In modern histology, cytology and embryology, a variety of research methods are used 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, preparation for studying in the microscope, the use of microscopic methods, as well as high-quality and quantitative analysis of images.
The objects of the study serve live and dead (fixed) cells and fabrics, and their images obtained in light and electron microscopes.
The main object of the study is histological preparationsFixed structures made from fixed structures. The drug may be smear(for example, blood smear, bone marrow, saliva, spinal fluid, etc.), printed (for example, spleen, thymus, liver), film from fabric (for example, connective or peritoneum, pleura, soft cerebral shell), thin slice. Most often for studying uses a selection of a tissue or organ. Histological preparations can be studied without special processing. For example, a cooked blood smear, an imprint, film or cutting of the organ can immediately be considered under a microscope. But due to the fact that structures have a weak contrast, they are poorly revealed in the usual light microscope and requires the use of special microscopes (phase-contrast, etc.). Therefore, specially treated preparations are used more often: fixed, enclosed in solid medium and painted.
The process of manufacturing a histological preparation For light and electron microscopy, the following main steps include:
- 1. Taking the material and its fixation,
- 2. Material seal
- 3. Preparation of sections,
- 4. Staining or contrasting sections.
For light microscopy, another stage is needed - conclusion of sections in balm or other transparent environments.
Fixation Provides 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 the organ is either immersed in the retainer (alcohol, formalin, solutions of heavy metals salts, osmium acid, special fixing mixtures), or heat treatment. Under the action of the retainer in the tissues and organs, complex physico-chemical changes occur. The most essential of them is the process of irreversible coagulation of proteins, as a result of which the vital activity is terminated, and the structures become dead, fixed. Fixation leads to a seal and decrease in the volume of pieces, as well as to improving the subsequent coloring of cells and tissues.
Seal The material necessary for the preparation of sections is carried out by impregnating the pre-dehydrated material of paraffin, cellidin, organic resins. A faster seal is achieved by using the freezing method of pieces, for example, in liquid carbon dioxide.
Cooking cuts happens on special devices - microtomes (for light microscopy) and ultramicrotomes (for electron microscopy). See link - devices for the manufacture of sections.
Coloring sections (in light microscopy) or spraying them with metal salts (in electron microscopy) are used to increase the contrast of the image of individual structures when viewed by them in the microscope. The coloring methods of histological structures are very diverse and selected depending on the tasks of the study. See Forum histological techniques.
Histological dyes (by chemical nature) divided into acidic, main and neutral. As an example, you can bring the most common dye hematoksilinwhich stains the core cells in purple, and sour dye - eosin, staining cytoplasm in pink-yellow color. The electoral affinity of structures to certain dyes is due to their chemical composition and physical properties. Structures that are well-colored with acidic dyes are called oxyfilic, and staining basic - basophilic. For example, cell cytoplasm is most often stained oxifically, and cell cores are painted basophilic.
Structures that perceive both sour and main dyes are neutrophilic (heterophilic). Painted preparations are usually dehydrated in alcohols of an increasing fortress and enlightened in xylene, benzene, toluene or some oils. For long-term preservation, the dehydrated histothological slice conclude between the substantive and coating glasses in Canadian balm or other substances. The finished histological preparation can be used to study under the microscope for many years.
For electronic microscopy, slices obtained on ultramicrothome are placed on special grids, contrast the salts of uranium, lead and other metals, after which they are browsing in the microscope and photograph. The resulting micrographs serve as an object of study along with histological drugs.
Objects of research divided into:
· Live (cells in a drop of blood, cells in culture and others);
· Dead or fixed, which can be taken from both a living organism (biopsy) and corpses.
In any case, after taking pieces, they are exposed to fixing solutions or freezing. Both scientific and curriculous objectives use fixed objects. Preparations prepared for studying under the microscope are prepared for studying under the microscope, are called histological preparations.
The histological preparation may be in the form of: (thin colored cutting of the organ or tissue; smear on the glass; print on the glass with a fault of the organ; thin film preparation).
The histological preparation of any form must meet the following requirements: (maintain the lifetime state of the structures; it is quite subtle and transparent to study it under a microscope in the transmitted light; be contrasting, that is, studied structures should be clearly defined under the microscope; drugs for light microscopy must be stored for a long time. Used to re-examine.)
These requirements are achieved when preparing the drug.
Research methods:
Light microscopy-Comcoping is the main method of studying drugs - used in biology for more than 300 years. Ultraviolet microscopy - This is a kind of light microscopy. In the ultraviolet microscope, shorter ultraviolet rays with a wavelength of about 0.2 μm are used. Fluorescent (luminescent) microscopy - fluorescence phenomena consist in the fact that atoms and molecules of a series of substances, absorbing short-wave rays, go to the excited state. Phase-contrast microscopy - This method is used to obtain contrast images of transparent and colorless objects invisible in conventional microscopation methods. Electronic microscopy. -The electron microscope uses the electron flow with a shorter than in a light microscope, wavelengths.
The main stages of cytological and histological analysis are the choice of the object of study, preparation for studying in the microscope, the use of microscopic methods, as well as high-quality and quantitative analysis of images.
Most often for studying uses a selection of a tissue or organ. Histological preparations can be studied without special processing. For example, a cooked blood smear, an imprint, film or cutting of the organ can immediately be considered under a microscope. But due to the fact that structures have a weak contrast, they are poorly revealed in the usual light microscope and requires the use of special microscopes (phase-contrast, etc.). Therefore, specially treated preparations are used more often: fixed, enclosed in solid medium and painted.
The process of manufacturing a histological preparation for light and electron microscopy includes the following main steps:
1. Taking the material and its fixation,
2. Material seal
3. Preparation of sections,
4. Staining or contrasting sections.
For light microscopy, another stage is needed - conclusion of sections in balm or other transparent environments.
Fixation ensures the prevention of decomposition processes, which contributes to the preservation of the integrity of the body structures. Male sample is either immersed in the retainer (alcohol, formalin, solutions of heavy metals salts, osmium acid, special fixing mixtures), or exposure to heat treatment
The seal of the material required for the preparation of sections is carried out by impregnating the pre-dehydrated material of paraffin, cellidine, organic resins. A faster seal is achieved by using the freezing method of pieces, for example, in liquid carbon dioxide.
Preparation of sections occurs on special devices - microtomes (for light microscopy) and ultramicrotomes (for electron microscopy).
Staining of 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 by them in the microscope. The coloring methods of histological structures are very diverse and selected depending on the tasks of the study.
Histological dyes (in chemical nature) are divided into acidic, basic and neutral. hematoksilinwhich color coders of cells in purple color and sour dye - eosin, staining cytoplasm in pink-yellow color. The electoral affinity of structures to certain dye is due to their chemical composition and physical properties. Structures, well-colored with acidic dyes, are called oxilic, and painting the main-elevated. For example, cell cytoplasm is most often stained oxifically, and cell cores are painted basophilic.
Structures that perceive both sour and main dyes are neutrophilic (heterophilic). Painted preparations are usually dehydrated in alcohols of an increasing fortress and enlightened in xylene, benzene, toluene or some oils. For long-term preservation, the dehydrated histothological slice conclude between the substantive and coating glasses in Canadian balm or other substances. The finished histological preparation can be used to study under the microscope for many years.
four) . Cell as a structural and functional unit of fabric. Definition. General plan for the structure of eukaryotic cells. Biological cell membranes, their structure, chemical composition and main functions.
The cell is an elementary structural, functional and genetic unit as part of all plant and animal organisms. The structure of the eukaryotic cell:
Cells forming animal and plant fabrics are significantly different in shape, sizes and inner structure. The cells of all types contain two main components, closely interconnected, cytoplasm and core. The kernel is separated from the cytoplasm of a porous membrane and contains nuclear juice, chromatin and nucleolo. The semi-liquid cytoplasm fills the entire cage and is permeated with numerous tubules. Outside it is covered with a cytoplasmic membrane.
Actually the body of the cell and its contents are separated from the external environment or from adjacent elements in multicellular organisms of the plasma membrane. The dust from the plasma membrane is a cell shell or wall, especially well-pronounced in plants. All internal contents of the cell, with the exception of the kernel, is called the cytoplasm. The cytoplasm of eukaryotic cells is not homogeneous in its structure and composition and includes: hyaloplasm, membrane and non-smalculated components. Membrane organelles are represented by two options: single-paved and two-paved. The first includes the organelles of the vacuolar system - the endoplasmic reticulum, the Machinery, Lizosoma, Peroxisoma, and other specialized vacuoles, as well as the plasma membrane. Two-grated organelles include mitochondria and plasts, as well as cell core. Nemembrained organelles belong to ribosomes, cell center of animal cells, as well as elements of the cytoskeleton (microtubule and microfilaments).
The term of the hyaloplasm is the main plasma or matrix of the cytoplasm, denotes a very important part of the cell, its true internal environment. Hyaloplasma is a complex colloid system that includes various biopolymers: proteins, nucleic acids, polysaccharides, etc. It contains enzymes involved in the synthesis of amino acids, nucleotides, fatty acids, in the metabolism of sugars .. The most important role of the hyaloplasma is that this environment unites all cellular structures and provides chemical interaction with each other. A large part of intracellular transport processes is carried out through the hyaloplasm: the transfer of amino acids, fatty acids, nucleotides, sugars. In the hyaloplasm there is a constant flow of ions to the plasma membrane and from it, to mitochondria, kernel and vacuoles. Galoplasm There is a deposition of spare products: glycogen, fat. In cytozole, proteins transported in different areas of cells are synthesized in the ribosomes located there, as well as all proteins of the cell nucleus, most of the proteins of mitochondria and plastids, main proteins peroxysis. The structure of cell membranes.
A total of all cell membranes (plasma, intracellular and membrane organoids) is that they are thin (6-10 nm) layers of lipoprotein nature (lipids in the complex with proteins), closed themselves
The three important principles of the structure of the membrane are exist:
Membranes are not homogeneous. The membranes surrounding intracellular organelles, and the plasma membrane differ in composition. Made the components of the membranes are in a state of continuous movement. The membrane resembles an ever-changing mosaic component of membranes extremely asymmetrical. There is a difference between the outer and inner layers of membranes on the relative number and qualitative composition of lipids. Proteins are located among lipids asymmetrically and have well-distinguishable outside and intracellular sections.
The most important functions of membranes are the following:
Membranes control the composition of the intracellular medium.
Membranes provide and facilitate the intercellular and intracellular information transmission.
Membranes provide fabric formation using intercellular contacts
Chemical composition of the cell.
Cells of living organisms are similar not only in their structure, but also by chemical composition. Similarities in the structure and chemical composition of cells indicate the unity of their origin.
The composition included in the cell substance is divided into organic and inorganic.
1.Norganic substances.
Water is of great importance in the vital cells of the cell. Many cell elements are contained in the form of ions. Most often, cations are found: K +, Na +, Ca2 + Mg2 +, and anions: H2PO4-, CL-, HCO3-.
Mineral salts (for example, calcium phosphate) can be included in the intercellular substance, mollusc shells and ensure the strength of these formations.
2. Organic substances.
Characteristic only for living. Organic compounds are presented in a cell by simple small molecules (amino acids, mono- and oligosaccharides, fatty acid, nitrogenous bases), and macromoleclasses of biopolymers (proteins, lipids, polysaccharides, nucleic acids). Molecules of biopolymers consist of repetitive low molecular weight compounds (monomers
Ministry of Education and Science of the Russian Federation
FGBOU VPO "Sakhalin State University"
Natural scientific faculty
Department of Ecology and Environmental Management
TEST
under the discipline "Histology and embryology of fish
on the topic "Methods of research in histology"
Student 2 courses
Terekhov Stepan Sergeevich
Scientific adviser,
art. Lecturer Department of Ecology and Environmental Management
A.V. Boyko
Yuzhno-Sakhalinsk
Introduction
Histology is the science of the characteristics of the organization, functions and the development of tissues and the tissue structure of the organs. The main object of studying histology is fabrics, which are phylogenetically established, topographically and functionally connected cellular systems and their derivatives, of 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 Basin Levenguka, when it was found to start the study of all the living, not visible earlier. The modern methods of study include the main, microscopation.
Objective: Consider research methods in histology, learn how to work with histological drugs and understand the process of studying tissues different ways Microscopic.
1. Methods of research in histology, the basis
Depending on the object of study, histology is divided into normal (studies tissue of a healthy organism) and pathological (pathogustology), which explores changes in tissues for diseases and damage (it is usually considered as a section of pathological anatomy). Due to the specifics of the object and research methods, they allocate neurogistology, as well as the doctrine of blood and blood formation, which has become theoretical basis of hematology. In addition, there are a number of directions in histology - a descriptive histology (description of tissues), comparative histology (comparison of tissues of various types of animals), evolutionary histology (patterns of tissue development in phylogenesis), environmental histology (studies tissue due to the effects of habitat), experimental Histology. In histology, numerous research methods are used - microscopy, experimental, fabric cultures (Afanasyev 1989).
The main subject of study of histology - cell complexes, component of the tissue, in their interaction with each other and with intermediate media. As part of morphology, histology is closely related to cytology, anatomy, embryology. The methodological basis of histology is a cell theory and evolutionary teaching. Histology is accepted for 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 (chemistry of fabrics) and histophysiology (tissue mechanisms) (Yushkantseva 2006).
2. Research methods
The methods of research in histology include the preparation of histological preparations, followed by their study using a light or electron microscope. Histological preparations are strokes, penprints of organs, thin sections of body pieces, possibly painted with a special dye, placed on a microscope-based glass, enclosed in a preservative environment and covered with coated glass.
In modern histology, cytology and embryology, a variety of research methods are used 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 the study, preparation for studying in the microscope, the use of microscopic methods, as well as high-quality and quantitative analysis of images.
The objects of the study serve live and dead (fixed) cells and fabrics, and their images obtained in light and electron microscopes.
The main object of the study is histological preparations prepared from fixed structures. The drug may be a smear (for example, blood smear, bone marrow, saliva, spinal fluid, etc.), imprint (for example, spleen, thymus, liver), tissue film (for example, connecting or peritoneum, pleura, soft cerebral shell) , thin cut. Most often for studying uses a selection of a tissue or organ. Histological preparations can be studied without special processing. For example, a cooked blood smear, an imprint, film or cutting of the organ can immediately be considered under a microscope. But due to the fact that structures have a weak contrast, they are poorly revealed in the usual light microscope and requires the use of special microscopes (phase-contrast, etc.). Therefore, specially treated drugs are used more often: fixed, enclosed in solid medium and painted (Yurina 1999).
The process of manufacturing a histological preparation for light and electron microscopy includes the following main steps:
taking the material and its fixation,
material seal
cooking cuts
staining or contrasting cuts.
For light microscopy, another stage is needed - conclusion of sections in balm or other transparent environments.
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 the organ is either immersed in the retainer (alcohol, formalin, solutions of heavy metals salts, osmium acid, special fixing mixtures), or heat treatment. Under the action of the retainer in the tissues and organs, complex physico-chemical changes occur. The most essential of them is the process of irreversible coagulation of proteins, as a result of which the vital activity is terminated, and the structures become dead, fixed. Fixation leads to a seal and decrease in the volume of pieces, as well as to improving the subsequent coloring of cells and tissues.
The seal of the material required for the preparation of sections is carried out by impregnating the pre-dehydrated material of paraffin, cellidine, organic resins. A faster seal is achieved by using the freezing method of pieces, for example, in liquid carbon dioxide.
The preparation of sections occurs on special devices - microtomes (for light microscopy) and ultramicrotomes (for electron microscopy).
Staining of 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 by them in the microscope. The coloring methods of histological structures are very diverse and selected depending on the tasks of the study.
Histological dyes (in chemical nature) are divided into acidic, basic and neutral. As an example, the most commonly used hematoxiline dye, which stains the cell kernels in purple color, and the sour dye - the eosin, painting the cytoplasm in pink-yellow color. The electoral affinity of structures to certain dye is due to their chemical composition and physical properties. Structures that are well-colored with acidic dyes are called oxilic, and painting basic - basophilic. For example, cell cytoplasm is most often stained oxifically, and cell cores are painted basophilic.
Structures that perceive both sour and main dyes are neutrophilic (heterophilic). Painted preparations are usually dehydrated in alcohols of an increasing fortress and enlightened in xylene, benzene, toluene or some oils. For long-term preservation, the dehydrated histothological slice conclude between the substantive and coating glasses in Canadian balm or other substances. The finished histological drug can be used to study under the microscope for many years (Yurina, Radotina 1999).
In electron microscopy, sections obtained on ultramicrothome are placed on special meshs, contrast the salts of uranium, lead and other metals, after which they are browsing in the microscope and photograph. The resulting micrographs serve as an object of study along with histological drugs.
3. Methods of microscopation of histological preparations
Microscopy may be light (using a light microscope) Ielectronic (using an electron microscope). Light microscopy can be carried out in the transmitted light when the light passes through a thin transparent histological preparation, or in the reflected light, when they are examined, for example, a thick or opaque object. Similarly, electron microscopy can be a transmission when the electron beam passes through the studied ultra-thin slice, or raster, or scanning, when the electron beam is reflected from the surface of the object under study. In the first case, the electron microscope is called transmission (TEM), and in the second - scanning (SEM).
1 Light microscopy
Microscopation is the main method of studying drugs - used in biology for more than 300 years. Modern microscopes are a variety of complex optical systems that have a high resolution and allowing the study of very thin parts of the structure of cells and tissues. The size of the smallest structure itself, which can be seen in the microscope is determined by the smallest resolution (D0). Basically it depends on the length of the light wave ?, and this dependence is approximately expressed by the formula D0 \u003d ? / 2. Thus, the lower the length of the light wave, the less resolved distance and the smaller the size of the structure can be seen in the drug (that is, above the "resolution" of the microscope). The concept of "an increase in the microscope" refers to its optical system and is expressed in the production of lens and eyepiece. However, the "resolution" of the microscope depends on the characteristics of the lens and does not depend on the eyepiece.
For the study of histological preparations, conventional light microscopes of various brands are more often used when natural or artificial light is used as a light source. The minimum wavelength of the visible part of the light spectrum corresponds to about 0.4 microns (purple spectrum). Therefore, for the conventional light microscope, the resolution is approximately 0.2 μm, and the overall increase (the product of the increase in the lens to increase the eyepiece) reaches 2000 times.
Measurement units used in histology: To measure structures in light microscopy, used mainly micrometers: 1 μm is 0.001 mm; Nanometers are used in electronic microscopy: 1 nm is 0.001 microns (Yushkantseva 2006)
2 ultraviolet microscopy
This is a kind of light microscopy. In the ultraviolet microscope, shorter ultraviolet rays with a wavelength of about 0.2 μm are used. The resolved distance here is approximately 0.1 microns. The image obtained in ultraviolet rays The image is converted to visible by registering on a photoflastic or by applying special devices (because fluorescent screen, or electronic optical converter) (Yurina 1999)
3 Fluorescent (luminescent) microscopy
The fluorescence phenomena consist in the fact that the atoms and molecules of the series of substances, absorbing short-wave rays, go to the excited state. The reverse transition from the excited state into normal occurs with the emission of light, but on the other, the larger wavelength. In a fluorescent microscope, mercury or xenon lamps of ultra-high pressure, having a high brightness in the range of 0.25-0.4 μm (near ultraviolet rays) and 0.4-0.5 μm (blue-purple, are used as sources of light to excite the fluorescence. rays). The light wavelength of the resulting fluorescence is always greater than the wavelength of the exciting light, so they are separated using light filters and study the image of the object only in the light of fluorescence. They distinguish their 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 processing drugs with special dyes - fluorochromas<#"justify">ARTIshevsky, A. A. Histology with histological technology
research (1999)
Antipchuk, Yu. P. Histology with the basics of embryology (1983)
Afanasyev Yu.I., Yurina N.A. - Histology, Cytology and Embryology (1989) "Medicine"
Afanasyev Yu.I., Yurina N.A. - Histology, Cytology and Embryology (2002) "Medicine"
Bykov V.L. Private Histology of Human "Sotis" 1999
Zavarzin A.A., Stroyva O.G. - Comparative histology. Tutorial "SPb" 2000
Kuznetsov S.L., Mushkambarov N.N. - Atlas on histology, cytology and embryology "Mia" 2002
Ryabov, K. P. Histology with the basics of embryology: Tutorial (1990)
Ulumbekov E.G., Chelyshev Yu.A. - Histology. Textbook for universities (1997) "Geotar" 1997
Chelywis Yu.A. - graphic tests in histology, cytology, embryology "KGMU" 2000
Yurina N.A., Radotina A.I. - Workshop on histology, cytology and embryology "UDN" 1999
Yushkantseva S.I., Bykov V.L. - Histology, cytology and embryology. Brief Atlas "P-2" 2006
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Methods of histological research. Modern histology has a wide arsenal of a variety of research methods. All these methods combine the requirement to apply a special device - microscope, and therefore all of them microscopic methods.
Depending on the state of the object under study, these methods are divided into living room (or subject), when living cells, fabrics, organs, and even whole organisms, and post-valve, when they explore dead fixed objects.
The formation of a post-dollar method, or the method of manufacturing a permanent histological preparation, occurred in parallel with the formation of the science of histology in the second half of the XIX century. It is called by the method of classical histology. This method is called histological or microscopic technology, requires a sufficiently difficult training of the object of the study. The latter is the subject for writing special, large enough benefits. A student who begins to study histology, it is necessary to familiarize himself with the basics of manufacturing histological preparations, in order to better understand these drugs and learn to analyze, "read", for it is precisely constant histological drugs are widely used both in the educational process and in scientific research.
The first stage in the manufacture of the drug - obtaining material. Already at this stage, as in all subsequent, unnecessary injury to the object should be avoided. Therefore, cutting a piece of organ or tissue, you need to take sharp scissors or blade, do not compress the tissue with a tweezers. The pieces are taken of small sizes - about 1 cm3 (better than 7x7x3 mm). The material should be fresh, it takes it necessary as soon as possible after the harness of the experimental animal or the death of a person.
The next stage is the fixation of the material, is carried out by immersing the piece taken piece into fixing liquids. The purpose of this stage is the fixing of histological structures and macromolecules in the place and condition in which they were in a lively object. Of course, the clamps determine certain changes in the lifetime state of the structures, but it is possible to reduce these changes to a minimum by the selection of special fixed agents. Locks are alcohols (ethyl, methyl), formalin solutions, salts of heavy metals, acids (acetic, picric, osmis). The various complex fixing mixtures are used more often, including these components in various ratios.
Third stage - dehydration of fixed material. For this, alcohols of different concentrations are used, gradually increase from 50-70 to 100 degrees. Dehydration is necessary for the next step - the object seal is carried out in paraffin, codifinities, synthetic resins. The overwhelming majority of these water substances are not mixed, and therefore it is necessary to carefully remove water from the fabric to impregnating material, and then soak it with xylene (toluene, benzene), that is, the substance, well dissolves paraffin, and also mixes with 100-degree ethyl alcohol. . After the impregnation of the object with liquid paraffin at a temperature of 55-5b ° C, it is solid at room temperature along with paraffin in special molds. So receive a paraffin unit. This procedure is called Pouring. The accelerated seal is achieved by freezing pieces of tissue with dry ice (carbon dioxide) or liquid nitrogen, however the structure of the studied histological objects is preserved in this case worse.
The material seal allows producing thin (5-7 μm thick), beerons (0.5-1 μm) Sections used for light microscopy for electron microscopy use ultra-thin sections (0.05-0.2 μM). Production of sections are carried out on special microtome instruments (for light microscopy) and ultramicrotomes (for electron microscopy). Thin, pevaton or ultra-thin sections are transparent for light rays or electron beam objects and make it possible to study them under the appropriate microscopes. In order to distinguish the structural details of the object, most of which do not have natural contrast, the resulting cut must be painted (for studying under a light microscope) or contrast (for electron microscopy).
In histology there are many methods of color of drugs and a lot of different dyes apply from the purpose of the study. Histological dyes on origin are divided into vegetable, animals and synthetic (aniline). An example of vegetable dyes is hematoxylin, obtained from the cords of a campash tree growing in Central America, and animal carmine, which is obtained from insects - Koshenyli. The absolute majority of dyes are synthetic - Eosin, Fuchsin, Azur.
The most important is the classification of histological dyes in chemical properties, since it is based on a number of concepts and terms that will meet throughout the course. So, according to chemical properties, histological dyes are divided into acidic, basic and neutral. The properties of acidic dyes are negotiated by groups-coxy, -HS03, -N2R03, these are the so-called anionic dyes. Sour dyes are painted cytoplasm cells, they are called cytoplasmic. An example of such dyes may be eosin (gives bright pink color), light green (gives green). Histological structures that can be painted with acidic dyes are called oxyfly (acidophilic, eosinophilic). This, for example, cytoplasmic granules of eosinophilic leukocytes, collagen fibers, etc.
The main dyes are cationic, the overwhelming majority of them in the composition of the molecule should have positively charged nitrogen atoms. Named dyes selectively color coders of cells and therefore they are called nuclear. An example is hematoxiline (colors in blue-purple color), carmine (in light red), saffranin (dark red), Azur II (in blue). Histological structures that can be painted with major dyes are called basophilic. These are granules in the cytoplasm of basophilic leukocytes, cell cores, etc.
Neutral dyes are formed in the case of a message aqueous solutions sour and main dyes, for example, eosine-acid methylene blue. In addition, neutral coloring mixtures should be distinguished when there are basic and sour dyes in the solution. Structures that simultaneously perceive both basic and acidic dyes are called neutro profile, or polychromatic films. An example is the granules of neutor profile leukocytes, the cytoplasm of polychromatophilic eryto-blasts. The ability of histological structures to change the color of the main dye is indicated by the term metacromasia. Metahromatic painted graininess of basophil leukocytes, an intercellular material of cartilage tissue, etc .. Preparations are usually painted, combining one sour and one main dye, which allows you to identify the kernel, cytoplasm and all basophilic and oximal structures. One of the most frequently combined dyes is hematoxylin and eosin.
In addition to acidic, basic and neutral dyes there are special used to identify certain substances or structures. For example, Sudan III stains fat substances into an orange color, and Orsyin - elastic fibers in brown.
Painted preparations usually dehydrates in alcohols, enlighten into xylene and poured by a thin layer of Canadian balm, covered with coating glass. After drying, the balm is obtained by a permanent drug, which can be used for a long time. For electron microscopy, the sections obtained on ultramicrotoms are placed on special messes, they contrast the salts of uranium or lead, browsed through the microscope and photograph. The resulting micrographs are an object of study simultaneously with histological preparations.
In addition to the described thin cuts, there are other types of histological preparations that are used much less frequently, only in some cases. These include strokes (blood, bone marrow, saliva, etc.), prints (liver, thymus, bladder mucous membrane), films ( connective tissue, pleura, peritoneum, soft cerebral shell), total drugs (embryos early stages development, sex cells).
Living rooms (fisted) Methods of study of cells or tissues make it possible to obtain information about how the processes of vital activity occur, trace the movement, separation, growth, cell interaction, their reaction to the action of various factors. Printermed research methods are the necessary addition to the data that is obtained by the method of classical histology on the structure of cells, tissues. Lifting studies are carried out in a living organism, that is, in vivo. For the study of living cells, methods of congratulatory and supplitular color are used. For this, special, non-toxic for living fabrics, dyes. With a congratulatory color, the dye is introduced into the body of a living animal and it selectively stains certain cells. So, the cells of the macrophage of the system are investigated under the condition of administration of tripan blue or lithium carmine. Supporter is not coloring - it is painting living cells, isolated from the body. So the lysosomes are found (neutral red dye), mitochondria (Janus green), blood reticulocytes (Blue diamond-cresil).
For congratulatory or subject, as well as post-high studies, unpainted histological objects use a number of special methods of light microscopy - phase-conventional, darkness, fluorescent.
The phase contrast method ensures the necessary contrast of the unpainted structures under study due to the special ring diaphragm, is placed in the condenser, and the so-called phase plate contained in the lens. This design of the light microscope optics allows you to convert phase changes in the light passing through the object into amplitude, which notices the eye as a change in brightness.
Exists many research methods, among which the following are allocated: monitoring alive germs with the use of film and video filming (used mainly in the experiment). For this, a special micrograph is used, connecting with the thermocamera in which the embryo develops. When studying the development of chicken embryo, for example, in the shell, you do the window, which is closed with a transparent plate. This method allowed to trace and clarify the dynamics of changes in the shape and size of the embryos in the development process.
Method of studying fixed slices embryos using light and electron microscopy, historadioavatography, histo and immunocytochemistry. These methods allow to analyze tissue and intracellular changes in the dynamics of the development of parts of the embryo. With the help of histo and immunocythochemical methods, biochemical processes occurring in embryos cells are investigated - synthesis of DNA, RNA, proteins, specific receptor proteins, etc. With the use of these methods, important information on cell and tissue differentiation in the development of embryos and fruits was obtained.
Marking methodSupposed in 1925 V. Vogt (1888-1941), allows to study the movement of cells in a developing embryo. For this, non-toxic markers for embryos are used (for example, neutral red, wood coal particles), as well as antibodies to certain proteins. When using antibodies, their property is used to connect with fluorescent dye and germ proteins. With the help of fluorescent microscopy, the dye distribution is traced and the dynamics of protein synthesis in the developing knob's tissues is investigated.
Methods of microsurgery Developed at the beginning of the 20th century by representatives of the Spemen school (1869-1941). They included: removing animal eggs shells, transplanting parts of one embryo to another, etc. These methods are also used to study the effects of destruction (for example, using a laser beam) parts of the embryo or its individual cells. Transplantation as a variety of microsurgery is used to identify cell migration paths and sources of tissue development. At the same time, the section of the embryo is transplanted, such as quail, in the same section of the chicken embryo in the place of the remote site. The core cores have a characteristic structure and therefore distinguishes from chicken nuclear cells.
Explantation - excision of a small section of the embryo and growing it on an artificial environment. With this method, you can receive information about the sources of the development of tissues from this section of the embryo and identify the histogenetic patterns of development.
Nuclear transplantation - Method allowing to cloning germs. For example, a cereal transplant from the cell epithelial cells The headstoves of a shore frog into a frog carton, the core of which was inactivated by the ultraviolet ray, led to the emergence of new individuals (Herdo's experiments). These experiments laid the foundation for cloning top vertebrates and contributed to the appearance (in 1997) the famous sheep dolly. Such embryological experiments convincingly showed that the somatic cell cores contain a complete set of genetic information for the development of a new organism.
The newest achievement experimental embryology The development of the method of extracorporeal fertilization was developed. The transplant of the embryos conceived in the test tube, in the uterus, is the basis of the treatment of infertility. In 1973, L. Shettles (USA) reached a pre-deputy egg from the ovarian of a fruitless woman and fertilized her spermatozoa her husband. So it was necessary to start the germ transplant technique for the purpose of treating infertility. However, only in 1978 in the UK as a result of a successful transplantation in the Makeup of a barren woman, a man's embryo in the stage of 8 blastomers after 2.5 days of cultivation appeared the first "tube" child in a mass of 2700.
