Research methods in histology, cytology and embryology. How is histological examination carried out: types, methods, features Histological method of studying cells

2. Objects of histology research

3. Preparation of histological preparations

4. Research methods

5. Historical stages in the development of histology

1. Histology the science of the microscopic and submicroscopic structure, development and vital activity of tissues of animal organisms. Consequently, histology studies one of the levels of organization of living tissue matter. The following are distinguished: hierarchical levels organization of living matter:

cellular;

fabric;

structural and functional units of organs;

organ level;

system level;

organismal level

Histology as an academic discipline, includes the following sections: cytology, embryology, general histology (studies the structure and functions of tissues), special histology (studies the microscopic structure of organs).

Main object The study of histology is the body of a healthy person and therefore this academic discipline is called human histology.

The main task histology consists of studying the structure of cells, tissues, organs, establishing connections between various phenomena, and establishing general patterns.

Histology, like anatomy, belongs to the morphological sciences, the main task of which is the study of the structures of living systems. Unlike anatomy, histology studies the structure of living matter at the microscopic and electron microscopic level. At the same time, the study of the structure of various structural elements is currently being carried out taking into account the functions they perform. This approach to the study of the structures of living matter is called histophysiological, and histology is often referred to as histophysiology. In addition, when studying living matter at the cellular, tissue and organ levels, not only the shape, size and location of the structures of interest are considered, but the composition of the substances forming these structures is often determined by the method of cyto- and histochemistry. Finally, the structures under study are usually considered taking into account their development, both in the prenatal (embryonic) period and throughout postembryonic ontogenesis. This is precisely why the need to include embryology in the histology course is connected.

Histology, like any science, has its own objects and methods their study. The direct objects of study are cells, fragments of tissues and organs, prepared in a special way for studying them under a microscope.

2. Objects of study are divided into:

living (cells in a drop of blood, cells in culture, etc.);

dead or fixed, which can be taken from either a living organism (biopsy) or from cadavers.

In any case, after taking the pieces, they are exposed to fixing solutions or freezing. Fixed objects are used for both scientific and educational purposes. Preparations prepared in a certain way and used for examination under a microscope are called histological preparations.

Histological specimen may be in the form:

a thin, stained section of an organ or tissue;

smear on glass;

imprint on glass from a broken organ;

thin film preparation.

A histological specimen of any form must meet the following requirements:

maintain the lifetime state of structures;

be thin and transparent enough to be examined under a microscope in transmitted light;

be contrasting, that is, the structures being studied must be clearly visible under a microscope;

preparations for light microscopy must be preserved for a long time and used for repeated study.

These requirements are achieved during the preparation of the drug.

3. The following are distinguished: stages of preparing a histological specimen

Taking material(a piece of tissue or organ) for preparing the drug. The following points are taken into account: the collection of material should be carried out as soon as possible after the death or slaughter of the animal, and, if possible, from a living object (biopsy), so that the structures of the cell, tissue or organ are better preserved; the collection of pieces should be done with a sharp instrument so as not to injure the tissue; the thickness of the piece should not exceed 5 mm so that the fixing solution can penetrate into the thickness of the piece; The piece must be marked (indicate the name of the organ, the number of the animal or the name of the person, the date of collection, and so on).

Fixing the material necessary to stop metabolic processes and preserve structures from decay. Fixation is most often achieved by immersing the piece in fixing liquids, which can be simple alcohols and formalin and complex Carnoy's solution, Zinker's fixative and others. The fixative causes denaturation of the protein and thereby stops metabolic processes and preserves the structures in their lifetime state. Fixation can also be achieved by freezing (cooling in a CO2 stream, liquid nitrogen, etc.). The duration of fixation is selected empirically for each tissue or organ.

Pouring pieces into sealing media(paraffin, celloidin, resins) or freezing for subsequent production of thin sections.

Preparation of sections on special instruments (microtome or ultramicrotome) using special knives. Sections for light microscopy are glued onto glass slides, and for electron microscopy they are mounted on special grids.

Staining of sections or contrasting them (for electron microscopy). Before staining the sections, the sealing medium is removed (dewaxing). The contrast of the studied structures is achieved by coloring. Dyes are divided into basic, acidic and neutral. The most widely used dyes are basic (usually hematoxylin) and acidic (eosin). Complex dyes are often used.

Clearing sections(in xylene, toluene), encapsulation in resins (balsam, polystyrene), covering with a coverslip.

After these sequential procedures, the drug can be studied under a light microscope.

For electron microscopy purposes There are some peculiarities in the preparation stages, but the general principles are the same. The main difference is that the histological preparation for light microscopy can be stored for a long time and reused. Sections for electron microscopy are used once. In this case, first, the objects of interest in the drug are photographed, and the structures are studied using electron diffraction patterns.

Made from fabrics of liquid consistency(blood, bone marrow and others) preparations are made in the form of a smear on a glass slide, which are also fixed, stained, and then studied.

From fragile parenchymal organs(liver, kidney and others) preparations are made in the form of an imprint of the organ: after a fracture or rupture of the organ, a glass slide is applied to the site of the organ fracture, onto which some free cells are glued. The preparation is then fixed, stained and examined.

Finally, from some organs(mesentery, pia mater) or from loose fibrous connective tissue, film preparations are made by stretching or crushing between two glasses, also with subsequent fixation, coloring and pouring into resins.

4. Main research method biological objects used in histology are microscopy, i.e., studying histological preparations using a microscope. Microscopy can be an independent method of study, but recently it is usually combined with other methods (histochemistry, historadiography and others). It should be remembered that different microscope designs are used for microscopy, allowing one to study different parameters of the objects being studied. The following are distinguished: types of microscopy:

light microscopy (resolution 0.2 µm) the most common type of microscopy;

ultraviolet microscopy (resolution 0.1 microns);

luminescent (fluorescent) microscopy to determine chemical substances in the structures in question;

phase contrast microscopy for studying structures in unstained histological preparations;

polarization microscopy to study mainly fibrous structures;

dark field microscopy for studying living objects;

incident light microscopy for studying thick objects;

electron microscopy (resolution up to 0.1-0.7 nm), its two varieties transmission (transmission) electron microscopy and scanning or raster microscopy provide an image of the surface of ultrastructures.

Histochemical and cytochemical methods allows you to determine the composition of chemical substances and even their quantity in the structures being studied. The method is based on carrying out chemical reactions with the reagent used and chemicals present in the substrate, with the formation of a reaction product (contrast or fluorescent), which is then determined by light or fluorescent microscopy.

Histoautoradiography method makes it possible to identify the composition of chemical substances in structures and the intensity of exchange based on the inclusion of radioactive isotopes in the structures under study. The method is most often used in animal experiments.

Differential centrifugation method allows you to study individual organelles or even fragments isolated from a cell. To do this, a piece of the organ under study is ground, filled with physiological solution, and then accelerated in a centrifuge at various speeds (from 2 to 150 thousand) and the fractions of interest are obtained, which are then studied by various methods.

Interferometry method allows you to determine the dry mass of substances in living or fixed objects.

Immunomorphological methods allows, using pre-conducted immune reactions, based on antigen-antibody interaction, to determine subpopulations of lymphocytes, determine the degree of foreignness of cells, carry out histological typing of tissues and organs (determine histocompatibility) for organ transplantation.

Cell culture method(in vitro, in vivo) growing cells in a test tube or in special capsules in the body and subsequent study of living cells under a microscope.

Units of measurement used in histology

To measure structures in light microscopy, micrometers are mainly used: 1 µm is 0.001 mm; Electron microscopy uses nanometers: 1 nm is 0.001 microns.

5. IN history of histology development conditionally there are three period:

Pre-microscopic period(from the 4th century BC to 1665) is associated with the names of Aristotle, Galen, Avicenna, Vesalius, Fallopius and is characterized by attempts to isolate heterogeneous tissues (hard, soft, liquid, etc.) in the body of animals and humans and the use anatomical preparation methods.

Microscopic period(from 1665 to 1950). The beginning of the period is associated with the name of the English physicist Robert Hooke, who, firstly, improved the microscope (it is believed that the first microscopes were invented at the very beginning of the 17th century), and secondly, used it for the systematic study of various objects, including biological ones and published the results of these observations in 1665 in the book “Micrography”, thirdly, he first introduced the term “cell” (“cellulum”). Subsequently, microscopes were continuously improved and used increasingly for the study of biological tissues and organs.

Particular attention was paid to studying the structure of the cell. Jan Purkinje described the presence of “protoplasm” (cytoplasm) and a nucleus in animal cells, and a little later R. Brown confirmed the presence of a nucleus in most animal cells. The botanist M. Schleiden became interested in the origin of cells by cytokenesis. The results of these studies allowed T. Schwan, based on their reports, to formulate the cell theory (1838-1839) in the form of three postulates:

all plant and animal organisms consist of cells;

all cells develop according to the general principle from the cytoblastema;

Each cell has independent vital activity, and the vital activity of the body is the sum of the activities of the cells.

However, soon R. Virchow (1858) clarified that cell development is carried out by dividing the original cell (any cell from a cell). The provisions of the cell theory developed by T. Schwan are still relevant today, although they are formulated differently.

Modern provisions of cell theory:

a cell is the smallest unit of living things;

the cells of animal organisms are similar in structure;

cell reproduction occurs by dividing the original cell;

multicellular organisms are complex ensembles of cells and their derivatives, united in systems of tissues and organs, interconnected by cellular, humoral and neural forms of regulation.

Further improvement of microscopes, especially the creation of achromatic lenses, made it possible to identify smaller structures in cells:

cell center Hertwig, 1875;

reticular apparatus or lamellar complex of Golgi, 1898;

Bend's mitochondria, 1898

Modern stage The development of histology begins in 1950 with the beginning of the use of the electron microscope to study biological objects, although the electron microscope was invented earlier (E. Ruska, M. Knoll, 1931). However, the modern stage of development of histology is characterized by the introduction of not only the electron microscope, but also other methods: cyto- and histochemistry, historadiography and other modern methods listed above. In this case, a complex of various techniques is usually used, which allows one to form not only a qualitative idea of the structures being studied, but also to obtain accurate quantitative characteristics. Various morphometric techniques are currently used especially widely, including automated systems for processing received information using computers.

LECTURE 2. Cytology. Cytoplasm

Histology – (“histos” in Greek – tissue, logis – study) This is the science of the structure, development and vital activity of tissues of multicellular organisms and humans. Objects that are the subject of this science are inaccessible to the naked eye. Therefore, the history of histology is closely connected with the history of the creation of such devices that make it possible to study the smallest objects with the naked eye. 2

The histology course is divided into the following sections: n 1. Cytology - the science of cells. n 2. Embryology is the science of development, from conception to the full formation of the organism. n 3. General histology is the science of the general patterns inherent in tissues. n 4. Particular histology - studies the structure and development of organs and systems.

CYTOLOGY – (Greek κύτος “cell” and λόγος - “teaching”, “science”) n A branch of biology that studies living cells, their organelles, their structure, functioning, processes of cell reproduction, aging and death. 4

EMBRYOLOGY n (from ancient Greek ἔμβρυον - embryo, embryo + -λογία from λόγος - teaching) is a science that studies the development of the embryo. 5

The history of the creation of the cell theory 1590. Jansen invented a microscope in which magnification was achieved by connecting two lenses. 1665 Robert Hooke first used the term cell. 1650 -1700. Anthony van Leeuwenhoek was the first to describe bacteria and other microorganisms. 1700 -1800. Many new descriptions and drawings of various tissues, mainly plant ones, have been published. In 1827, Karl Baer discovered the egg in mammals. 1831 -1833. Robert Brown described the nucleus in plant cells. 1838 -1839. Botanist Matthias Schleiden and zoologist Theodor Schwann combined the ideas of different scientists and formulated the cell theory, which postulated that the basic unit of structure and function in living organisms is the cell. 1855 Rudolf Virchow showed that all cells are formed as a result of cell division.

The history of the creation of cell theory 1665. Examining a section of cork under a microscope, the English scientist and physicist Robert Hooke discovered that it consists of cells separated by partitions. He called these cells "cells"

The history of the creation of cell theory In the 17th century, Leeuwenhoek designed a microscope and opened the door to the microworld for people. A variety of ciliates, rotifers and other tiny living creatures flashed before the eyes of the amazed researchers. It turned out that they are everywhere - these tiny organisms: in water, manure, in the air and dust, in the ground and gutters, in rotting waste of animal and plant origin.

The history of the creation of cell theory 1831 -1833. Robert Brown described the nucleus in plant cells. In 1838, the German botanist M. Schleiden drew attention to the nucleus and considered it to be the maker of the cell. According to Schleiden, a nucleolus condenses from the granular substance, around which a nucleus is formed, and around the nucleus a cell, and the nucleus may disappear during the formation of the cell.

The history of the creation of the cell theory The German zoologist T. Schwann showed that animal tissues are also composed of cells. He created a theory stating that cells containing nuclei constitute the structural and functional basis of all living things. The cellular theory of structure was formulated and published by T. Schwann in 1839. Its essence can be expressed in the following provisions: 1. The cell is the elementary structural unit of the structure of all living beings; 2. Cells of plants and animals are independent, homologous to each other in origin and structure. Each cell functions independently of the others, but together with everyone else. 3. All cells arise from structureless intercellular substance. (Error!) 4. The vital activity of the cell is determined by the membrane. (Error!)

History of the creation of the cell theory In 1855, the German physician R. Virchow made a generalization: a cell can only arise from a previous cell. This led to the recognition of the fact that the growth and development of organisms is associated with cell division and their further differentiation, leading to the formation of tissues and organs.

The history of the creation of the cell theory by Karl Baer Back in 1827, Karl Baer discovered the egg in mammals and proved that the development of mammals begins with a fertilized egg. This means that the development of any organism begins with one fertilized egg; the cell is the unit of development.

History of the creation of the cell theory 1865 The laws of heredity were published (G. Mendel). 1868 Nucleic acids were discovered (F. Miescher) 1873 Chromosomes were discovered (F. Schneider) 1874 Mitosis was discovered in plant cells (I. D. Chistyakov) 1878 Mitotic division of animal cells was discovered (W. Fleming, P. I. Peremezhko) 1879 Fleming - behavior of chromosomes during division. 1882 Meiosis was discovered in animal cells (W. Fleming) 1883 It was shown that in germ cells the number of chromosomes is half that in somatic cells (E. Van Beneden) 1887 Meiosis was discovered in plant cells (E. Strassburger ) 1898 Golgi discovered the reticular apparatus of the cell, the Golgi apparatus. 1914 The chromosomal theory of heredity was formulated (T. Morgan). 1924 A natural scientific theory of the origin of life on Earth was published (A.I. Oparin). 1953 Ideas about the structure of DNA were formulated and its model was created (D. Watson and F. Crick). 1961 The nature and properties of the genetic code are determined (F. Crick, L. Barnett, S. Benner).

Basic provisions of modern cell theory 1. A cell is an elementary living system, a unit of structure, vital activity, reproduction and individual development of organisms. 2. The cells of all living organisms are homologous, the same in structure and origin. 3. Cell formation. New cells arise only by dividing pre-existing cells. 4. Cell and organism. A cell can be an independent organism (prokaryotes and unicellular eukaryotes). All multicellular organisms are made up of cells. 5. Cell functions. The cells carry out: metabolism, irritability and excitability, movement, reproduction and differentiation. 6. Cell evolution. Cellular organization arose at the dawn of life and went through a long path of evolutionary development from non-nuclear forms (prokaryotes) to nuclear ones (eukaryotes).

METHODS FOR MICROSCOPY OF HISTOLOGICAL PREPARATIONS 1. Light microscopy. 2. Ultraviolet microscopy. 3. Fluorescent (luminescent) microscopy. 4. Phase contrast microscopy. 5. Dark field microscopy. 6. Interference microscopy 7. Polarization microscopy. 8. Electron microscopy. 17

Microscope n This optical instrument allows you to observe small objects. Image magnification is achieved by a system of objective lenses and eyepiece. The mirror, condenser and diaphragm direct the light flow and regulate the illumination of the object. The mechanical part of the microscope includes: a tripod, a stage, macro- and micrometer screws, and a tube holder. 18

Special microscopy methods: - phase contrast microscope - (for studying living, unpainted objects) - microscopy allows you to study living and unpainted objects. When light passes through painted objects, the amplitude of the light wave changes, and when light passes through unpainted objects, the phase of the light wave changes, which is used to obtain high-contrast images in phase-contrast and interference microscopy. - dark-field microscope (for studying living unpainted objects). A special condenser is used to highlight the contrasting structures of the unpainted material. Dark-field microscopy allows you to observe living objects. The observed object appears as if it were illuminated in a dark field. In this case, the rays from the illuminator fall on the object from the side, and only scattered rays enter the microscope lenses. 19

Special methods of microscopy: fluorescent microscopy (for studying living, unpainted objects) microscopy is used to observe fluorescent (luminescent) objects. In a fluorescence microscope, light from a powerful source passes through two filters. One filter stops light in front of the sample and transmits light of the wavelength that excites fluorescence from the sample. Another filter allows light of the wavelength emitted by the fluorescent object to pass through. Thus, fluorescent objects absorb light of one wavelength and emit in another region of the spectrum. - ultraviolet ability of m-pa) mik-p (increases the resolution - polarization mik-p (for studying objects with an ordered arrangement of molecules - skeletal muscles, collagen fibers, etc.) microscopy - image formation of unstained anisotropic structures ( for example, collagen fibers and myofibrils).20

Special microscopy methods - interference microscopy (to determine the dry residue in cells, determine the thickness of objects) - microscopy combines the principles of phase-contrast and polarization microscopy and is used to obtain a contrast image of unpainted objects. Special interference optics (Nomarski optics) have found application in differential interference contrast microscopes. B. Electron microscopy: -transmission (study of objects through transmission) -scanning (study of the surface of objects) Theoretically, the resolution of transmission EM is 0.002 nm. The actual resolution of modern microscopes approaches 0.1 nm. For biological objects, the EM resolution in practice is 2 nm. 21

Special microscopy techniques A transmission electron microscope consists of a column through which electrons emitted by a cathode filament pass in a vacuum. A beam of electrons, focused by ring magnets, passes through the prepared sample. The nature of electron scattering depends on the density of the sample. Electrons passing through the sample are focused, observed on a fluorescent screen, and recorded using a photographic plate. A scanning electron microscope is used to obtain a three-dimensional image of the surface of the object under study. The cleavage method (freezing-cleavage) is used to study the internal structure of cell membranes. The cells are frozen at liquid nitrogen temperature in the presence of a cryoprotectant and used for making chips. The cleavage planes pass through the hydrophobic middle of the lipid bilayer. The exposed inner surface of the membranes is shaded with platinum, and the resulting replicas are studied in a scanning EM. 22

Special (non-microscopic) methods: 1. Cyto- or histochemistry - the essence is the use of strictly specific chemical reactions with a light final product in cells and tissues to determine the amount of various substances (proteins, enzymes, fats, carbohydrates, etc.). Can be applied at the light or electron microscope level. 2. Cytophotometry - the method is used in combination with 1 and makes it possible to quantitatively evaluate proteins, enzymes, etc. identified by the cytohistochemical method. 3. Autoradiography - substances containing radioactive isotopes of chemical elements are introduced into the body. These substances are included in metabolic processes in cells. The localization and further movements of these substances in the organs are determined on histological preparations by radiation, which is captured by a photographic emulsion applied to the preparation. 4. X-ray structural analysis - allows you to determine the amount of chemical elements in cells and study the molecular structure of biological microobjects. 24 5. Morphometry - measurement of biol sizes. structures at the cellular and subcellular level.

Special (non-microscopic) methods 6. Microurgy - carrying out very fine operations with a micromanipulator under a microscope (transplanting nuclei, introducing various substances into cells, measuring biopotentials, etc.) 6. Method of cultivating cells and tissues - in nutrient media or in diffusion chambers, implanted into various tissues of the body. 7. Ultracentrifugation - fractionation of cells or subcellular structures by centrifugation in solutions of varying densities. 8. Experimental method. 9. Method of tissue and organ transplantation. 25

Fixation preserves the structure of cells, tissues and organs, prevents their bacterial contamination and enzymatic digestion, stabilizes macromolecules by chemical cross-linking. 32

Fixing liquid formalin, alcohols, glutaraldehyde - The most common fixatives; Cryofixation - Better preservation of structures is ensured by instant freezing of samples in liquid nitrogen (– 196 °C); Lyophilization - small pieces of tissue are rapidly frozen, stopping metabolic processes. Dehydration - a standard procedure for removing water - dehydration in alcohols of increasing strength (from 70 to 60%). Filling – makes the fabric durable, prevents it from being crushed and wrinkled when cutting, and makes it possible to obtain sections of standard thickness. The most common embedding medium is paraffin. Celloidin, plastic media and resins are also used. 33

Dehydration prepares the fixed tissue for penetration of embedding media. Water from living tissue, as well as water from fixative mixtures (most fixatives are aqueous solutions) must be completely removed after fixation. The standard procedure for removing water is dehydration in alcohols of increasing strength from 60° to 100°. 34

Pouring is a necessary procedure prior to preparing sections. Filling makes the fabric durable, prevents it from being crushed and wrinkled when cutting, and makes it possible to obtain thin sections of standard thickness. The most common embedding medium is paraffin. Celloidin, plastic media and resins are also used. 35

Rotary microtome. 40 n Blocks containing a piece of an organ are secured in a movable object holder. When it is lowered, serial sections remain on the knife; they are removed from the knife and mounted on a glass slide for subsequent processing and microscopy.

Methods for staining histological sections: n Nuclear (main): n hematoxylin – stains n n n n nuclei blue; iron hematoxylin; Azur II (in purple); carmine (in red); safranin (in red); methyl blue (blue); toluidine (blue); thionin (blue). n Cytoplasmic- (acidic): n eosin – pink; n erythrosine; n orange "G"; n sour magenta – red; n picric acid - yellow; n Congo – red – to red 44

SPECIAL Methods for staining histological sections n Sudan III – staining lipids and fats orange; n osmic acid – coloring of lipids and fats black; n orcein - coloring of elastic fibers brown; n silver nitrate – impregnation of nerve elements in a dark brown color. 45

Cell structures: n OXYPHILIAn the ability to be stained pink with acidic dyes n Basophilian the ability to be stained blue with basic dyes n Neutrophilia - n the ability to be stained violet with acidic and basic dyes. 47

Cell n is an elementary living system consisting of cytoplasm, nucleus, membrane and is the basis for the development, structure and life of animal and plant organisms.

The glycocalyx is a supra-membrane complex consisting of saccharides associated with proteins and saccharides associated with lipids. Functions n Reception (hormones, cytokines, mediators and antigens) n Intercellular interactions (irritability and recognition) n Parietal digestion (microvilli of intestinal border cells)

Functions of the cytolemma: - delimiting; - active and passive transport of substances in both directions; - receptor functions; - contact with neighboring cells.

Histology is the science of the microscopic and submicroscopic structure, development and vital activity of tissues of animal organisms.

The following are distinguished: hierarchical levels organization of living matter:

cellular;
fabric;
structural and functional units of organs;
organ level;
system level;
organismal level

Objects of histology research

Objects of research are divided into:

living (cells in a drop of blood, cells in culture, etc.);
dead or fixed, which can be taken from either a living organism (biopsy) or from cadavers.

Histological specimen

Histological specimen may be in the form:

a thin, stained section of an organ or tissue;
smear on glass;
imprint on glass from a broken organ;
thin film preparation.

A histological specimen of any form must meet the following requirements:

maintain the lifetime state of structures;
be thin and transparent enough to be examined under a microscope in transmitted light;
be contrasting, that is, the structures being studied must be clearly visible under a microscope;
preparations for light microscopy must be preserved for a long time and used for repeated study.

These requirements are achieved during the preparation of the drug.

Stages of preparing a histological specimen

Taking material(a piece of tissue or organ) for preparing the drug.

Fixing the material necessary to stop metabolic processes and preserve structures from decay.

Pouring pieces into sealing media(paraffin, celloidin, resins) or freezing for subsequent production of thin sections.

Preparation of sections on special instruments (microtome or ultramicrotome) using special knives.

Staining of sections or contrasting them (for electron microscopy).

Clearing sections(in xylene, toluene), encapsulation in resins (balsam, polystyrene), covering with a coverslip.

For electron microscopy purposes There are some peculiarities in the preparation stages, but the general principles are the same.

Made from fabrics of liquid consistency(blood, bone marrow and others) preparations are made in the form of a smear on a glass slide, which are also fixed, stained, and then studied.

Research methods in histology

The main method for studying biological objects used in histology is microscopy, i.e., studying histological preparations using a microscope. Microscopy can be an independent method of study, but recently it is usually combined with other methods (histochemistry, historadiography and others). It should be remembered that different microscope designs are used for microscopy, allowing one to study different parameters of the objects being studied. The following are distinguished: types of microscopy:

light microscopy (resolution 0.2 µm) the most common type of microscopy;
ultraviolet microscopy (resolution 0.1 microns);
luminescent (fluorescent) microscopy to determine chemical substances in the structures in question;
phase contrast microscopy for studying structures in unstained histological preparations;
polarization microscopy to study mainly fibrous structures;
dark field microscopy for studying living objects;
incident light microscopy for studying thick objects;
electron microscopy (resolution up to 0.1-0.7 nm), its two varieties transmission (transmission) electron microscopy and scanning or raster microscopy provide an image of the surface of ultrastructures.

Interferometry method allows you to determine the dry mass of substances in living or fixed objects.

Cell culture method(in vitro, in vivo) growing cells in a test tube or in special capsules in the body and subsequent study of living cells under a microscope.

Units of measurement used in histology

To measure structures in light microscopy, micrometers are mainly used: 1 µm is 0.001 mm; Electron microscopy uses nanometers: 1 nm is 0.001 microns.

Historical stages in the development of histology

In the history of the development of histology, conditionally there are three period:

all plant and animal organisms consist of cells;
all cells develop according to the general principle from the cytoblastema;
Each cell has independent vital activity, and the vital activity of the body is the sum of the activities of the cells.

Modern provisions of cell theory:

a cell is the smallest unit of living things;
the cells of animal organisms are similar in structure;
cell reproduction occurs by dividing the original cell;
multicellular organisms are complex ensembles of cells and their derivatives, united in systems of tissues and organs, interconnected by cellular, humoral and neural forms of regulation.
Further improvement of microscopes, especially the creation of achromatic lenses, made it possible to identify smaller structures in cells:
cell center Hertwig, 1875;
reticular apparatus or lamellar complex of Golgi, 1898;
Bend's mitochondria, 1898

Modern stage development of histology

begins in 1950 with the beginning of the use of the electron microscope to study biological objects, although the electron microscope was invented earlier (E. Ruska, M. Knoll, 1931). However, the modern stage of development of histology is characterized by the introduction of not only the electron microscope, but also other methods: cyto- and histochemistry, historadiography and other modern methods listed above. In this case, a complex of various techniques is usually used, which allows one to form not only a qualitative idea of the structures being studied, but also to obtain accurate quantitative characteristics. Various morphometric techniques are currently used especially widely, including automated systems for processing received information using computers.

Objects of research are divided into:

· living (cells in a drop of blood, cells in culture, etc.);

· dead or fixed, which can be taken from either a living organism (biopsy) or from cadavers.

A histological specimen can be in the form of: (a thin stained section of an organ or tissue; a smear on glass; an imprint on glass from a fracture of an organ; a thin film preparation).

A histological preparation of any form must meet the following requirements: (preserve the vital state of the structures; be thin and transparent enough to be studied under a microscope in transmitted light; be contrasting, that is, the structures being studied must be clearly defined under the microscope; preparations for light microscopy must be preserved for a long time and used for re-learning.)

These requirements are achieved during the preparation of the drug.

Research methods:

Light microscopy-Microscopy, the main method for studying drugs, has been used in biology for more than 300 years. Ultraviolet microscopy- This is a type of light microscopy. An ultraviolet microscope uses shorter ultraviolet rays with a wavelength of about 0.2 microns. Fluorescence (luminescence) microscopy- The phenomena of fluorescence consist in the fact that atoms and molecules of a number of substances, absorbing short-wave rays, go into an excited state. Phase contrast microscopy- This method is used to obtain high-contrast images of transparent and colorless objects that are invisible with conventional microscopy methods. Electron microscopy-An electron microscope uses a stream of electrons with shorter wavelengths than a light microscope.

The main stages of cytological and histological analysis are the selection of an object of study, its preparation for examination under a microscope, the use of microscopy methods, as well as qualitative and quantitative image analysis.

Most often, a section of tissue or organ is used for study. Histological preparations can be studied without special processing. For example, a prepared blood smear, print, film or organ section can be immediately examined under a microscope. But due to the fact that the structures have weak contrast, they are poorly visible in a conventional light microscope and the use of special microscopes (phase contrast, etc.) is required. Therefore, specially processed preparations are more often used: fixed, enclosed in a solid medium and colored.

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. material compaction,

3. preparation of sections,

4. staining or contrasting sections.

For light microscopy, one more step is required - enclosing sections in balm or other transparent media.

Fixation ensures the prevention of decomposition processes, which helps preserve the integrity of the organ structures. A small sample is either immersed in a fixative (alcohol, formaldehyde, solutions of heavy metal salts, osmic acid, special fixative mixtures) or subjected to heat treatment

The compaction of the material necessary for preparing sections is carried out by impregnating the previously dehydrated material with paraffin, celloidin, and organic resins. Faster compaction is achieved by using the method of freezing the pieces, for example, in liquid carbon dioxide.

The sections are prepared using special devices - microtomes(for light microscopy) and ultramicrotomes(for electron microscopy).

Staining sections (in light microscopy) or sputtering them with metal salts (in electron microscopy) is used to increase the contrast of the image of individual structures when viewing them under a microscope. Methods for staining histological structures are very diverse and are selected depending on the objectives of the study.

Histological dyes (according to their chemical nature) are divided into acidic, basic and neutral. Common dye hematoxylin, which stains cell nuclei purple, and acidic dye - eosin, staining the cytoplasm pink-yellow. The selective affinity of structures for certain dyes is determined by their chemical composition and physical properties. Structures that stain well with acidic dyes are called oxyphilic, and those that stain with basic dyes are called basophilic. For example, the cytoplasm of cells is most often stained oxyphilic, and the nuclei of cells are stained 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, the dehydrated histological section is enclosed between a slide and cover glass in Canada balsam or other substances. The finished histological specimen can be used for study under a microscope for many years.

4) . The cell as a structural and functional unit of tissue. Definition. General plan of the structure of eukaryotic cells. Biological cell membranes, their structure, chemical composition and main functions.

A cell is an elementary structural, functional and genetic unit within all plant and animal organisms. Structure of a eukaryotic cell:

The cells that form the tissues of animals and plants vary significantly in shape, size and internal structure. Cells of all types contain two main components that are closely related to each other - the cytoplasm and the nucleus. The nucleus is separated from the cytoplasm by a porous membrane and contains nuclear sap, chromatin and the nucleolus. Semi-liquid cytoplasm fills the entire cell and is penetrated by numerous tubules. On the outside it is covered with a cytoplasmic membrane.

The cell body itself and its contents are separated from the external environment or from neighboring elements in multicellular organisms by a plasma membrane. Outside the plasma membrane is the cell membrane or wall, which is especially pronounced in plants. All the internal contents of the cell, with the exception of the nucleus, are called cytoplasm. The cytoplasm of eukaryotic cells is not homogeneous in its structure and composition and includes: hyaloplasm, membrane and non-membrane components. Membrane organelles come in two varieties: single-membrane and double-membrane. The first include organelles of the vacuolar system - the endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes and other specialized vacuoles, as well as the plasma membrane. Double-membrane organelles include mitochondria and plastids, as well as the cell nucleus. Non-membrane organelles include ribosomes, the cellular center of animal cells, as well as cytoskeletal elements (microtubules and microfilaments).
The term hyaloplasm, the main plasma or matrix of the cytoplasm, refers to a very important part of the cell, its true internal environment. Hyaloplasm is a complex colloidal system that includes various biopolymers: proteins, nucleic acids, polysaccharides, etc. Enzymes involved in the synthesis of amino acids, nucleotides, fatty acids, and sugar metabolism are localized in it. The most important role of hyaloplasm is that this environment unites all cellular structures and ensures their chemical interaction with each other. Most of the intracellular transport processes are carried out through the hyaloplasm: the transfer of amino acids, fatty acids, nucleotides, and sugars. In the hyaloplasm there is a constant flow of ions to and from the plasma membrane, to the mitochondria, nucleus and vacuoles. In the hyaloplasm, storage products are deposited: glycogen, fat. In the cytosol, on the ribosomes located there, proteins are synthesized that are transported to different parts of the cell, as well as all the proteins of the cell nucleus, most of the proteins of mitochondria and plastids, and the main proteins of peroxisomes. Structure of cell membranes.
A common feature of all cell membranes (plasma, intracellular and membrane organelles) is that they are thin (6-10 nm) layers of lipoprotein nature (lipids in complex with proteins), closed on themselves

There are three important principles of membrane structure:
The membranes are not homogeneous. The membranes surrounding intracellular organelles and the plasma membrane differ in composition. Many membrane components are in a state of continuous movement. The membrane resembles an ever-changing mosaic. The components of the membranes are extremely asymmetrical. There is a difference in the relative quantity and qualitative composition of lipids between the outer and inner layers of membranes. Proteins are located asymmetrically among lipids and have clearly distinguishable extra- and intracellular regions.

The most important functions of membranes are the following:

Membranes control the composition of the intracellular environment.

Membranes provide and facilitate intercellular and intracellular transmission of information.

Membranes provide tissue formation through intercellular contacts

Chemical composition of the cell.
The cells of living organisms are similar not only in their structure, but also in their chemical composition. The similarity in the structure and chemical composition of cells indicates the unity of their origin.

Based on their composition, the substances entering the cell are divided into organic and inorganic.
1.Inorganic substances.
Water is of great importance in the life of a cell. Many elements in cells are contained in the form of ions. The most common cations are: K+, Na+, Ca2+ Mg2+, and anions: H2PO4-, Cl-, HCO3-.
Mineral salts (for example, calcium phosphate) can be part of the intercellular substance and shells of mollusks and provide the strength of these formations.
2. Organic substances.
Characteristic only of living things. Organic compounds are represented in the cell by simple small molecules (amino acids, mono- and oligosaccharides, fatty acids, nitrogenous bases), and macromolecules of biopolymers (proteins, lipids, polysaccharides, nucleic acids). Biopolymer molecules consist of repeating low molecular weight compounds (monomers

Cell functions. The cell has various functions: cell division, metabolism and

Main Objects of research are histological preparations, and the main research method is microscopy.

The histological specimen must be sufficiently transparent (thin) and contrast. It is made from both living and dead (fixed) structures. The specimen can be a suspension of cells, a smear, an imprint, a film, a total mount, or a thin section.

The process of manufacturing histological preparations for microscopic studies includes the following main stages: 1) taking material and fixing it; 2) material compaction; 3) preparation of sections; 4) staining or contrasting sections; 5) conclusion of sections.

For staining, special histological dyes with different pH values are used: acidic, neutral and basic. The structures stained by them are, respectively, called oxyphilic, neutrophilic (heterophilic) and basophilic.

What methods does histological science use? They are quite numerous and varied:

Microscoping.

Light microscopy. Modern microscopes have high-resolution capabilities. Resolution is determined by the smallest distance (d) between two adjacent points that can be seen separately. This distance depends on the light wavelength (λ) and is expressed by the formula: d = 1/2 λ.

The minimum wavelength of the visible part of the spectrum is 0.4 microns. Consequently, the resolution of a light microscope is 0.2 μm, and the total magnification reaches 2500 times.

Ultraviolet microscopy . The wavelength of ultraviolet light is 0.2 microns, therefore, the resolution of an ultraviolet microscope is 0.1 microns, but since ultraviolet radiation is invisible, a fluorescent screen is needed to observe the object under study.

Fluorescence (luminescence) microscopy. Short-wave (invisible) radiation, absorbed by a number of substances, excites their electrons, which emit light with a longer wavelength, becoming the visible part of the spectrum. In this way, we achieve an increase in the resolution of the microscope.

Phase contrast microscopy allows you to emit unpainted objects.

Polarization microscopy used to study the architectonics of histological structures, for example, collagen fiber.

Electron microscopy makes it possible to study objects magnified tens of thousands of times.

Microphotography and microcinema . These methods make it possible to study fixed objects in photographs and living microscopic objects in motion.

Methods of qualitative and quantitative research.

Histo –and cytochemistry , including quantitative, allows for a qualitative analysis of the objects under study at the tissue, cellular and subcellular levels.

Cytospectrophotometry It makes it possible to study the quantitative content of certain biological substances in cells and tissues based on the absorption of light of a certain wavelength by the dye associated with them.

Differential centrifugation allows you to separate the contents of cells that differ in their mass.

Radiography It is based on the inclusion of a radioactive label (for example, radioactive iodine, H³-thymidine, etc.) in the metabolic process.

Morphometry allows you to measure the areas and volumes of cells, their nuclei and organelles using eyepieces and object micrometers and special grids.

Application of computers for automatic processing of digital material.

Tissue culture method is the maintenance of the viability and division of cells and tissues outside the body. For this purpose, special containers with a nutrient medium are used, in which all the necessary conditions for the life of cells are created. Using this method, it is possible to study the differentiation and functional formation of cells, the patterns of their malignant degeneration and development of the tumor process, intercellular interaction, damage to cells and tissues by viruses and microorganisms, the effect of drugs on metabolic processes in cells and tissues, etc.

Intravital (vital) staining used to study the phenomena of phagocytosis and activity of macrophages, filtration capacity of renal tubules, etc.

Tissue transplantation method. This method is used to study the behavior of cells and their morphofunctional state when they are transplanted into another organism. For example, this method is used to maintain the life of animals exposed to lethal doses of radiation.

Micromanipulation. This method has been used in molecular biology, genetic engineering, as well as in cloning, when using a micromanipulator the nucleus is removed from an egg with a haploid set of chromosomes and the nucleus of a somatic cell with a diploid set of chromosomes is transplanted into it.