondria, ribosomes and polysomes, lysosomes, cell center, microbodies, microtubules, microfilaments. Special purpose organelles (present in cells that perform specialized functions): cilia and flagella (cell movement), microvilli, synaptic vesicles, myofibrils.

Cell inclusions- these are non-permanent formations, either arising or disappearing in the process of cell life, i.e. are products of cellular metabolism. Most often they are found in the cytoplasm, less often in organelles or in the nucleus. Inclusions are mainly represented by granules (polysaccharides: glycogen in animals, starch in plants; less often proteins - in the cytoplasm of eggs), drops (lipids) and crystals (calcium oxalate). Cell inclusions also include some pigments - yellow and brown lipofuscin (accumulates during cell aging), retinin (part of the visual pigment), hemoglobin, melanin, etc.

Core. The main function of the nucleus is the storage of hereditary information. The components of the nucleus are the nuclear membrane, nucleoplasm (nuclear juice), nucleolus (one or two), clumps of chromatin (chromosomes). The nuclear membrane of a eukaryotic cell separates the hereditary material (chromosomes) from the cytoplasm, in which various metabolic reactions take place. The nuclear envelope consists of 2 biological membranes. At certain intervals, both membranes merge with each other, forming pores are holes in the nuclear membrane. Through them, metabolism with the cytoplasm occurs.

basis nucleoplasm make up proteins, including fibrillar ones. It contains the enzymes necessary for the synthesis of nucleic acids and ribosomes. Nuclear sap also contains RNA.

Nucleoli- this is the place of assembly of ribosomes, these are non-permanent structures of the nucleus. They disappear at the beginning of cell division and reappear towards its end. In the nucleolus, an amorphous part and a nucleolar filament are distinguished. Both components are built from filaments and granules, consisting of proteins and RNA.

Chromosomes. Chromosomes are made up of DNA surrounded by two types of proteins: histone(main) and nonhistone(sour). Chromosomes can be in two structural and functional states: spiralized And despiralized. A partially or completely decondensed (despiralized) state is called a working state, because in this state, the processes of transcription and reduplication occur. Inactive state - in a state of metabolic rest at their maximum condensation, when they perform the function of distribution and transfer of genetic material to daughter cells.

IN interphase Chromosomes are represented by a ball of thin threads, which are distinguishable only under an electron microscope. During division, the chromosomes shorten and thicken, they are spiralized and are clearly visible under a microscope (best of all in the metaphase stage). At this time, the chromosomes consist of two chromatids connected by a primary constriction, which divides each chromatid into two sections - the shoulders.

According to the location of the primary constriction, several types of chromosomes are distinguished:

1. metacentric or equal arms (both arms of the chromosome have the same length);
2. submetacentric or unequal arms (the arms of the chromosome differ somewhat in size);
3. acrocentric(one arm is very short).

cell metabolism.

This is one of the basic properties of living things. Metabolism is possible due to the fact that living organisms are open systems, i.e. There is a constant exchange of matter and energy between the organism and the environment. Metabolism proceeds in all organs, tissues and cells, providing self-renewal of the morphological structures and chemical composition of the cytoplasm.

Metabolism consists of two processes: assimilation (or plastic exchange) and dissimilation (or energy exchange). Assimilation(plastic exchange) - the totality of all biosynthesis processes taking place in living organisms. Dissimilation(energy metabolism) - the totality of all processes of decomposition of complex substances into simple ones with the release of energy, taking place in living organisms.

According to the method of assimilation and depending on the type of energy used and the starting materials, organisms are divided into autotrophs (photosynthetics and chemosynthetics) and heterotrophs. Autotrophs- These are organisms that independently synthesize organic substances, using the energy of the Sun for this ( photoautotrophs) or the energy of oxidation of inorganic substances ( chemoautotrophs). Autotrophs include plants, bacteria, blue-green. Heterotrophs- These are organisms that receive ready-made organic substances along with food. These include animals, fungi, bacteria.

The role of autotrophs in the circulation of substances is enormous: 1) they transform the energy of the Sun into the energy of chemical bonds of organic substances, which is used by all other living beings on our planet; 2) saturate the atmosphere with oxygen (photoautotrophs), which is necessary for most heterotrophs to obtain energy by oxidizing organic substances. Heterotrophs also play an important role in the cycle of substances: they release inorganic substances (carbon dioxide and water) used by autotrophs.

Dissimilation. All heterotrophic organisms receive energy as a result of redox reactions, i.e. those in which electrons are transferred from electron donors-reductors to electron acceptors - oxidizers.

The energy exchange aerobic organisms consists of three stages:

1. preparatory, which passes in the gastrointestinal tract or in the cell under the action of lysosome enzymes. During this stage, all biopolymers decompose to monomers: proteins decompose first to peptides, then to amino acids; fats - to glycerol and fatty acids; carbohydrates - to monosaccharides (to glucose and its isomers).
2. anoxic(or anaerobic), which takes place in the matrix of the cytoplasm. This stage is called glycolysis. Under the action of enzymes, glucose is broken down to two PVC molecules. In this case, 4 H atoms are released, which are accepted by a substance called NAD + (nicotinamide adenine dinucleotide). At the same time, NAD + is restored to NAD * H (this stored energy will later be used for ATP synthesis). Also, due to the breakdown of glucose, 4 ATP molecules are formed from ADP. At the same time, 2 ATP molecules are consumed during the chemical reactions of glycolysis, so the total ATP yield after glycolysis is 2 ATP molecules.
3. oxygen that takes place in the mitochondria. Two molecules of PVC enter the enzymatic ring “conveyor”, which is called the Krebs cycle or the tricarboxylic acid cycle. All enzymes of this cycle are located in mitochondria.

Once in the mitochondria, PVC is oxidized and converted into an energy-rich substance - acetyl coenzyme A(it is a derivative of acetic acid). Further, this substance reacts with Pike, forming citric acid (citrate), coenzyme A, protons (accepted by NAD +, which turns into NAD * H) and carbon dioxide. Subsequently, citric acid is oxidized and again turns into PEA, which reacts with a new molecule of acetyl coenzyme A, and the whole cycle is repeated anew. During this process, energy is stored in the form of ATP and NAD*H.

The next stage is the conversion of the energy stored in NAD * H into the energy of ATP bonds. During this process, electrons from NAD*H move along a multi-step electron transport chain to the final acceptor, molecular oxygen. When electrons move from step to step, energy is released, which is used to convert ADP to ATP. Since in this process oxidation is associated with phosphorylation, the whole process is called oxidative phosphorylation(This process was discovered by the Russian scientist V.A. Engelhardt; it occurs on the inner membrane of mitochondria). At the end of this process, water is formed. During the oxygen stage, 36 ATP molecules are formed.

Thus, the end products of the breakdown of glucose are carbon dioxide and water. With the complete breakdown of one glucose molecule, 38 ATP molecules are released. With a lack of oxygen in the cell, glucose is oxidized with the formation of lactic acid (for example, with intensive muscle work - running, etc.). As a result, only two ATP molecules are formed.

It should be noted that not only glucose molecules can serve as an energy source. Fatty acids are also oxidized in the cell to acetyl coenzyme A, which enters the Krebs cycle; at the same time, NAD + is restored to NAD * H, which is involved in oxidative phosphorylation. With an acute shortage of glucose and fatty acids in the cell, many amino acids undergo oxidation. They also form acetyl coenzyme A or organic acids involved in the Krebs cycle.

At anaerobic dissimilation method there is no oxygen stage, and the energy metabolism in anaerobes is called "fermentation". The end products of dissimilation during fermentation are lactic acid (lactic acid bacteria) or ethyl alcohol (yeast). With this type of metabolism, 2 ATP molecules are released from one glucose molecule.

Thus, aerobic respiration is almost 20 times more energetically beneficial than anaerobic respiration.

Photosynthesis. Life on Earth is completely dependent on plant photosynthesis, which supplies organic matter and O 2 to all organisms. Photosynthesis converts light energy into chemical bond energy.

Photosynthesis- this is the formation of organic substances from inorganic with the participation of solar energy. This process was discovered by K.A. Timiryazev in the 19th century. The total photosynthesis equation: 6CO 2 + 6H 2 O \u003d C 6 H 12 O 6 + 6O 2.

Photosynthesis is carried out in plants that have plastids - chloroplasts. Chloroplasts have two membranes, inside - a matrix. They have a well-developed inner membrane, which has folds, between which there are bubbles - thylakoids. Some of the thylakoids are stacked in groups called grains. Granas contain all photosynthetic structures; in the stroma surrounding the thylakoids, there are enzymes that reduce carbon dioxide to glucose. The main pigment of chloroplasts is chlorophyll, similar in structure to human heme. Chlorophyll contains a magnesium atom. Chlorophyll absorbs blue and red rays of the spectrum and reflects green ones. Other pigments may also be present: yellow carotenoids and red or blue phycobilins. Carotenoids are masked by chlorophyll; they absorb light not available to other pigments and transfer it to chlorophyll.

The chloroplasts contain two photosystems of different structure and composition: photosystem I and II. Photosystem I has a reaction center, which is a chlorophyll molecule complexed with a specific protein. This complex absorbs light with a wavelength of 700 nm (which is why it is called the P700 photochemical center). Photosystem II also has a reaction center, the photochemical center P680.

Photosynthesis has two stages: light and dark.

light stage. The energy of light is absorbed by chlorophyll and puts it into an excited state. An electron in the P700 photochemical center absorbs light, moves to a higher energy level and is transferred to NADP + (nicotinamide adenine dinucleotide phosphate), reducing it to NADP*H. In the chlorophyll molecule of photosystem I, “holes” remain - unfilled places for electrons. These "holes" are filled with electrons coming from photosystem II. Under the action of light, the chlorophyll electron in the photochemical center P680 also enters an excited state and begins to move along the chain of electron carriers. Ultimately, this electron comes to photosystem I, filling the free places in it. In this case, the electron loses part of the energy that is spent on the formation of ATP from ADP.

Also in chloroplasts, under the action of sunlight, water is split - photolysis, at which electrons are formed (they enter photosystem II and take the place of electrons that have gone into the carrier chain), protons (NADP + are accepted) and oxygen (as a by-product):

2H 2 O \u003d 4H + + 4e - + O 2

Thus, as a result of the light stage, energy is accumulated in the form of ATP and NADP * H, as well as the formation of oxygen.

dark stage. Does not require light. A carbon dioxide molecule reacts with 1,5 ribulose diphosphate (this is a derivative of ribose) with the help of enzymes. An intermediate compound C 6 is formed, which is decomposed by water into two molecules of phosphoglyceric acid (C 3). From these substances, fructose is synthesized through complex reactions, which is then converted into glucose. These reactions require 18 ATP molecules and 12 NADP*H molecules. Plants produce starch and cellulose from glucose. The fixation of CO 2 and its conversion into carbohydrates is cyclic and is called Calvin cycle.

The importance of photosynthesis for Agriculture large - the crop yield depends on it. In photosynthesis, the plant uses only 1-2% of solar energy, so there is a huge prospect of increasing yields through the selection of varieties with higher photosynthetic efficiency. To increase the efficiency of photosynthesis, the following are used: artificial lighting (additional illumination with fluorescent lamps on cloudy days or in spring and autumn) in greenhouses; lack of shading of cultivated plants, observance of the necessary distances between plants, etc.

Chemosynthesis. This is the process of formation of organic substances from inorganic substances using the energy obtained from the oxidation of inorganic substances. This energy is stored in the form of ATP. Chemosynthesis was discovered by the Russian microbiologist S.N. Vinogradsky in the 19th century (1889-1890). This process is possible in bacteria: sulfur bacteria (oxidize hydrogen sulfide to sulfur and even to sulfuric acid); nitrifying bacteria (oxidize ammonia to nitric acid).

DNA replication(doubling of DNA). As a result of this process, two double helixes of DNA are formed, which are no different from the original (maternal) one. First, with the help of a special enzyme (helicase), the DNA double helix is ​​untwisted at the points of origin of replication. Then, with the participation of the enzyme DNA polymerase, the synthesis of daughter DNA chains occurs. On one of the chains, the process goes on continuously - this chain is called the leader. The second strand of DNA is synthesized in short fragments ( fragments of Okazaki), which are "stitched" together with the help of special enzymes. This chain is called lagging or lagging.

The region between two points where the synthesis of daughter chains begins is called replicon. Eukaryotes have many replicons in their DNA, while prokaryotes have only one replicon. In each replicon you can see replication fork- that part of the DNA molecule that has already unraveled.

Replication is based on a number of principles:

1. complementarity (A-T, C-G) antiparallelism. Each strand of DNA has a specific orientation: one end carries an OH group attached to the 3" carbon in the sugar deoxyribose, at the other end of the chain there is a phosphoric acid residue in the 5" position of the sugar. The two DNA strands are oriented in opposite directions, i.e. antiparallel. The enzyme DNA polymerase can move along the template chains in only one direction: from their 3' ends to 5' ends. Therefore, in the process of replication, the simultaneous synthesis of new chains proceeds antiparallel.
2. semi-conservative. Two daughter helices are formed, each of which preserves (preserves) one of the halves of the maternal DNA unchanged
3. discontinuity. In order for new strands of DNA to form, the parent strands must be fully untwisted and stretched out, which is impossible; therefore, replication starts simultaneously in several places.

protein biosynthesis. An example of plastic metabolism in heterotrophic organisms is protein biosynthesis. All the main processes in the body are associated with proteins, and in each cell there is a constant synthesis of proteins characteristic of this cell and necessary in a given period of the cell's life. Information about a protein molecule is encrypted in a DNA molecule using triplets or codons.

Genetic code is a system for recording information about the sequence of amino acids in proteins using the sequence of nucleotides in mRNA.

Code properties:

1. Tripletity - each amino acid is encrypted by a sequence of three nucleotides. This sequence is called a triplet or codon.
2. Degeneracy or redundancy - each amino acid is encrypted by more than one codon (from 2 to 6). The exceptions are methionine and tryptophan - each of them is encoded by one triplet.
3. Unambiguous - each codon codes for only one amino acid.
4. Between the genes there are "punctuation marks" - these are three special triplets (UAA, UAG, UGA), each of which does not encode amino acids. These triplets are found at the end of each gene. There are no "punctuation marks" within the gene.
5. Universality - the genetic code is the same for all living beings of the planet Earth.

In protein biosynthesis, three stages are distinguished - transcription, post-transcriptional processes and translation.

Transcription- this is the process of synthesis of mRNA, carried out by the enzyme RNA polymerase. Occurs in the nucleus. Transcription is carried out according to the rule of complementarity. The length of the mRNA corresponds to one or more genes. There are 4 stages in the transcription process:

1. binding of RNA polymerase to a promoter (this is the site for enzyme attachment).
2. initiation - the beginning of synthesis.
3. elongation - the growth of an RNA chain; the sequential attachment of nucleotides to each other in the order in which the complementary nucleotides of the DNA strand are. Its speed is up to 50 nucleotides per second.
4. termination - completion of the synthesis of pre-i-RNA.

post-transcriptional processes. After the formation of pre-mRNA, maturation or processing of mRNA begins. In this case, intron regions are removed from the RNA molecule, followed by the connection of exonic regions (this process is called splicing). After that, the mature mRNA leaves the nucleus and goes to the site of protein synthesis (to the ribosomes).

Broadcast- this is the synthesis of polypeptide chains of proteins, performed by the mRNA template in ribosomes.

The amino acids required for protein synthesis are delivered to the ribosomes via tRNA. The transfer RNA molecule has the shape of a clover leaf, on top of which there is a sequence of three nucleotides that are complementary to the nucleotides of the codon in the mRNA. This sequence is called anticodon. The enzyme (codase) recognizes tRNA and attaches the corresponding amino acid to it (the energy of one ATP molecule is spent).

Protein biosynthesis begins with the fact (in bacteria) that the AUG codon, located in the first place in the copy from each gene, occupies a place on the ribosome in the donor site, and a t-RNA carrying formylmethionine (this is an altered form of the amino acid methionine) is attached to it. After completion of protein synthesis, formylmethionine is cleaved from the polypeptide chain.

The ribosome has two sites for binding two tRNA molecules: donor And acceptor. The tRNA with the amino acid enters the acceptor site and attaches to its mRNA codon. The amino acid of this t-RNA attaches the growing protein chain to itself, and a peptide bond arises between them. The tRNA, to which the growing protein is attached, moves along with the mRNA codon to the donor site of the ribosome. A new t-RNA with an amino acid comes to the vacated acceptor site, and everything repeats anew. When one of the punctuation marks appears on the ribosome, none of the amino acid tRNAs can occupy the acceptor site. The polypeptide chain breaks off and leaves the ribosome.

Cells of different tissues of the body produce different proteins (amylase - cells of the salivary glands; insulin - cells of the pancreas, etc.). At the same time, all cells of the body were formed from one fertilized egg by repeated division using mitosis, i.e. have the same genetic makeup. These differences are related to the fact that different DNA regions are transcribed in different cells; different mRNAs are formed, according to which proteins are synthesized. Cell specialization is determined not by all genes, but only by those from which the information was read and implemented into proteins. Thus, in each cell only a part of the hereditary information is realized, and not all information as a whole.

Regulation of gene activity in the synthesis of individual proteins on the example of bacteria (the scheme of F. Jacob and Zh Monod).

It is known that until sugar is added to the nutrient medium where bacteria live, there are no enzymes in the bacterial cell that are necessary for its breakdown. But a few seconds after the addition of sugar, all the necessary enzymes are synthesized in the cell.

Enzymes involved in the same chain of transformation of the substrate into the final product are encoded in one after another structural genes one operon. Operon- This is a group of genes that carry information about the structure of proteins necessary to perform one function. Between the structural genes and the promoter (the landing site for RNA polymerase) there is a site called operator. It is so called because it is from it that the synthesis of mRNA begins. A special protein interacts with the operator - repressor (suppressor). While the repressor is on the operator, mRNA synthesis cannot begin.

When a substrate enters the cell, the cleavage of which requires proteins encoded in the structural genes of the given operon, one of the substrate molecules interacts with the repressor. The repressor loses the ability to interact with the operator and moves away from him; the synthesis of i-RNA begins and the formation of the corresponding proteins on the ribosome. As soon as the last substrate molecule is converted into the final substance, the released repressor will return to the operator and block the synthesis of mRNA.

References:

1. Y. Chentsov "Introduction to Cell Biology" (2006)
2. V.N. Yarygin (editor) "Biology" (in two volumes, 2006)
3. O.V. Alexandrovskaya et al. "Cytology, Histology and Embryology" (1987)
4. A.O. Ruvimsky (editor) "General Biology" (textbook for grades 10-11 with in-depth study biology) - in my opinion, this is one of the best textbooks on general biology for applicants, although not without flaws.

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 methods studies allow studying tissues not only as a whole, but also isolating individual cell types from them to study their vital activity for a long time, isolating individual cell organelles and their macromolecules (for example, DNA), and studying 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 high-contrast images of transparent and colorless living objects that are 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. Next on internal surfaces metals (platinum, palladium, uranium) are deposited on the 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. Thus, 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 engraftment donor cells that form 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 egg sea ​​urchin 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 the researcher to have a new approach to conducting it, to have the skills to compose algorithms for the research process, the accuracy of reasoning, and, ultimately, to increase 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 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.

The main methods of cytological studies are light and electron microscopy, i.e., the use of light and electron microscopes that allow you to see the external and internal structure of cells.

Light microscopes make it possible, among other things, to observe living cells (usually unicellular organisms, blood cells are used for this). However, the resolution of light microscopes is not as high as that of electron microscopes. The resolution of a magnifying device is the minimum distance between two points that can be seen separately. For light microscopes, this distance is measured in hundreds of nanometers, and for electron microscopes, in tens and units of nanometers. If the former uses a luminous flux (the resolution is inversely proportional to the wavelength), then the latter uses a flux of electrons.

There are two types of electron microscopes - transmission and scanning. The resolution of the former is somewhat higher, but with the help of the latter, a three-dimensional image can be obtained. For transmission microscopes, very thin sections are prepared through which the electron beam passes. In scanning microscopes, an electron beam is reflected from an object.

Also used in cytology fluorescence microscopy method, which lies in the fact that certain coloring substances are added to living cells, which, when combined with various components of the cell, begin to glow. Thus, cell structures (chloroplasts, microtubules, etc.) can be observed in a light microscope.

In addition to microscopy, other research methods are used in modern cytology. Cytochemical method allows you to study chemical composition cells. This method is based on chemical reactions certain substances. By adding reagents to cells, it is possible to detect the presence of DNA, certain proteins, etc. in them, as well as to determine their amount.

Radio autography method involves the introduction of a substance containing labeled (radioactive) atoms. After some time, the labeled molecules are included in the biopolymers of the cell, and they can be used to track the course of metabolic processes in the cell.

Since the 20s of the XX century, such a method of cytological research as centrifugation (or method of fractionating cell structures). It is based on the fact that cell structures have different masses and are deposited at different rates during centrifugation. Thus, if the cells are destroyed, then after the centrifuge the mixture will be divided into fractions, where at the bottom there will be heavier structures (usually cell nuclei), and at the top - lighter ones.

Relatively new is cell culture method, which allows growing identical cells (colonies) from one or more initial cells outside the body under specially created conditions. This method makes it possible to study the properties of its cells separately from the body, to conduct cytological, genetic and other studies.

A new method of cytological research is microsurgery method. With the help of a micromanipulator connected to a microscope, various components are extracted or introduced from cells, substances are introduced.

Murmansk State Technical University

Department of Biology

Report on the topic:

"Research Methods in Cytology"

Completed:

1st year student

Faculty of Technology

Departments of Biology

Serebryakova Lada Vyacheslavovna

Checked:

Murmansk2001

Plan:

1. What does cytology study.

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

3. Research methods used in cytology.

4. Fractionation of cells.

5. Radio autography.

6. Determination of the duration of some stages of the cell cycle by radioautography.

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 has 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 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, 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 the 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 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 representation 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 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 the 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 makes it possible for 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, by which biochemists can isolate relatively pure fractions of cells containing certain organelles, and thus study individual metabolic reactions of interest to them; 3) radio autography, 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 opportunity 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, 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 on bottom centrifuge tubes at different centrifugation speeds, depending on the size, density and shape of the organelles. The resulting precipitate can be sampled and examined. Large, dense 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, the nuclei settle out, while other cell organelles remain in suspension. At higher speeds, mitochondria and lysosomes are precipitated, and with long centrifugation and very high speeds, even such small particles 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.

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

Autoradiography is a relatively new method that has immensely expanded the possibilities 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. For radioautography, in particular, isotopes of those elements that are used by the cell or can bind to substances used by the cell, and which can be administered to animals or added to cultures in amounts that do not interfere with normal cellular metabolism. Since a radioactive isotope (or a 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 detection of radioactivity. One way to detect radioactivity is based on its ability to act on photographic film like light; but radioactive radiation penetrates the black paper used to shield the film from light, and has the same effect on the film as light does.

In order to be able to detect radiation emitted by radioactive isotopes on preparations intended for study using light or electron microscopes, the preparations are covered in a dark room with a special photographic emulsion, after which they are 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 emulsion lying above them, in which dark "grains" appear under the action of the emitted radiation. Thus, they receive radio autographs (gr. radio- radiant autos- myself and grapho- write).

Initially, histologists had only a few radioactive isotopes; for example, radioactive phosphorus was used in many of the early studies using autoradiography. Later, much more of these isotopes were used; The radioactive isotope of hydrogen, tritium, has found especially wide application.

Radio autography has been 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 receives from food; they serve as building blocks for building tissues and are incorporated into the 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 of 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 injecting tritium-thymidine into the cells and then making autographs, 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 course of mitosis and set 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 impulse label: 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 absorption of the labeled thymidine by the cells. In this case, the label is included only in those cells that, during a short stay in the 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 just entered the S-period to those that have almost completed it during the time of exposure to tritium-thymidine. In a sample taken immediately after the 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 label exposure; 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 produce a radioautograph for each successive sample, then there will come a moment when the label will begin 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 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, consequently, the label will be found in their mitotic chromosomes. Thus, the interval between 1) the time when the labeled thymidine was removed from the culture, and 2) the time of the 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 moment the label is introduced into the medium will be the first to enter mitosis, then, consequently, in those cells in which the S-period begins immediately before the label is removed, the labeled mitotic chromosomes will appear last. Therefore, if we could determine the interval between the entry into mitosis of the cells labeled first and the cells labeled last, we would establish the duration of the S-period. However, although it is easy to establish the time when labeled mitotic chromosomes first appear, the time of entry into mitosis of the cells labeled last cannot be determined (this is prevented by a very large number of labeled dividing cells in the last samples). Therefore, the duration of the S-period has to be determined in a different way.

In the study of radioautographs of successive samples of cells taken at regular intervals, it is found 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 last. 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 label 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. It is not difficult to determine the period by determining the interval between: 1) the moment when 50% of the mitotic cells in the culture are labeled, and 2) the time after which the culture no longer contains 50% of the labeled cells.

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

Continuing to take cell samples from the culture, one can find that the labeled mitotic figures disappear completely at some point, and then reappear. Such dividing cells are daughter cells derived from those mother cells that have included the label, being in the S-period at the time of exposure to tritium-thymidine. These mother cells went into the S-period, divided, and then went through the second interphase and the second division, that is, they went through one complete cycle and part of the next. The time it takes 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 segment between those points of successive ascending curves in which 50% of the mitotic figures contain the label.

Literature.

A. Ham, D. Kormak "Histology", volume 1 Moscow "MIR" 1982;

M.G. Abramov "Clinical Cytology" Moscow "MEDICINE" 1974;

Yu.S. Chentsov "General Cytology"