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Histology - (Greek "histos" - tissue, logis - teaching) This is the science of the structure, development and vital activity of tissues of multicellular organisms and humans. The objects that are the subject of this science are inaccessible to the naked eye. Therefore, the history of histology is closely related to the history of the creation of such devices that allow you to study the smallest objects with the naked eye. 2
The course of histology is conventionally divided into the following sections: n 1. Cytology - the science of the cell. n 2. Embryology is the science of development, from inception to complete formation of an organism. n 3. General histology - the science of general patterns inherent in tissues. n 4. Private histology - studies the structure, development of organs and systems.
CYTOLOGY - (Greek κύτος "cell" and λόγος - "teaching", "science") n Section 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 provided by connecting two lenses. 1665 year. Robert Hooke first used the term cell. 1650-1700 years. Anthony van Leeuwenhoek was the first to describe bacteria and other microorganisms. 1700-1800 years. Many new descriptions and drawings of various fabrics, mainly vegetable, have been published. In 1827, Karl Baer discovered the egg cell in mammals. 1831-1833 years. Robert Brown described the nucleus in plant cells. 1838-1839 years. Botanist Mathias Schleiden and zoologist Theodor Schwann combined the ideas of different scientists and formulated the cellular theory, which postulated that the cell is the basic unit of structure and function in living organisms. 1855 year. Rudolf Virchow showed that all cells are formed as a result of cell division.
The history of the creation of the cell theory 1665. Examining a section of a cork under a microscope, the English scientist, physicist Robert Hooke discovered that it consists of cells separated by partitions. He called these cells "cells"
The history of the creation of the cell theory In the 17th century, Leeuwenhoek designed a microscope and opened the door for people to the microcosm. A variety of ciliates, rotifers and other tiny animals flashed before the eyes of the astonished researchers. It turned out that they are everywhere - these tiny organisms: in water, manure, air and dust, in the ground and gutters, in rotting animal and plant waste.
The history of the creation of the cell theory 1831-1833 years. Robert Brown described the nucleus in plant cells. In 1838 the German botanist M. Schleiden drew attention to the nucleus and considered it the originator of the cell. According to Schleiden, the nucleolus condenses from the granular substance, around which the nucleus is formed, and around the nucleus - the cell, and the nucleus may disappear during the formation of the cell.
The history of the creation of the cell theory German zoologist T. Schwann showed that animal tissues also consist of cells. He created the theory that nucleated cells represent 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. A cell is an elementary structural unit of the structure of all living things; 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 shell. (Error!)
The history of the creation of the cell theory In 1855, the German physician R. Virchow made a generalization: a cell can arise only from a previous cell. This led to the realization that the growth and development of organisms are 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 Karl Baer Back in 1827, Karl Baer discovered an egg in mammals, 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 a unit of development.
The history of the creation of the cell theory 1865 The laws of heredity are published (G. Mendel). 1868 Nucleic acids discovered (F. Misher) 1873 Chromosomes were discovered (F. Schneider) 1874 Mitosis in plant cells was discovered (ID Chistyakov) 1878 Mitotic division of animal cells was discovered (V. Fleming, P I. Peremezhko) 1879 Fleming - the behavior of chromosomes during division. 1882 Meiosis in animal cells was discovered (W. Fleming) 1883 It was shown that the number of chromosomes in germ cells is two times less than in somatic cells (E. Van Beneden) 1887 Meiosis in plant cells is discovered (E. Strasburger ) 1898 Golgi discovered the mesh apparatus of the cell, the Golgi apparatus. 1914 The chromosomal theory of heredity is formulated (T. Morgan). 1924 The natural-scientific theory of the origin of life on Earth is published (A.I. Oparin). 1953 The concept of the structure of DNA is formulated and its model is created (D. Watson and F. Crick). 1961 The nature and properties of the genetic code are determined (F. Crick, L. Barnet, S. Benner).
The main provisions of modern cell theory 1. A cell is an elementary living system, a unit of structure, life, reproduction and individual development organisms. 2. The cells of all living organisms are homologous, unified in structure and origin. 3. Cell formation. New cells only arise 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. The cellular organization arose at the dawn of life and went a long way of evolutionary development from nuclear-free forms (prokaryotes) to nuclear (eukaryotes).
METHODS OF MICROSCOPING HISTOLOGICAL PREPARATIONS 1. Light microscopy. 2. Ultraviolet microscopy. 3. Fluorescent (luminescent) microscopy. 4. Phase contrast microscopy. 5. Dark field microscopy. 6. Interference microscopy 7. Polarizing 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 an eyepiece. The mirror, condenser and diaphragm direct the light flux and dim the object's illumination. The mechanical part of the microscope includes: a tripod, a stage, macro- and micrometric screws, a tube holder. eighteen
Special methods of microscopy: - phase contrast microscope - (for studying live unpainted objects) - microscopy allows you to study living and unpainted objects. When light passes through colored objects, the amplitude of the light wave changes, and when light passes through uncolored objects, the phase of the light wave changes, which is used to obtain a high-contrast image in phase contrast and interference microscopy. - dark-field microscope (for studying live 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 looks like it is lit 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 luminescent microscopy (for studying living unpainted objects) microscopy is used to observe fluorescent (luminescent) objects. In a fluorescent microscope, light from a powerful source passes through two filters. One filter blocks light in front of the sample and transmits light of the wavelength that excites the fluorescence of the sample. Another filter transmits light of the wavelength emitted by the fluorescent object. Thus, fluorescent objects absorb light at one wavelength and emit in a different region of the spectrum. - ultraviolet ability of m-pa) mic-p (increases the resolution - polarizing mic-p (for examining objects with an orderly arrangement of molecules - skeleton musk-ra, collagen fibers, etc.) microscopy - image formation of uncolored anisotropic structures ( e.g. collagen fibers and myofibrils) 20
Special microscopy methods - interference microscopy (for determining the dry residue in cells, determining the thickness of objects) - microscopy combines the principles of phase contrast and polarization microscopy and is used to obtain a contrast image of unstained objects. Special interference optics (Nomarski optics) have found application in differential interference contrast microscopes. C. Electron microscopy: -transmission (study of objects in the transmission) -scanning (study of the surface of objects) Theoretically, the resolution of the transmission EM is 0.002 nm. The real resolution of modern microscopes is approaching 0.1 nm. For biological objects, EM resolution in practice is 2 nm. 21
Special Microscopic Methods A transmission electron microscope consists of a column through which electrons emitted by a cathode filament pass in a vacuum. An electron beam focused by ring magnets passes through the prepared sample. The nature of the scattering of electrons depends on the density of the sample. The 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 an object under study. The chipping method (freezing-chipping) is used to study internal structure cell membranes. The cells are frozen at liquid nitrogen temperature in the presence of a cryoprotectant and used for making chips. 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 examined in scanning EM. 22
Special (non-microscopic) methods: 1. Cyto- or histochemistry - the essence lies in the use of strictly specific chemical reactions with a light end product in cells and tissues to determine the amount of various substances (proteins, enzymes, fats, carbohydrates, etc.). Can be applied at the level of a light or electron microscope. 2. Cytophotometry - the method is used in combination with 1 and makes it possible to quantify the 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 involved in metabolic processes in cells. Localization, further movements of these substances in the organs are determined on histopreparations 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, to study the molecular structure of biological micro-objects. 24 5. Morphometry - measurement of the size of biol. structures at the cellular and subcellular level.
Special (non-microscopic) methods 6. Microurgy - carrying out very delicate operations with a micromanipulator under a microscope (transplanting nuclei, introducing various substances into cells, measuring biopotentials, etc.) 6. The method of culturing cells and tissues - in nutrient media or in diffusion chambers, implanted in various tissues of the body. 7. Ultracentrofugation - fractionation of cells or subcellular structures by centrifugation in solutions of various densities. 8. Experimental method. 9. Method of tissue and organ transplantation. 25
Fixation preserves the structure of cells, tissues and organs, prevents bacterial contamination and enzymatic digestion, stabilizes macromolecules by chemically linking them. 32
Fixing liquid formalin, alcohols, glutaraldehyde - The most common fixatives; Cryofixation - Instant freezing of samples in liquid nitrogen (- 196 ° С) provides the best preservation of structures; Lyophilization - small pieces of tissue are quickly frozen, stopping metabolic processes. Dehydration - the standard procedure for removing water is dehydration in alcohols, increasing strength (from 70 to 60%). Filling - makes the fabric strong, prevents it from crushing and creasing during cutting, makes it possible to obtain cuts of standard thickness. The most common embedding medium is paraffin wax. Also used are celloidin, plastic media and resins. 33
Dehydration prepares the fixed tissue for embedding media to enter. Living tissue water, as well as the water of fixative mixtures (most fixatives - aqueous solutions) after fixation must be completely removed. Standard procedure removal of water - dehydration in alcohols increasing from 60 ° to 100 ° strength. 34
Pouring is a necessary procedure prior to preparing the slices. Pouring makes the fabric strong, prevents it from crushing and wrinkling when cutting, and makes it possible to obtain thin cuts of standard thickness. The most common embedding medium is paraffin wax. Celloidin, plastic media and resins are also used. 35
Rotary microtome. 40 n Blocks containing a piece of organ are fixed 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 of staining histosections: n Nuclear (basic): n hematoxylin - stains n n n n nuclei blue; iron hematoxylin; azure II (in purple); carmine (in red); safranin (in red); methyl blue (in blue); toluidine (in blue); thionine (in blue). n Cytoplasmic - (acidic): n eosin - pink; n erythrosine; n orange "G"; n sour fuchsin - red; n picric acid - to yellow; n Congo - red - into red 44
SPECIAL Histosection staining methods n Sudan III - orange staining of lipids and fats; n osmic acid - black color of lipids and fats; n orsein - brown color of elastic fibers; n silver nitrate - impregnation of nerve elements in a dark brown color. 45
Cell structures: n OXYPHILY n the ability to stain pink with acidic dyes n Basophilia n the ability to stain blue with basic dyes n Neutrophilia - n the ability to stain purple with acidic and basic dyes. 47
1
Cell n is an elementary living system consisting of cytoplasm, nucleus, membrane and is the basis for the development, structure and life of animals and plant organisms.
Glycocalyx is a supramembrane 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: - dividing; - active and passive transport of substances in both directions; - receptor functions; -contact with neighboring cells.
In modern histology, cytology and embryology, a variety of research methods are used, which make it possible to comprehensively study the processes of development, structure and function of cells, tissues and organs.
The main stages of cytological and histological analysis are the choice of the object of study, its preparation for examination in a microscope, the use of microscopic methods, as well as the qualitative and quantitative analysis of images.
The objects of research are living and dead (fixed) cells and tissues, and their images obtained in light and electron microscopes.
The main object of research is histological preparations made from fixed structures. The drug can be smear(for example, a smear of blood, bone marrow, saliva, cerebrospinal fluid, etc.), imprint(e.g. spleen, thymus, liver), film tissue (eg, connective or peritoneum, pleura, pia mater), thin slice... Most often, a section of tissue or organ is used for examination. Histological specimens can be studied without special treatment. For example, a prepared blood smear, print, film, or section of an organ can be viewed immediately 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: fixed, enclosed in a solid medium and colored.
The process of making a histological specimen for light and electron microscopy includes the following main steps:
- 1. taking the material and fixing it,
- 2.sealing material,
- 3.preparation of slices,
- 4. staining or contrasting sections.
For light microscopy, one more stage is required - the conclusion of the sections in balm or other transparent media.
Fixation ensures the prevention of decomposition processes, which contributes to the preservation of 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 fixing mixtures), or subjected to heat treatment. Under the action of the fixative, complex physicochemical changes occur in tissues and organs. The most important 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 compaction and a decrease in the volume of the pieces, as well as to an improvement in the subsequent staining of cells and tissues.
Sealing material required 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 piece freezing method, for example, in liquid carbon dioxide.
Preparation of slices occurs on special devices - microtomes(for light microscopy) and ultramicrotomes(for electron microscopy). See link - slicing devices.
Dyeing sections (in light microscopy) or their sputtering with metal salts (in electron microscopy) are used to increase the contrast of the image of individual structures when examining them in a microscope. Methods for staining histological structures are very diverse and are selected depending on the objectives of the study. See forum histological techniques.
Histological dyes (by chemical nature) are subdivided into acidic, basic and neutral. The most commonly used dye can be cited as an example. hematoxylin, which stains the nuclei of cells in purple, and an acidic dye - eosin, staining the cytoplasm in a pink-yellow color. The selective affinity of structures for certain dyes is due to their chemical composition and physical properties... Structures that stain well with acidic dyes are called oxyphilic, and colored with the main ones - basophilic... For example, the cytoplasm of cells is most often stained oxyphilically, and cell nuclei are stained basophilically.
Structures that accept both acidic and basic dyes are neutrophilic (heterophilic). Colored preparations are usually dehydrated in alcohols of increasing strength and clarified in xylene, benzene, toluene, or some oils. For long-term preservation, a dehydrated histological section is enclosed between a microscope and a cover slip in Canadian balm or other substances. The finished histological specimen can be used for examination under a microscope for many years.
For electron microscopy, sections obtained on an ultramicrotome are placed on special grids, contrasted with salts of uranium, lead and other metals, after which they are examined in a microscope and photographed. The obtained micrographs serve as an object of study along with histological preparations.
MINISTRY OF EDUCATION AND SCIENCE OF THE RUSSIAN FEDERATION
FSBEI HPE "SAKHALIN STATE UNIVERSITY"
FACULTY OF NATURAL SCIENCES
DEPARTMENT OF ECOLOGY AND NATURE MANAGEMENT
TEST
in the discipline "Histology and embryology of fish
on the topic "Research methods in histology"
2nd year student
Terekhov Stepan Sergeevich
Supervisor,
Art. Lecturer at the Department of Ecology and Nature Management
A.V. Boyko
Yuzhno-Sakhalinsk
Introduction
Histology is the science of the peculiarities of the organization, functions and development of tissues and the tissue structure of organs. The main object of the study of histology is tissues, which are phylogenetically formed, topographically and functionally related cellular systems and their derivatives, from which organs are formed. In order to understand how it all works and works, mankind needed to go through a lot, starting with the lenses of Anthony Vann Leeuwenhoek, when the beginning of the study of all living things, which had not been seen before, was laid. To modern methods of study, include the main one, microscopy.
Purpose of the work: To consider research methods in histology, to learn how to work with histological preparations and to understand the process of studying tissues by various methods of microscopy.
1. Research methods in histology, the basis
Depending on the object of study, histology is divided into normal (studies the tissues of a healthy body) and pathological (pathological histology), which examines changes in tissues in diseases and injuries (it is usually considered as a section of pathological anatomy). Due to the specifics of the object and research methods, neurohistology is distinguished, as well as the doctrine of blood and hematopoiesis, which has become theoretical basis hematology. In addition, a number of areas in histology are distinguished - descriptive histology (description of tissues), comparative histology (comparison of tissues of various animal species), evolutionary histology (patterns of tissue development in phylogenesis), ecological histology (studies tissues in connection with the impact of environmental conditions), experimental histology. In histology, numerous research methods are used - microscopy, experimental, tissue culture (Afanasyev 1989).
The main subject of the study of histology is the complexes of cells that make up tissues, in their interaction with each other and with intermediate media. As part of morphology, histology is closely related to cytology, anatomy, and embryology. The methodological basis of histology is cell theory and evolutionary teaching. Histology is usually divided into general (studies the general patterns of development, structure and function of tissues) and private (studies the microscopic structure of individual organs and systems of the body). Special sections of histology are histochemistry (tissue chemistry) and histophysiology (mechanisms of tissue activity) (Yushkantseva 2006).
2. Research methods
Research methods in histology include the preparation of histological preparations with their subsequent study using a light or electron microscope. Histological preparations are smears, organ prints, thin sections of pieces of organs, possibly stained with a special dye, placed on a microscope slide, enclosed in a preservative medium and covered with a cover glass.
In modern histology, cytology and embryology, a variety of research methods are used, which make it possible to comprehensively study the processes of development, structure and function of cells, tissues and organs.
The main stages of histological analysis are the choice of the object of study, its preparation for examination in a microscope, the use of microscopic methods, as well as the qualitative and quantitative analysis of images.
The objects of research are living and dead (fixed) cells and tissues, and their images obtained in light and electron microscopes.
The main object of the study is histological preparations prepared from fixed structures. The drug can be a smear (for example, a smear of blood, bone marrow, saliva, cerebrospinal fluid, etc.), an imprint (for example, spleen, thymus, liver), a film of tissue (for example, connective or peritoneum, pleura, pia mater) , thin section. Most often, a section of tissue or organ is used for examination. Histological specimens can be studied without special treatment. For example, a prepared blood smear, print, film, or section of an organ can be viewed immediately 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: fixed, enclosed in a solid medium and colored (Yurina 1999).
The manufacturing process of a histological specimen for light and electron microscopy includes the following main stages:
taking material and fixing it,
compaction of material,
preparation of slices,
staining or contrasting sections.
For light microscopy, one more stage is required - the conclusion of the sections in balm or other transparent media.
Fixation ensures the prevention of decomposition processes, which contributes to the preservation of 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 fixing mixtures), or subjected to heat treatment. Under the action of the fixative, complex physicochemical changes occur in tissues and organs. The most important 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 compaction and a decrease in the volume of the pieces, as well as to an improvement in the subsequent staining of cells and tissues.
Compaction of the material required 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 piece freezing method, 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 spraying them with metal salts (in electron microscopy) is used to increase the contrast of the image 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 dyes (by chemical nature) are subdivided into acidic, basic and neutral. As an example, the most commonly used dye, hematoxylin, which stains cell nuclei in a purple color, and an acidic dye, eosin, which stains the cytoplasm in a pink-yellow color, can be cited. The selective affinity of structures for certain dyes is due to their chemical composition and physical properties. Structures that stain well with acidic dyes are called oxyphilic, and those that stain with basic ones are called basophilic. For example, the cytoplasm of cells is most often stained oxyphilically, and cell nuclei are stained basophilically.
Structures that accept both acidic and basic dyes are neutrophilic (heterophilic). Colored preparations are usually dehydrated in alcohols of increasing strength and clarified in xylene, benzene, toluene, or some oils. For long-term preservation, a dehydrated histological section is enclosed between a microscope and a cover slip in Canadian balm or other substances. The finished histological specimen can be used for examination under a microscope for many years (Yurina, Radostina 1999).
In electron microscopy, sections obtained on an ultramicrotome are placed on special grids, contrasted with salts of uranium, lead and other metals, after which they are viewed in a microscope and photographed. The obtained micrographs serve as an object of study along with histological preparations.
3. Methods of microscopy of histological preparations
Microscopy can be light (using a light microscope) and electronic (using an electron microscope). Light microscopy can be performed in transmitted light, when light passes through a thin transparent histological specimen, or in reflected light, when examining, for example, a thick or opaque object. Similarly, electron microscopy can be transmission, when an electron beam passes through an ultrathin section of interest, or scanning, or scanning, when an electron beam is reflected from the surface of an object under study. In the first case, the electron microscope is called transmission (TEM), and in the second, scanning (SEM).
1 Light microscopy
Microscopy, the main method for studying drugs, has been used in biology for over 300 years. Modern microscopes are a variety of complex optical systems with high resolution and allow you to study very fine details of the structure of cells and tissues. The size of the smallest structure that can be seen in a microscope is determined by the smallest resolvable distance (d0). It mainly depends on the wavelength of the light. ?, and this dependence is approximately expressed by the formula d0 = ? / 2. Thus, the shorter the light wavelength, the smaller the resolved distance and the smaller the structures can be seen in the preparation (ie, the higher the "resolution" of the microscope). Magnification of a microscope refers to its optical system and is expressed as the product of the objective and eyepiece magnifications. However, the "resolution" of the microscope depends on the characteristics of the objective and does not depend on the eyepiece.
To study histological preparations, conventional light microscopes of various brands are often used, when natural or artificial light is used as a source of illumination. The minimum wavelength of the visible part of the light spectrum corresponds to approximately 0.4 µm (violet spectrum). Therefore, for a conventional light microscope, the resolvable distance is approximately 0.2 µm, and the total magnification (product of the objective magnification times the eyepiece magnification) reaches 2000 times.
Units of measurement used in histology: For the measurement of structures in light microscopy, micrometers are mainly used: 1 µm is 0.001 mm; electron microscopy uses nanometers: 1 nm is 0.001 microns (Yushkantseva 2006)
2 Ultraviolet microscopy
This is a type of light microscopy. An ultraviolet microscope uses shorter ultraviolet rays with a wavelength of about 0.2 μm. The permitted distance here is approximately 0.1 µm. An image, which is invisible to the eye, obtained in ultraviolet rays, is converted into a visible one by recording on a photographic plate or by using special devices (since a luminescent screen, or an image converter) (Yurina 1999)
3 Fluorescence (luminescence) microscopy
The phenomenon of fluorescence consists in the fact that atoms and molecules of a number of substances, absorbing short-wave rays, pass into an excited state. The reverse transition from an excited state to a normal one occurs with the emission of light, but with a different, longer wavelength. In a fluorescence microscope, mercury or ultra-high pressure xenon lamps are used as light sources for exciting fluorescence, which have high brightness in the spectral region 0.25-0.4 microns (near ultraviolet rays) and 0.4-0.5 microns (blue-violet rays). The light wavelength of the induced fluorescence 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 fluorescence light. 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. Secondary fluorescence occurs when drugs are processed with special dyes - fluorochromes<#"justify">Artishevsky A.A. Histology with the technique of histological
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The main Research objects are histological preparations, and the main research method is microscopy.
The histological specimen should be sufficiently transparent (thin) and contrasting. It is made from both living and dead (fixed) structures. The preparation can be a cell suspension, smear, imprint, film, total preparation and thin section.
The process of making histological preparations for microscopic examinations includes the following main stages: 1) taking the material and fixing it; 2) material compaction; 3) preparation of slices; 4) staining or contrasting sections; 5) the conclusion of the sections.
For staining, special histological dyes with different pH values are used: acidic, neutral and basic. The structures that they color are, respectively, called oxyphilic, neutrophilic (heterophilic) and basophilic.
What methods does histological science use? They are quite numerous and varied:
Microscopy.
Light microscopy. Modern microscopes have high-resolution capabilities. Resolution is defined as the smallest distance (d) between two adjacent points that can be seen separately. This distance depends on the length of the light wave (λ) and is expressed by the formula: d = 1/2 λ.
The minimum wavelength of the visible part of the spectrum is 0.4 µm. Therefore, the resolution of the 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 the ultraviolet microscope is 0.1 microns, but since ultraviolet radiation is invisible, a luminescent screen is required 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. Thus, they seek to increase the resolution of the microscope.
Phase contrast microscopy allows you to emit unpainted objects.
Polarizing microscopy It is used to study the architectonics of histological structures, for example, collagen fibers.
Electron microscopy makes it possible to study objects magnified tens of thousands of times.
Microphotography and microcinema ... These methods allow studying 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 bound by them.
Differential centrifugation allows you to separate the contents of cells that differ in their mass.
Radiography 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 an eyepiece - and object micrometers and special grids.
Computer application 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, special containers with a nutrient medium are used, in which all the necessary conditions for the vital activity of cells are created. Using this method, one can study the differentiation and functional formation of cells, the patterns of their malignant transformation and the development of a 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 is used to study the phenomena of phagocytosis and the activity of macrophages, the filtration capacity of the renal tubules, etc.
Tissue transplantation method... This method is used to study the behavior of cells and their morphological and functional 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 found application 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.
This method is basic, or classic. For their manufacture, the object of study is immersed in fixing liquids, which denature proteins and stabilize certain structures and compounds to be studied. The most common fixative is formalin. It cross-links proteins with methylene bridges, causing them to denature. After fixing and rinsing in water, the object of study can be cut into thin plates, after freezing it on a special freezing microtome - a device with which histological sections are made. To freeze an object, liquid carbon dioxide or an electric freezing unit is most often used. However, with this method of processing the material, rather thick histological sections are obtained. To make thinner sections, up to 2 microns thick, the object of study must be impregnated with a substance that would make it denser. Such substances are paraffin, gelatin and celloidin. After fixing and washing, the object is sequentially immersed in alcohols of increasing concentration - from 50 to 100 degrees for its dehydration and impregnated with gelatin, paraffin or celloidin. Once the object has been impregnated and compacted, it can be cut with a microtome.The histological sections are then stained with specially selected dyes, most of which selectively stain the structural components of cells and tissues. All dyes can be divided into three groups:
After staining, histological sections are quickly dehydrated in alcohols, clarified in xylene or toluene, transferred to a glass slide, covered with a thin layer of Canadian balsam or polystyrene and covered with a cover glass. Balm, polystyrene and glass have the same refractive index of light, and light rays are minimally scattered when passing through the preparation.
