Autoradiography. Radioautography Method of autoradiography in cytology
Autoradiography autoradiography, radioautography, a method for studying the distribution of radioactive substances in an object under study by imposing a photographic emulsion sensitive to radioactive radiation on the object. The radioactive substances contained in the object seem to be photographing themselves (hence the name). The A. method is widely used in physics and technology, in biology and medicine - everywhere where isotope tracers are used. After developing and fixing the photographic emulsion, an image is obtained on it that displays the distribution under study. There are several ways to apply photographic emulsion to an object. A photographic plate can be directly applied to the polished surface of the sample, or a warm liquid emulsion can be applied to the sample, which, when solidified, forms a layer tightly adjacent to the sample and is examined after exposure and photo processing. The distribution of radioactive substances is studied by comparing the blackening density of the film from the test and reference sample (the so-called macroradiography). The second method consists in counting traces formed by ionizing particles in a photographic emulsion using an optical or electron microscope (microradiography). This method is much more sensitive than the first one. Transparency and X-ray emulsions are used to obtain macroautographs, and special fine-grained emulsions are used for microautographs. A photographic image of the distribution of radioactive substances in the object under study, obtained by the A. method, is called an autoradiogram, or radioautograph. On the rice. 12
and 3
examples of autoradiograms are given. The A. method can detect the presence of radioactive elements in various ores, the distribution of natural radioactive elements in the tissues of plant and animal organisms, and so on. The introduction of compounds labeled with radioisotopes into the body and further examination of tissues and cells by the method of A. makes it possible to obtain precise data on the particular cells or cellular structures in which certain processes occur, certain substances are localized, and to establish the time parameters of a number of processes. So, for example, the use of radioactive phosphorus and A. made it possible to detect the presence of an intensive metabolism in a growing bone; the use of radioiodine and A. made it possible to clarify the patterns of activity of the thyroid gland; the introduction of labeled compounds - precursors of protein and nucleic acids, and A. helped to understand the role of certain cellular structures in the exchange of these vital compounds. The A. method makes it possible to determine not only the localization of a radioisotope in a biological object, but also its quantity, since the number of reduced grains of silver in the emulsion is proportional to the number of particles acting on it. Quantitative analysis of macroautographs is carried out by the usual methods of photometry (See Photometry) ,
and microautographs - by counting under a microscope silver grains or traces-tracks that have arisen in the emulsion under the action of ionizing particles. A. begin to successfully combine with electron microscopy (See Electron microscopy). See also Radiography. Lit.: Boyd D. A. Autoradiography in biology and medicine, trans. from English, M., 1957; Zhinkin L. N., The use of radioactive isotopes in histology, in the book: Radiotracers in histology, L., 1959, p. 5-33; Perry R., Quantitative autoradiography, Methods in Cell Physiology, 1964, v. I, ch. 15, p. 305-26. N. G. Khrushchov. Rice. 2. Autoradiogram (print) showing the distribution of phosphorus (32 P) in tomato leaves. The plant was previously placed in a solution containing radioactive phosphorus. Light areas correspond to elevated concentrations of the radioactive isotope; it can be seen that phosphorus is concentrated at the stem and in the vascular parts of the leaves. Rice. 1. Microradiogram of a nickel sample. The diffusion of tin labeled with the radioactive isotope 113 Sn in nickel is studied. The distribution of radioactive tin shows that diffusion mainly occurs along the grain boundaries of nickel.
Great Soviet Encyclopedia. - M.: Soviet Encyclopedia. 1969-1978 .
Synonyms:See what "Autoradiography" is in other dictionaries:
- (from auto ... and radiography) a method of recording the distribution of radioactive substances in an object. A film with a radiation-sensitive emulsion is applied to the surface (cut). Radioactive substances, as it were, take pictures of themselves ... ... Big Encyclopedic Dictionary
- (radioautography), a method for measuring the distribution of radioact. c c in the object under study (according to their own radiation), consisting in applying a layer of nuclear photographic emulsion on it. The distribution is determined by the density of blackening developed ... ... Physical Encyclopedia
A method for studying the distribution of radioactive substances (isotopes) in an object or compounds under study. It consists in imposing on an object (or, for example, a chromatogram) a photographic emulsion sensitive to radioactive radiation and obtaining an imprint, ... ... Dictionary of microbiology
Exist., number of synonyms: 4 autoradiography (2) macroautoradiography (1) ... Synonym dictionary
Autoradiography. See radioautograph. (Source: "English Russian Explanatory Dictionary of Genetic Terms". Arefiev V.A., Lisovenko L.A., Moscow: VNIRO Publishing House, 1995) ... Molecular biology and genetics. Dictionary.
autoradiography- Method for studying the distribution of radioact. components in the sample under study by their own radiation by imposing on the sample sensitive to the radioactive act. emulsion radiation. The distribution is determined by the density of blackening developed ... ... Technical Translator's Handbook
Autoradiography- * autoradiography * autoradiography see ... Genetics. encyclopedic Dictionary
- (from auto ... and radiography), a method of recording the distribution of radioactive substances in an object. A film with a radiation-sensitive emulsion is applied to the surface (cut). Radioactive substances, as it were, take pictures of themselves ... ... encyclopedic Dictionary
Books
- Autoradiography in Biology and Medicine, J. Boyd, The book belongs to one of the creators of the autoradiography method. The first eight chapters are devoted to the theory of the question. They consider the theory of the photographic process, properties and features ... Category: Fundamentals of medical knowledge Publisher:
Radio autography is a relatively new method that has immensely expanded the possibilities of both light and electron microscopy. This is a highly modern method, due to the development of nuclear physics, which made it possible to obtain radioactive isotopes of various elements. For radioautography, in particular, isotopes of those elements that are used by the cell or can bind to substances used by the cell, and which can be administered to animals or added to cultures in amounts that do not interfere with normal cellular metabolism. Since a radioactive isotope (or a substance labeled with it) participates in biochemical reactions in the same way as its non-radioactive counterpart, and at the same time emits radiation, the path of isotopes in the body can be traced using various methods for detecting radioactivity. One way to detect radioactivity is based on its ability to act on photographic film like light; but radioactive radiation penetrates the black paper used to shield the film from light, and has the same effect on the film as light does.
In order to be able to detect radiation emitted by radioactive isotopes on preparations intended for study using light or electron microscopes, the preparations are covered in a dark room with a special photographic emulsion, after which they are left for some time in the dark. Then the slides are developed (also in the dark) and fixed. Areas of the drug containing radioactive isotopes affect the emulsion lying above them, in which dark "grains" appear under the action of the emitted radiation. Thus, they receive radio autographs (from the Greek. radio- radiant autos- himself and grapho- write).
At first, histologists had only a few radioactive isotopes; for example, many of the early studies using autoradiography used radioactive phosphorus. Much more of these isotopes were later used; The radioactive isotope of hydrogen, tritium, has found particularly widespread use.
Autoradiography has been and still is very widely used to study where and how certain biochemical reactions occur in the body.
Chemical compounds labeled with radioactive isotopes that are used to study biological processes are called precursors. Precursors are usually substances similar to those that the body receives from food; they serve as building blocks for building tissues and are incorporated into the complex components of cells and tissues in the same way that unlabeled building blocks are incorporated into them. The tissue component into which the labeled precursor is incorporated and which emits radiation is called the product.
Cells grown in culture, although of the same type, will be at different stages of the cell cycle at any given time, unless special care is taken to synchronize their cycles. However, by injecting tritium-thymidine into the cells and then making autographs, it is possible to determine the duration of the various stages of the cycle. The time of onset of one stage - mitosis - can be determined without labeled thymidine. To do this, a sample of cells from the culture is kept under observation in a phase-contrast microscope, which makes it possible to directly monitor the course of mitosis and set its timing. The duration of mitosis is usually 1 hour, although in some cell types it takes up to 1.5 hours.
Radio autography method
Radio autography, definition, history.
The method of autoradiography is based on the introduction of a compound "labeled" with a radioactive atom into the object under study and the identification of the place of its inclusion by photographic registration of radiation. The basis for obtaining an image is the effect of ionizing particles formed during the decay of a radioactive atom on a nuclear photographic emulsion containing silver halide crystals.
The discovery of the method of autoradiography is directly related to the discovery of the phenomenon of radioactivity. In 1867, the first observation was published on the effect of uranium salts on silver halides (Niepce de St.Victor). In 1896, Henry Becquerel observed the illumination of a photographic plate with uranium salts without prior exposure to light. This experiment is considered the moment of the discovery of the phenomenon of radioactivity. Autoradiography applied to biological material was first used by Lacassagne and Lattes (Lacassagne, Lattes 1924) in the 1920s; the histological block from various organs of animals after the introduction of isotopes was pressed with its flat side to the X-ray plate and exposed. A histological section was prepared in advance and subjected to a standard staining procedure. The resulting autograph was studied separately from the cut. This method makes it possible to estimate the intensity of isotope incorporation into a biological sample. In the 1940s, Leblond used autoradiography to demonstrate the distribution of the iodine isotope in sections of the thyroid gland (Leblond C.P. 1943).
The first attempts to combine autoradiography with electron microscopy were made in the 1950s (Liquir-Milward, 1956). Electron microscopic autoradiography is a special case of conventional autoradiography, in which silver grains are also counted and their distribution taken into account. The peculiarity of the method is the use of a very thin layer of emulsion. At present, a resolution of about 50 nm has been achieved, which is 10-20 times higher than with light microscopy.
At present, the autoradiography method has been supplemented with the possibility of automatically estimating the number of silver grains using video analyzers. Often, to amplify the tag signal (as a rule, these are high-energy isotopes), various types of scintillators are used, deposited on plates (phosphor-coated intensifying screen), or impregnated into an emulsion (PPO) - in this case, photon emission illuminates a conventional photographic plate or film.
Photographic principle of obtaining an image, photographic emulsion
In a radiographic study, the role of a nuclear decay detector is performed by a photographic emulsion, in which, when an ionizing particle passes, a latent image remains, which is then revealed during development, similarly to the processing of ordinary photographic film.
Photo emulsion is a suspension of silver halide microcrystals in gelatin. Microcrystals have structural defects called sensitivity centers. According to the Gurney-Mott model, these disturbances in the ionic lattice of a crystal are able to capture electrons released when an alpha or beta particle passes through the conduction band of the crystal, as a result of which the ion is converted into an atom. The resulting latent image can be revealed by a procedure that converts activated silver halide crystals into grains of metallic silver (this process is called chemical development). Any agent with sufficient reducing activity can be used as a developer (typically metol, amidol, or hydroquinone are used in photography and autoradiography). After the exposure of the exposed crystals, the remaining silver halide microcrystals are removed from the emulsion with a fixative (usually hyposulfite). Nuclear photographic emulsions are characterized by resolution (grain) and sensitivity. The first is determined by the size of silver salt microcrystals and is inversely proportional to the latter. The photographic emulsion is characterized by reduced sensitivity to visible light, but work with it, however, must be done in the dark to exclude the appearance of artifacts.
The emulsion can be applied to the drug in the form of a finished film with a substrate or by immersing the drug in a heated liquid emulsion - this way a thin uniform layer is obtained, which is developed in the usual way. Before applying the emulsion for light microscopy, the slide is usually stained with the desired histological stain, but paler than usual, to enable the counting of silver grains in all areas. The drug is exposed for a certain time, then it is developed.
Isotopes used in autoradiography.
In autoradiography, depending on the objectives of the study and the available materials, various isotopes can be used. The image created by an ionizing particle on a nuclear photographic emulsion depends on the energy of the particle and the type of its interaction with matter.
Alpha particles emitted by identical radioactive nuclei have the same energy ( E) and the same path length ( R) , connected by the following relation:
R = kE3/2
Where k – a constant characterizing the medium in which the particles propagate. The range of particles in the heart is determined by its density and elemental composition. The Bragg-Klymen relation makes it possible to estimate the range in a substance with atomic mass A and density by the range of alpha particles in air (R0) d:
R= 0.0003 (R0 / d) A1/2
Since the ionizing power of alpha particles is very high, this facilitates the photographic registration of the isotope distribution, and also allows the use of non-emulsion materials for registration. The trace of alpha particles emitted by one source, on autographs, looks like a beam of straight segments, usually 15-50 microns long, emanating from one point, which allows you to accurately localize the site of the inclusion of a radioactive label. However, alpha particles are emitted by isotopes with large atomic numbers, which limits their use as a biological label.
Tracks of alpha particles are often observed in histological radiographs as an artifact - the result of self-radiation of isotopes in the glass slide.
Beta radiation is characterized by a continuous spectrum of the initial energy of particles - from zero to E max determined for each isotope. The shapes of the spectrum differ significantly. Thus, the most probable energy of particles emitted by the tritem is 1/7 of E max, 14C - about ¼, 32P - about 1/3. The maximum energy of beta radiation of various isotopes varies from 18 keV to 3.5 MeV - in a much wider range than alpha radiation. As a rule, the maximum energy is higher for short-lived isotopes.
The passage of beta particles and monoenergetic electrons through matter is accompanied by two main types of interaction. When interacting with an orbiting electron, the particle can transfer to it energy sufficient to ionize the atom (remove the electron from the orbit). In rare cases, this energy is so high that the track of the released electron can be observed. Due to the equality of the masses of the particle and the electron, there is a deviation from the initial motion. Interaction of the second type, with atomic nuclei, leads to the appearance of bremsstrahlung X-rays. Although the latter is not registered by the emulsion, the act of interaction of the particle with the nucleus can be detected by a sharp break in the trajectory.
Repeated interaction with orbiting electrons leads to a curvature of the trajectory, which usually looks like a winding line, especially in the final part, when the particle's speed decreases and the ionizing power increases. The length of the trajectory noticeably exceeds the distance from the start to the end point of the track - the run. For this reason, even monoenergetic electrons are characterized by the presence of a range of ranges limited from above by R max, which is typical for this radiation. Because of the lower ionization losses, beta particles are more difficult to detect than alpha particles. They do not form continuous tracks (except for the softest radiation of tritium - however, in this case, the probability of passing through more than one emulsion crystal is small), the density and number of developed crystals vary within different limits. The range of a beta particle in another element can be estimated from the formula:
R = RA1 (Z/A)A1/ (Z/A)
In a wide range of values of E max the maximum mileage is related to the maximum energy by the relationship:
R m= 412 E max 1.265 – 0.0954 ln E max
The difference in the ranges, ionization ability, and density of the developed emulsion crystals for particles with different energies can be used to discriminate the distribution of elements if their isotopes differ significantly in E max, as in the case of tritium and 14C. Discrimination of the distribution of two isotopes is carried out by applying two emulsion layers to the sample, the first layer registers predominantly soft radiation, the second - hard. According to some works, different isotopes can be reliably separated from the size of the developed emulsion crystals - crystals affected by the beta particle of tritium, which has a higher ionization power, are larger.
Internal conversion electrons are formed when a gamma quantum with a very low radiation energy is absorbed and an electron is removed from the inner shell of an atom. These electrons are similar to soft beta particles, but unlike the latter, they are monoenergetic. The presence of internal conversion electrons allows the use of isotopes such as 125I.
At present, the most commonly used isotopes emitting beta particles. As a rule, tritium is used for labeling in histological studies. The first autographs using tritium were made back in the 1950s (Fitzgerald et al. 1951), but its widespread use began after tritium-labeled thymidine was obtained at the Brookhaven Laboratory. Since hydrogen is part of all organic substances, using tritium, you can get a variety of compounds that carry a radioactive label. The lower the energy of the emitted particle, the shorter the track left by it when moving in a photographic emulsion, and the more accurately it is possible to localize the location of the tagged atom. The path length of tritium beta particles is about 1-2 μm, the most probable energy is 0.005 MeV, and the track consists in most cases of a single silver grain, which makes it possible to localize the radiation source not only in relatively large cellular structures, such as the nucleus, but also in individual chromosomes.
The introduction of "labeled" metabolites into the body makes it possible to trace the incorporation of the isotope into the cells of animal tissues, which makes it possible to study a variety of biochemical processes in a living organism.
Obtaining absolute data - the concentration of the labeled substance in the object under study is rarely the goal of radioautographic research, for this it is necessary to know a number of conditions, the determination of which is difficult. Therefore, quantitative radioautographic studies are usually carried out by comparing the concentration of silver grains over the test object and the control, while the control data is conveniently taken as one, or 100%.
Characteristics of some isotopes used
in radioautography of biological objects
Radio autography method
Radio autography, definition, history.
The method of autoradiography is based on the introduction of a compound "labeled" with a radioactive atom into the object under study and the identification of the place of its inclusion by photographic registration of radiation. The basis for obtaining an image is the effect of ionizing particles formed during the decay of a radioactive atom on a nuclear photographic emulsion containing silver halide crystals.
The discovery of the method of autoradiography is directly related to the discovery of the phenomenon of radioactivity. In 1867, the first observation was published on the effect of uranium salts on silver halides (Niepce de St.Victor). In 1896, Henry Becquerel observed the illumination of a photographic plate with uranium salts without prior exposure to light. This experiment is considered the moment of the discovery of the phenomenon of radioactivity. Autoradiography applied to biological material was first used by Lacassagne and Lattes (Lacassagne, Lattes 1924) in the 1920s; the histological block from various organs of animals after the introduction of isotopes was pressed with its flat side to the X-ray plate and exposed. A histological section was prepared in advance and subjected to a standard staining procedure. The resulting autograph was studied separately from the cut. This method makes it possible to estimate the intensity of isotope incorporation into a biological sample. In the 1940s, Leblond used autoradiography to demonstrate the distribution of the iodine isotope in sections of the thyroid gland (Leblond C.P. 1943).
The first attempts to combine autoradiography with electron microscopy were made in the 1950s (Liquir-Milward, 1956). Electron microscopic autoradiography is a special case of conventional autoradiography, in which silver grains are also counted and their distribution taken into account. The peculiarity of the method is the use of a very thin layer of emulsion. At present, a resolution of about 50 nm has been achieved, which is 10-20 times higher than with light microscopy.
At present, the autoradiography method has been supplemented with the possibility of automatically estimating the number of silver grains using video analyzers. Often, to amplify the tag signal (as a rule, these are high-energy isotopes), various types of scintillators are used, deposited on plates (phosphor-coated intensifying screen), or impregnated into an emulsion (PPO) - in this case, photon emission illuminates a conventional photographic plate or film.
Photographic principle of obtaining an image, photographic emulsion
In a radiographic study, the role of a nuclear decay detector is performed by a photographic emulsion, in which, when an ionizing particle passes, a latent image remains, which is then revealed during development, similarly to the processing of ordinary photographic film.
Photo emulsion is a suspension of silver halide microcrystals in gelatin. Microcrystals have structural defects called sensitivity centers. According to the Gurney-Mott model, these disturbances in the ionic lattice of a crystal are able to capture electrons released when an alpha or beta particle passes through the conduction band of the crystal, as a result of which the ion is converted into an atom. The resulting latent image can be revealed by a procedure that converts activated silver halide crystals into grains of metallic silver (this process is called chemical development). Any agent with sufficient reducing activity can be used as a developer (typically metol, amidol, or hydroquinone are used in photography and autoradiography). After the exposure of the exposed crystals, the remaining silver halide microcrystals are removed from the emulsion with a fixative (usually hyposulfite). Nuclear photographic emulsions are characterized by resolution (grain) and sensitivity. The first is determined by the size of silver salt microcrystals and is inversely proportional to the latter. The photographic emulsion is characterized by reduced sensitivity to visible light, but work with it, however, must be done in the dark to exclude the appearance of artifacts.
The emulsion can be applied to the drug in the form of a finished film with a substrate or by immersing the drug in a heated liquid emulsion - this way a thin uniform layer is obtained, which is developed in the usual way. Before applying the emulsion for light microscopy, the slide is usually stained with the desired histological stain, but paler than usual, to enable the counting of silver grains in all areas. The drug is exposed for a certain time, then it is developed.
Isotopes used in autoradiography.
In autoradiography, depending on the objectives of the study and the available materials, various isotopes can be used. The image created by an ionizing particle on a nuclear photographic emulsion depends on the energy of the particle and the type of its interaction with matter.
Alpha particles emitted by identical radioactive nuclei have the same energy ( E) and the same path length ( R) , connected by the following relation:
R = kE 3/2
Where k – a constant characterizing the medium in which the particles propagate. The range of particles in the heart is determined by its density and elemental composition. The Bragg-Klymen relation makes it possible, by the range of alpha particles in air (R 0), to estimate the range in a substance with atomic mass A and density d:
R= 0.0003 (R0 / d) A 1/2
Since the ionizing power of alpha particles is very high, this facilitates the photographic registration of the isotope distribution, and also allows the use of non-emulsion materials for registration. The trace of alpha particles emitted by one source, on autographs, looks like a beam of straight segments, usually 15-50 microns long, emanating from one point, which allows you to accurately localize the site of the inclusion of a radioactive label. However, alpha particles are emitted by isotopes with large atomic numbers, which limits their use as a biological label.
Tracks of alpha particles are often observed in histological radiographs as an artifact - the result of self-radiation of isotopes in the glass slide.
The passage of beta particles and monoenergetic electrons through matter is accompanied by two main types of interaction. When interacting with an orbiting electron, the particle can transfer to it energy sufficient to ionize the atom (remove the electron from the orbit). In rare cases, this energy is so high that the track of the released electron can be observed. Due to the equality of the masses of the particle and the electron, there is a deviation from the initial motion. Interaction of the second type, with atomic nuclei, leads to the appearance of bremsstrahlung X-rays. Although the latter is not registered by the emulsion, the act of interaction of the particle with the nucleus can be detected by a sharp break in the trajectory.
Repeated interaction with orbiting electrons leads to a curvature of the trajectory, which usually looks like a winding line, especially in the final part, when the particle's speed decreases and the ionizing power increases. The length of the trajectory noticeably exceeds the distance from the start to the end point of the track - the run. For this reason, even monoenergetic electrons are characterized by the presence of a range of ranges limited from above by R max, which is typical for this radiation. Because of the lower ionization losses, beta particles are more difficult to detect than alpha particles. They do not form continuous tracks (except for the softest radiation of tritium - however, in this case, the probability of passing through more than one emulsion crystal is small), the density and number of developed crystals vary within different limits. The range of a beta particle in another element can be estimated from the formula:
R = R A1 (Z/A) A1 / (Z/A)
In a wide range of values of E max the maximum mileage is related to the maximum energy by the relationship:
R m= 412 E max 1.265 – 0.0954 ln E max
The difference in the ranges, ionization ability and density of the developed emulsion crystals for particles with different energies can be used to discriminate the distribution of elements if their isotopes differ significantly in E max, as in the case of tritium and 14 C. Discrimination of the distribution of two isotopes is carried out by applying on a sample of two emulsion layers, the first layer registers predominantly soft radiation, the second - hard. According to some works, different isotopes can be reliably separated from the size of the developed emulsion crystals - crystals affected by the beta particle of tritium, which has a higher ionization power, are larger.
Internal conversion electrons are formed when a gamma quantum with a very low radiation energy is absorbed and an electron is removed from the inner shell of an atom. These electrons are similar to soft beta particles, but unlike the latter, they are monoenergetic. The presence of internal conversion electrons allows the use of isotopes such as 125 I.
At present, the most commonly used isotopes emitting beta particles. As a rule, tritium is used for labeling in histological studies. The first autographs using tritium were made back in the 1950s (Fitzgerald et al. 1951), but its widespread use began after tritium-labeled thymidine was obtained at the Brookhaven Laboratory. Since hydrogen is part of all organic substances, using tritium, you can get a variety of compounds that carry a radioactive label. The lower the energy of the emitted particle, the shorter the track left by it when moving in a photographic emulsion, and the more accurately it is possible to localize the location of the tagged atom. The path length of tritium beta particles is about 1-2 μm, the most probable energy is 0.005 MeV, and the track consists in most cases of a single silver grain, which makes it possible to localize the radiation source not only in relatively large cellular structures, such as the nucleus, but also in individual chromosomes.
The introduction of "labeled" metabolites into the body makes it possible to trace the incorporation of the isotope into the cells of animal tissues, which makes it possible to study a variety of biochemical processes in a living organism.
Obtaining absolute data - the concentration of the labeled substance in the object under study is rarely the goal of radioautographic research, for this it is necessary to know a number of conditions, the determination of which is difficult. Therefore, quantitative radioautographic studies are usually carried out by comparing the concentration of silver grains over the test object and the control, while the control data is conveniently taken as one, or 100%.
Characteristics of some isotopes used
in radioautography of biological objects
Beta-particles of radioactive phosphorus are capable of flying distances of up to several millimeters in a nuclear emulsion, the track consists of dozens of rarely located silver particles - for example, radioactive phosphorus can only be used to study the distribution of the isotope in tissues, localization in individual cell structures cannot be established.
Radioactive sulfur and carbon can be used to localize the isotope in individual cells, provided they are large or spaced sufficiently apart, which can be achieved in blood smears or cell suspensions.
Resolution and method errors, method errors.
geometric error– due to the fact that the emitted particle can be directed at any angle to the surface of the photolayer. Consequently, the silver grain in the photolayer may not be located exactly above the radioactive atom, but more or less displaced depending on the direction of particle motion and the path length (energy).
photo bug arises due to the fact that a grain of silver, consisting of thousands of metal atoms, is much larger than a radioactive atom. Thus, the localization of a smaller object has to be judged based on the position of the larger one.
When using tritium, which is characterized by low energy (mileage) of emitted particles and nuclear photographic emulsions with low grain size, the resolution of the autoradiography method lies within the resolution of optical systems - 1 μm. Thus, these errors do not have a significant impact on the result.
To achieve better resolution, it is necessary to reduce the thickness of the cut, the emulsion layer and the distance between them. The specimen should be slightly underexposed.
Auto absorption effect: The number of silver grains depends on the degree of absorption of radiation by cellular structures, due to the low range and low energy of beta particles, their absorption in tissues is quite large, which can lead to loss of the mark, so the question of the thickness of the sections becomes important. It has been shown that the number of silver grains is proportional to tissue radioactivity only at a slice thickness of not more than 5 microns.
The relative number of beta particles that have passed through the absorber layer with a thickness X can be estimated according to Baer's law -
N x/N 0 = e - m x
Where m is the absorption coefficient (the reciprocal of the layer thickness, during the passage of which the number of particles decreases in e once. The value of the absorption coefficient can be approximately estimated from the value of R m(maximum range), known for all isotopes, using the relation m R m= 10, which is valid for not too hard radiation.
If in a layer of unit thickness per unit time there are n particles moving towards the surface, then in a sample with a thickness X surface will reach N particles:
Background and artifacts: An error in the measurements can also be introduced by mechanical influences - scratches, emulsion cracks leading to the formation of a latent image and background radiation, which must be taken into account when processing autographs. The background is taken into account by counting the number of silver grains in the empty area of the preparation. Errors are also introduced as a result of histological processing of sections - wiring for alcohols (dehydration), paraffin embedding, staining. These procedures can affect the size and ratio of cellular structures.
Radiation effect of labeled metabolites: Due to the low radiation energy, tritium causes significant ionization in the cell, much greater than the radiative effect of carbon beta particles. As a result, with prolonged action of a labeled compound, for example, 3 H-thymidine, cells are destroyed and die, leading to tissue growth arrest. First of all, spermatogenesis is disturbed. There is evidence of mutagenic and carcinogenic effects of labeled metabolites. The observed cytological changes consist in disruption of the passage of the mitotic cycle by cells, changes in cell ploidy, and the appearance of chromosomal aberrations. But, apparently, the damaging effect of the isotope on cells can noticeably affect the results of the study only under conditions of a long experiment.
Quantification of radioactivity
As a rule, not the absolute, but the relative amount of the included isotope is determined in the experiment. The degree of inclusion of the label can be assessed in two ways - densitometrically - which is more applicable to macroautographs and direct counting of silver grains over objects. This time-consuming procedure can currently be performed using a computer. A digital image of a histological preparation is processed by special software in order to automatically highlight cells and cellular structures on it and count the number of silver grains. If the question of quantitative assessment arises, it is necessary to involve the concept of efficiency. Most often, efficiency is understood as the number of silver grains formed during the registration of one radioactive decay. The effectiveness of the method is influenced by many factors, primarily the thickness of the object and the emulsion.
In studies using a scintillation counter, a high correlation was found between the average number of disintegrations per minute and the number of silver grains. According to Hunt (Hunt and Foote, 1967), the formation of one grain in the emulsion used in the experiment corresponds to 5.8 radioactive decays, i.e., the efficiency of the method is 17.8%.
To quantify tritium in macroscopic preparations, samples with standard activity, which are mounted on the same autograph, can be used.
An accurate assessment of the radioactivity of the compared biological objects is very difficult.
A classic example of a radioautographic study is the work on the accumulation of 32 P in the DNA of horse bean root cells (Howard and Pelc, 1953). In this experiment, the division of the mitotic cycle into four periods (mitosis - M, G 1 - presynthetic period, S - DNA synthesis, premitotic period G 2) was shown for the first time, that the period of DNA synthesis occupies a limited part of the interphase, being separated in time from the beginning and end of mitosis. The data of Howard and Pelk later found confirmation in more precise experiments using a specific DNA precursor, 3 H-thymidine.
Methods for assessing protein synthesis. The most common precursors for assessing total protein synthesis in radioautographic studies are 3 H-leucine, 3 H-methionine, 3 H-phenylalanine. For example, the synthesis of total protein in the brain of rats during the first weeks of postnatal development was studied using a leucine label (Pavlik and Jakoubek, 1976). To study the synthesis of histones and their effect on the regulation of transcription, the basic amino acids 3 H-lysine and 3 H-arginine are used, and 3 H-tryptophan is used to study the synthesis of acidic proteins. The inclusion density of the amino acid label corresponds to the intensity of protein synthesis, and therefore reflects the functional activity of the neuron. The radioautographic method makes it possible to compare the characteristics of protein synthesis in various animal tissues under experimental exposure, and allows us to trace the dynamics of changes at the level of individual cell types and cellular structures (nucleus, cell body, neuron processes - axonal transport).
Currently, autoradiography is often used to study the brain in studies using radioligands for certain receptors. Thus, maps of the distribution of various receptors in the brain structures of animals and humans were constructed.
Autoradiography is also used to visualize gels in biochemistry and in combination with immunoassays (RIA).
References:
1. Epifanova O.I. et al. Radio autograph M., Higher School, 1977
2. Sarkisov D.S. Perov Yu.L. Microscopic technique M.: "Medicine", 1996
3.Rogers A.W. Practical autoradiography, Amersham UK, 1982
4.Bokshtein S.Z. Ginzburg S.S. et al. Electron-microscopic autoradiography in metal science M., "Metallurgy"
Autoradiogram a fiya, autoradiography, autoradiography , a method for studying the distribution of radioactive substances in an object under study by imposing a photographic emulsion sensitive to radioactive radiation on the object. The radioactive substances contained in the object taking pictures of themselves(hence the name). The method of autoradiography is widely used in physics and technology, in biology and medicine, wherever isotope tracers are used.
After developing and fixing the photographic emulsion, an image is obtained on it that displays the distribution under study. There are several ways to apply photographic emulsion to an object. A photographic plate can be directly applied to the polished surface of the sample, or a warm liquid emulsion can be applied to the sample, which, when solidified, forms a layer tightly adjacent to the sample and is examined after exposure and photo processing. The distribution of radioactive substances is studied by comparing blackening density of the film from the test and reference sample(the so-called macroradiography).
Second method consists in counting the traces formed by ionizing particles in a photographic emulsion, using optical or electron microscope (microradiography). This method is much more sensitive than the first one. Transparency and X-ray emulsions are used to obtain macroautographs, and special fine-grained emulsions are used for microautographs.
A photographic image of the distribution of radioactive substances in the object under study, obtained by autoradiography, is called autoradiogram or radioautograph.
The introduction of compounds labeled with radioisotopes into the body and further examination of tissues and cells by autoradiography allows:
- get accurate information about which ones cells or cellular structures, certain processes occur,
- localized substances,
- set the time parameters for a number of processes.
For example, the use of radioactive phosphorus and autoradiography made it possible to detect the presence of an intensive metabolism in the growing bone; the use of radioiodine and autoradiography made it possible to clarify the patterns of activity of the thyroid gland; the introduction of labeled compounds - precursors of protein and nucleic acids, and autoradiography helped to clarify the role of certain cellular structures in the exchange of these vital compounds. The method of autoradiography makes it possible to determine not only the localization of a radioisotope in a biological object, but also its quantity, since the number of reduced silver grains of the emulsion is proportional to the number of particles affecting it. Quantitative Analysis macroautographs are carried out by the usual methods of photometry, and microautographs - by counting under a microscope silver grains or traces-tracks that have arisen in the emulsion under the action of ionizing particles. Autoradiography is beginning to be successfully combined with electron microscopy
