Methods of light and electron microscopy
1. Light microscopy
light electron microscope interference
A light microscope can increase the resolving power of the human eye by about 1000 times. This is the "useful" magnification of the microscope. When using the visible part of the light spectrum, the final resolution limit of the light microscope is 0.2-0.3 microns. Next, light microscopy techniques will be discussed.
1 Dark field method (ultramicroscopy)
The dark field method in transmitted light is based on the Tyndall effect, since it is similar to dust particles from a beam of light, with side illumination, tiny particles glow, the reflected light from which enters the microscope objective. The light from the illuminator and mirror is directed to the preparation by a dark field condenser. After leaving the condenser, the main part of the light rays, which did not change its direction when passing through the transparent preparation, does not enter the objective. The image in the microscope is formed with the help of only a small part of the rays scattered by the microparticles of the preparation located on the glass slide. In the field of view on a dark background, light images of the elements of the preparation structure are visible, which differ from the environment in the refractive index. For large particles, only bright edges are visible, scattering light rays.
The method of ultramicroscopy is based on the same principle - preparations in ultramicroscopes are illuminated perpendicular to the direction of observation. With this method, extremely small particles can be detected, the dimensions of which lie far beyond the resolution of the most powerful microscopes. With the help of immersion ultramicroscopes, it is possible to register the presence of particles up to 2 in size in the preparation. 10-9m. But the shape and exact dimensions these particles cannot be determined using this method. Ultramicroscopes are mainly used in colloid chemistry. 2 Bright field method and its variations The bright field method in transmitted light is used in the study of transparent preparations with light-absorbing particles and details included in them. In the absence of the preparation, the beam of light from the condenser, passing through the lens, gives a uniformly illuminated field near the focal plane of the eyepiece. In the presence of an absorbent element in the preparation, partial absorption and partial scattering of the light incident on it occurs, which causes the appearance of the image. The oblique illumination method is a variation of the previous method. The difference between them is that the light is directed at the object at a large angle to the direction of observation, this helps to reveal the "relief" of the object due to the formation of shadows. The bright field method in reflected light is used in the study of opaque light-reflecting objects. Lighting is produced from above, through the lens, which simultaneously plays the role of a condenser. In the image created in the plane by the lens together with the tube lens, the structure of the preparation is visible due to the difference in the reflectivity of its elements; in a bright field, inhomogeneities are also distinguished, scattering the light incident on them. 1.3 Phase contrast microscopy method Most of the cellular structures differ little in the refractive index of light, the absorption of rays from each other and the environment. In order to study such components, one has to change the illumination (with a loss of image clarity) or use special methods and devices. Phase-contrast microscopy is one such method. It is widely used in the vital study of cells. The essence of the method is that even with very small differences in the refractive indices of different elements of the drug, the light wave passing through them undergoes different phase changes. Invisible directly neither to the eye nor to the photographic plate, these phase changes are converted by a special optical device into changes in the amplitude of the light wave, i.e., into changes in brightness that are already visible to the eye or are recorded on the photosensitive layer. In the resulting visible image, the distribution of brightness (amplitudes) reproduces the phase relief. The resulting image is called phase contrast. Objects can appear dark against a light background (positive phase contrast) or light against a dark background (negative phase contrast). 4 Interference contrast method (interference microscopy) The method of interference contrast is similar to the previous one - they are both based on the interference of rays that have passed through the microparticle and passed it. A beam of parallel light rays from the illuminator splits into two streams, entering the microscope. One of the obtained beams is directed through the observed particle and acquires changes in the oscillation phase, the other - bypassing the object along the same or additional optical branch of the microscope. In the ocular part of the microscope, both beams reconnect and interfere with each other. As a result of interference, an image will be built, on which sections of the cell with different thicknesses or different densities will differ from each other in terms of contrast. The interference contrast method is often used in conjunction with other microscopy methods, in particular, observation in polarized light. Its use in combination with ultraviolet microscopy makes it possible, for example, to determine the content of nucleic acids in the total dry mass of an object. 5 Polarizing microscopy Polarizing microscopy is a method of observing in polarized light objects that have isotropy, i.e. ordered orientation of submicroscopic particles. A polarizer is placed in front of the condenser of a polarizing microscope, which transmits light waves with a certain plane of polarization. After the preparation and the lens, an analyzer is placed, which can transmit light with the same plane of polarization. If the analyzer is then rotated by 90o with respect to the first one, no light will pass through. In the event that between such crossed prisms there is an object that has the ability to polarize light, it will be seen as glowing in a dark field. Using a polarizing microscope, one can verify, for example, the oriented arrangement of micelles in the plant cell wall. 6 Vital study of cells Light microscopy enables us to observe living cells. For short observation, the cells are usually placed in a liquid medium on a glass slide. But for a longer time, other methods are needed. Often, special cameras are used for long-term observation (flat vials with holes covered with thin glasses, or collapsible flat cameras). Cells are studied in media specially selected for them. Usually, for protozoa, these are balanced salt solutions with additions of microorganisms and other protozoa that serve as food for the object of study. Free cells of multicellular organisms can be studied in blood plasma or in special synthetic media. Cell culture is used to study animal cells and tissues. Cell culturing is a process by which in vitro single cells (or a single cell) are artificially grown under controlled conditions for further study. A simpler version of this method is that a small piece of living tissue is placed in a chamber with a nutrient solution, and after a while, cell growth and division begin to proceed along the periphery. The second way to obtain a cell culture is to treat a piece of tissue with trypsin or chelaton (which will lead to cell dissociation), and then place it in a vessel with a nutrient extract, where, after the cells are lowered to the bottom and attached, the culture begins to grow and multiply. For cell culture, it is preferable to use embryonic material, which has a greater ability to divide than an adult. During cell culture, special conditions of sterility, temperature, pH should be observed. Plant cells can be cultured in the same way. To do this, they are treated with enzymes that dissolve cell membranes, and the separated contents are placed in a special environment where they grow and divide. Together with cell culture, microfilming and microphotography are usually used, with the help of which many cellular processes are recorded. 6.2 Microsurgery method The microsurgery method is used to study living cells. This is a method of surgical intervention and influence on the cell, incl. removal or implantation of individual organelles. This method also involves the transplantation of organelles from cell to cell, the introduction of large macromolecules into the cell. Typically, the micromanipulator is combined with a microscope, which serves to monitor the progress of the surgical intervention. Microsurgical instruments are glass hooks, needles, capillaries, which are made at "microforges". During microoperations, the cell is placed in a special chamber, where instruments are also inserted. AT recent times Laser microbeams and UV spectrum microbeams are also widely used. They are used to target cells during the study. 6.3 Fluorescence microscopy method A method for obtaining an enlarged image using the glow of excited atoms and molecules of the sample. In a fluorescent microscope, the sample is irradiated with light at a higher frequency, and the image is obtained in the optical spectrum. The image of the fluorescent preparation can be photographed with a specialized digital camera, which allows taking long-exposure photographs. Quite often, substances capable of fluorescence are used or fluorescent agents are injected into the cell. One type of fluorescence microscopy is confocal microscopy, a technique that allows imaging from a certain depth in the middle of a specimen. 1.6.4 Confocal scanning light microscopy method A confocal light scanning microscope is used to obtain a three-dimensional reconstruction of the object. With its help, a series of successive sections of a cell taken from different depths is obtained. After that, a special computer program brings these images together and we can get a three-dimensional, three-dimensional image of the object. 7 Study of fixed cells Many data on the structure and functioning of the cell were obtained precisely on fixed cells. Fixation is a complex process aimed at killing a cell, stopping the activity of cellular enzymes, disintegrating components, avoiding the loss of structures and substances, as well as their acquisition if they were absent in a living cell. But, unfortunately, there is no fixer yet that would satisfy all these requirements. 7.1 Cyto methods chemical analysis This is a series of staining techniques, main goal which is to identify specific chemicals of the cell. There are a number of staining techniques that directly reveal certain substances. Such cytochemical reactions are subject to the following requirements: the specificity of dye binding, the immutability of the localization of the substance. An example of such a reaction is a reaction to DNA - Felgen. 7.2 Cytophotometric method This method allows quantitative measurement of the final product of the cytochemical reaction. It is based on determining the number of substances by their absorption of light of a certain wave. The intensity of the absorption of rays is proportional to the concentration of the substance at the same thickness of the object. For this method, cytophotometer microscopes are used (a sensitive photometer is located behind their lenses). This method is widely used in determining the amount of DNA after the Feulgen reaction. It is also possible to quantify not only light-absorbing substances, but also light-emitting ones. This is the basis of fluorometry. 7.3 Method of immunochemical research (immunochemical reactions using fluorescent antibodies) The method has high sensitivity and specificity. There are methods of direct and indirect immunofluorescence, in the first case, the antigen binds to an antibody conjugated with a fluorescent label (fluorochrome, for example, FITC or TRITC), in the second, the antigen binds to an unconjugated specific antibody (primary antibody). The fluorescent label is conjugated with a secondary antibody specific to the Fc-fragment of the primary antibody.The indirect method is more sensitive than the direct method.The location of the desired proteins in the cell is found by the glow of the fluorochrome.But in order for the labeled antibodies to penetrate the cell, it is necessary to make the membrane more permeable.Usually this is achieved by fixing the cells and partial extraction of lipids from membranes. 7.4 Autoradiography method This method is used to detect the localization of biopolymer synthesis sites, determine the pathways for the transfer of substances in a cell, and monitor migration or cell properties. For this, registration of substances labeled with isotopes is used. The method repeats the Becquerel method. A precursor of one of the substances in which one of the atoms is replaced by a radioactive isotope is introduced into the medium with the cells. In the process of synthesis, the labeled atom will get into the molecule itself, and it will be possible to register it using a photographic emulsion. The accuracy of this method depends on the size of the AgBr grain and on the energy of the particle. Therefore, a special fine-grained photographic emulsion and low-energy isotopes are used for the autoradiography method. Autographs cannot be used to study water-soluble compounds, as atoms can be lost. 7.5 Molecular hybridization method The use of the previous method in conjunction with the method of autoradiography gives us the opportunity to localize on the chromosome places with a certain nucleotide sequence or even the location of certain genes. To do this, a solution with a labeled nucleic acid is applied to a preparation with denatured DNA in the composition of chromosomes or nuclei. In the process of DNA renaturation, a hybrid of the labeled nucleic acid and the complementary region of the original DNA is formed. The place of such a connection is recorded radioautographically. The method is also used for staining nucleic acids with fluorochromes. This means that we can determine the position of any DNA sequence. Since the issue of increasing resolution has always been acute, scientists have come to the conclusion that in light microscopy, resolution can only be increased by using a light source that emits waves with the shortest wavelength. Such a source can be a hot filament that emits a stream of electrons that can be focused by passing through a magnetic field. Based on this, a light microscope was created. Currently, a distinction is made between transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Next, the methods of electron microscopy will be considered. 1 Contrasting corpuscular objects There are several contrast methods. I'll look at two of them: negative contrast and metal shading. Negative contrasting is performed using FVA, ammonium molybdenum acid, uranyl acetate. after applying the solution to the object under study, then applying it to the substrate films and drying, the object under study will be immersed in a high-density amorphous substance. This will cause them to appear as white objects against a dark background in pictures. Solutions can also penetrate deep into the object under study and reveal hidden structures. There is also a positive contrast method. In this case, salts of heavy metals are used, which increase the electron density of the object, thus increasing its contrast. Shading by metals - during the thermal evaporation of metals, their particles fly apart and deposit on the object under study in the form of a layer whose thickness varies depending on the direction of the particles' flight. In places where the particle beam shields, the space will be darker. 2.2 Ultramicrotomy Ultramicrotomy is a set of techniques for obtaining ultra-thin sections using ultratomes, or ultramicrotomes. Ultramicrotomes - devices for automatic preparation of ultrathin tissue sections of programmed thickness (5-10 nm). This is necessary because the electron beam, passing through thicker objects, is absorbed, causes heating and leads to deformation of the preparation. Their procedure is quite similar to that for light microscopy. Cells are fixed first, often using GA (glutaraldehyde) followed by OsO 4, although they can also be used separately. Then carry out the wiring with the last stage - immersion in xylene. After the preparation is poured with epoxy resins (epon). Now the drug, enclosed in solid blocks, can be cut with glass or diamond knives to make sections so small that the thermal supply of the object is used. The preparation is then stained, usually with uranyl acetate and lead nitrate, which contrast positively. This manufacturing technique has opened up enormous possibilities for the application of electron microscopy in almost all areas of biology and medicine. It is possible to carry out research by radioautography using ultra-fine-grained emulsions and huge exposures. As well as studies without fixation and conclusion in epon by cryoultramicrotomy. In this case, the object is instantly frozen to the temperature of liquid nitrogen, the water passes into a glassy state, and the processes are instantly inhibited. The sections obtained in this way are used in immunochemical studies, etc. To study the structure of membrane components, the freeze-shear method is used. The method is similar to the previous one: the object is instantly cooled to the temperature of liquid nitrogen, and then chipped off with a chilled knife. The surface of the layer is covered with a layer of metal and carbon, and then the object is dissolved and a replica is obtained from the cleavage surface. In this case, it is possible to study the surface relief of the membranes. 3 High voltage microscopy method The accelerating voltage of such a microscope is 1-3 million volts. This voltage allows us to view objects of greater thickness (1-10 microns), and using stereoscopic imaging, we can obtain information about the 3D organization of intracellular structures with high resolution. 4 Method of scanning (raster) electron microscopy When using this method, the object is covered with a thin layer of evaporated metal, reflected from which, the probe (electron beam) enters the receiving device, from where the signal is transmitted further. In this case, an almost three-dimensional image of the surface of the object under study is obtained, due to the very large depth of focus. You can also get information about the chemical composition of cells. 3. Light and electron microscopes 1 The structure of a light microscope and its main characteristics To understand the principle of operation of a light microscope, it is necessary to consider its structure. The main instrument of biology is an optical system, which consists of a tripod, lighting and optical parts. The tripod includes a shoe; object stage with a slide holder and two screws that move the stage in two perpendicular directions; tube, tube holder; macro- and microscrews that move the tube in the vertical direction. To illuminate the object, natural diffused or artificial illumination is used, which is carried out by means of a microscope permanently mounted in a shoe or an illuminator connected through a bar. The lighting system also includes a mirror with flat and concave surfaces and a condenser located under the stage and consisting of 2 lenses, an iris diaphragm and a flip-down frame for filters. The optical part includes a set of objectives and eyepieces that allow you to study cells at different magnifications. The principle of operation of a light microscope is that a beam of light from an illumination source is collected in a condenser and directed to an object. After passing through it, the light rays enter the lens system of the objective. They build a primary image, which is enlarged with the help of eyepiece lenses. In general, the lens and eyepiece give a reverse virtual and magnified image of the object. The main characteristics of any microscope are resolution and contrast. Resolution is the minimum distance at which two points can be seen separately by a microscope. ,
where λ - illuminator wavelength, α - the angle between the optical axis of the lens and the most deviating beam falling into it is the refractive index of the medium. The shorter the wavelength of the beam, the finer details we can observe through a microscope. And the higher the numerical aperture of the lens (n , the higher the resolution of the lens. A light microscope can increase the resolving power of the human eye by about 1000 times. This is the "useful" magnification of the microscope. When using the visible part of the light spectrum, the final resolution limit of the light microscope is 0.2-0.3 microns. However, it should be noted that light microscopy allows us to see particles that are smaller than the resolution limit. This can be done using the "Dark Field" or "Ultramicroscopy" method. Rice. 1 Light microscope: 1 - tripod; 2 - subject table; 3 - nozzle; 4 - eyepiece; 5 - tube; 6 - lens changer; 7 - microlens; 8 - condenser; 9 - mechanism for moving the condenser; 10 - collector; 11 - lighting system; 12 - focusing mechanism of the microscope. 2 The structure of the electron microscope The main part of the electron microscope is a hollow vacuum cylinder (the air is evacuated to exclude the interaction of electrons with its components and the oxidation of the cathode filament). A high voltage is applied between the cathode and anode to further accelerate the electrons. In a condenser lens (which is an electromagnet, like all lenses of an electron microscope), the electron beam is focused and hits the object under study. The transmitted electrons form an enlarged primary image on the objective lens, which is magnified by the projection lens, and is projected onto a screen that is coated with a luminescent layer to glow when electrons hit it. Rice. 2. Electron microscope: 1 - electron gun; 2 - anode; 3 - coil for gun alignment; 4 - gun valve; 5 - 1st condenser lens; 6 - 2nd condenser lens; 7 - coil for tilting the beam; 8 - condenser 2 diaphragms; 9 - objective lens; 10 - sample block; 11 - diffraction diaphragm; 12 - diffractive lens; 13 - intermediate lens; 14 - 1st projection lens; 15 - 2nd projection lens; 16 - binocular (magnification 12); 17 - vacuum block of the column; 18 - camera for 35 mm roll film; 19 - screen for focusing; 20 - chamber for records; 21 - main screen; 22 - ion sorption pump. Bibliography Educational literature .Yu.S. Chentsov, Introduction to Cell Biology, edition 4 Internet sites .#"justify">. #"justify">. #"justify">. #"justify">. #"justify">. #"justify">. http://immunologja.ru/267/
Light microscopy
The light microscope, the main instrument of biology, is an optical system consisting of a capacitor and an objective. The beam of light from the light source is collected in a condenser and directed to the object (Fig. 1). After passing through the object, the light rays enter the lens system of the objective; they build the primary image, which is magnified by the eyepiece lenses. The main optical part of the microscope, which determines its main capabilities, is the lens. In modern microscopes, lenses are interchangeable, which allows you to study cells at different magnifications. Main characteristic microscope as an optical system is the resolution. Images given by the lens can be magnified many times by using a strong eyepiece or, for example, by projecting onto a screen (up to 105 times). It is calculated that the resolution of the lens, i.e. the minimum distance between two points that are visible separately will be equal to
where? is the wavelength of the light used to illuminate the object; n is the refractive index of the medium; ? - the angle between the optical axis of the lens and the most deviating beam entering the lens. The resolution of a microscope depends on the wavelength - the smaller it is, the smaller the detail we can see, and on the numerical aperture of the objective (n sin ?) - the higher it is, the higher the resolution. Typically, light microscopes use light sources in the visible region of the spectrum (400-700 nm), so the maximum resolution of the microscope in this case may not be higher than 200-350 nm (0.2-0.35 microns). If ultraviolet light (260-280 nm) is used, the resolution can be increased to 130-140 nm (0.13-0.14 µm). This will be the limit of the theoretical resolution of the light microscope, determined by the wave nature of light. Thus, all that a light microscope can give as an auxiliary device to our eye is to increase its resolution by about 1000 times (the human naked eye has a resolution
capacity is about 0.1 mm, which is equal to 100 microns). This is the "useful" magnification of the microscope, above which we will only increase the contours of the image without revealing new details in it. Therefore, when using the visible region of light, 0.2-0.3 µm is the ultimate resolution limit of the light microscope.
But still, in a light microscope, particles smaller than 0.2 microns can be seen. This is the "dark field" method, or, as it used to be called, the "ultramicroscopy" method. Its essence is that, like dust particles in a beam of light (Tyndall effect), tiny particles (less than 0.2 microns) glow in a cell under side illumination, the reflected light from which enters the microscope lens. This method has been successfully used in the study of living cells.
If untreated living or dead cells are viewed in transmitted light, then only large details are distinguished in them due to the fact that they have a different refractive index and absorption of light rays than the environment. Big
some of the cellular components differ little in these properties both from the medium (water or tissue solutions) and from each other and, therefore, are hardly noticeable and do not contrast. To study them, one has to change the illumination (while losing image clarity) or apply special methods and devices. One of these methods is phase-contrast microscopy, which is widely used to observe living cells. It is based on the fact that individual sections of a generally transparent cell, although slightly, still differ from each other in density and light refraction. Passing through them, the light changes its phase, but our eye does not catch such a change in the phase of the light wave, since it is sensitive only to changes in light intensity. The latter depends on the amplitude of the light wave. In a phase-contrast microscope, a special plate is mounted in the lens, passing through which the light beam experiences an additional phase shift of oscillations. When constructing an image, the rays that are already interacting
in one phase or in antiphase, but with different amplitudes; this creates a light-dark contrast image of the object.
A similar technique is used in the interference microscope. It is designed so that a beam of parallel light rays from the illuminator is divided into two streams. One of them passes through the object and acquires changes in the oscillation phase, the other goes bypassing the object. In the prisms of the objective, both streams reconnect and interfere with each other. As a result of interference, an image will be built, on which sections of the cell with different thicknesses or different densities will differ from each other in terms of contrast. In this device, by measuring phase shifts, it is possible to determine the concentration and mass of dry matter in the object.
With the help of a polarizing microscope, objects that have the so-called isotropy are studied, i.e. ordered orientation of submicroscopic particles (for example, fission spindle fibers, myofibrils, etc.). In such a microscope, in front of the condenser
a polarizer is placed that transmits light waves with a certain plane of polarization. After the preparation and the lens, an analyzer is placed, which can transmit light with the same plane of polarization. The polarizer and analyzer are prisms made from Icelandic spar (Nicol prisms). If the second prism (analyzer) is then rotated by 90o with respect to the first, then no light will pass through. In the case when there is an object with birefringence between such crossed prisms, i.e. the ability to polarize light, it will be seen as glowing in a dark field. Using a polarizing microscope, one can verify, for example, the oriented arrangement of micelles in the plant cell wall.
Vital (lifetime) study of cells
A light microscope allows you to see living cells. For short-term observation, the cells are simply placed in a liquid medium on a glass slide; if you need long-term observation of the cells, then
special cameras are used. These are either flat bottles with holes covered with thin glasses, or collapsible flat chambers. As objects, one can use free-living cells of protozoa and other unicellular organisms, blood cells, or dissociated tissue cells of multicellular organisms of both animal and plant origin. In any of these cases, cells are studied in specially selected media. Free-living unicellular organisms are considered and studied in the same environments in which they live in natural conditions or are cultivated in the laboratory. So, for some protozoa, artificial environments have been created in which they grow and multiply. Usually these are balanced salt solutions with additions of microorganisms or other protozoa that serve as food for this type of organism.
Blood cells or other free cells of multicellular organisms can be studied in a plasma drop or in special synthetic media.
To study the cells of organs and tissues of animals,
cell culture method. A simpler version of this method is that a small piece of living tissue is placed in a chamber filled with a nutrient medium (a mixture of blood plasma with an embryonic extract or a mixture of a synthetic medium with the addition of blood plasma). After some time, cell division and growth begin on the periphery of such a piece. In another case, the cut piece of tissue is lightly treated with a solution of the enzyme trypsin or helaton - versen, which leads to its dissociation, to the complete separation of cells from each other. Then, such a suspension of washed cells is placed in a vessel with a nutrient medium, where they sink to the bottom, attach to the glass and begin to multiply, forming first colonies, and then a continuous cell layer. This is how single-layer cell cultures grow, which are very convenient for in vivo observations. It is best to use embryonic material to obtain primary cultures from animal tissues; cultures from cells of adult organisms grow very poorly.
When cultivating cells outside the body, in addition to changing the medium, it is important to maintain the required temperature (about 20° for cold-blooded and about 37° for warm-blooded). A prerequisite for cell culture is sterility. There are a number of long-term cultured cells; These are special cell strains that have adapted for decades to growth outside the body. For the most part, these are cells of tumor origin or significantly altered cells that have acquired the properties of tumor cells.
Now the method of culturing cells outside the body is widely used not only for cytological, but also for genetic, virological and biochemical studies.
Plant cells can also be grown in culture. To do this, pieces of tissue are treated with enzymes that dissolve cell membranes. Separated cell bodies, protoplasts, are placed in a culture medium where they divide and form zones of multiplied cells.
Observations on living cells are usually recorded as
photographs taken with special microscope attachments. Living cells can also be filmed. In some cases, such microfilming provides very important information. Using accelerated or slow motion filming (time-lapse filming), one can see in detail the course of such important processes as cell division, phagocytosis, cytoplasmic flow, cilia beating, etc.
Now, with the development of computer technology, with the help of special television cameras, it is possible to obtain an image of cells directly on a computer monitor, record them in the computer's memory, process them in every possible way and receive prints on color or black-and-white printers. It is also possible to use such computer video equipment for time-lapse shooting of moving objects.
In the study of living cells, methods of microsurgery and surgical intervention on cells are used. With the help of a micromanipulator device, cells are cut, parts are removed from them, substances are injected (microinjection), etc. The micromanipulator is combined with a conventional
microscope in which the progress of the operation is observed. Microsurgical instruments are glass hooks, needles, capillaries, which have microscopic dimensions and are made on special devices - "microforges". During micromanipulation, cells are placed in special chambers, into which instruments are also inserted. So, with the help of a micromanipulator, it was possible to transplant nuclei from one amoeba strain to another and prove that it is the cell nucleus that determines the physiological characteristics of the cell as a whole. With the help of a micromanipulator, it was possible to inject colloidal gold into an amoeba cell, and then study the distribution of its particles in the cytoplasm and nucleus.
With the help of such microsurgical instruments, it is possible to turn mitotic spindles in cells, pull out individual chromosomes, and introduce labeled antibodies or various protein molecules into a living cell. In addition to the mechanical effect on cells in microsurgery, ultraviolet microbeams have been widely used recently.
light or laser microbeams. This makes it possible to almost instantly inactivate individual parts of a living cell. Thus, for example, it is possible to inactivate one of the nucleoli and follow the fate of the second, intact one. In this case, it is shown that the second nucleolus takes on an additional load and “works for two”. With the help of microbeams, it is possible to hit a part of the mitotic chromosome or a section of the division spindle. It turned out that damage to the centromere of the chromosome removes the latter from the process of divergence of chromosomes to the poles of the cell during mitosis. Recently, devices with a laser microbeam have been used, which makes it possible to very accurately dose the amount of energy at the point of injury and use very short (nanoseconds) radiation pulses.
When studying living cells, they try to stain them with the help of so-called vital dyes. These are dyes of an acidic nature (trypan blue, lithium carmine), used at a very high dilution (1: 200,000), therefore,
the influence of the dye on the vital activity of the cell is minimal. When staining living cells, the dye is collected in the cytoplasm in the form of granules, while in damaged or dead cells diffuse staining of the cytoplasm and nucleus occurs.
In the study of living cells, fluorescent dyes and fluorescence microscopy are widely used. Its essence lies in the fact that a number of substances have the ability to glow (fluoresce, luminesce) when they absorb light energy. The fluorescence spectrum is always shifted towards longer wavelengths with respect to the radiation that excites fluorescence. So, for example, isolated chlorophyll glows red when illuminated in ultraviolet rays. This principle is used in fluorescence microscopy: viewing fluorescent objects in the short wavelength zone. Typically, such microscopes use filters that provide illumination in the blue-violet region. There are ultraviolet fluorescent microscopes.
Some pigments (chlorophylls, bacterial pigments), vitamins (A and B2), hormones have their own fluorescence. If we examine plant cells with a fluorescent microscope, then against a dark blue background, brightly glowing red grains inside the cell will be visible - these are chloroplasts.
Fluorescence microscopy can be used by adding fluorochromes (fluorescent substances) to living cells. This method is similar to vital staining in that very low dye concentrations (1 x 10-4–1 x 10-5) are also used here. Many fluorochromes selectively bind to certain cell structures, causing their secondary luminescence. For example, fluorochrome acridine orange binds selectively to nucleic acids. Moreover, when binding in the monomeric form with DNA, it fluoresces in green, and in the dimeric form with RNA it glows in red. Observing living cells stained with acridine orange, it is clear that their nuclei have a green glow, and the cytoplasm
and the nucleoli glow red. Thus, in a living cell, using this method, one can see the localization (and in some cases calculate the amount) of certain chemicals. There are fluorochromes that selectively bind to lipids, mucus, keratin, etc.
Fluorochrome-labeled antibodies can also be injected into living cells. For example, fluorochrome-bound antibodies to the protein tubulin introduced into a cell bind to microtubules, which can now be observed in living cells with a fluorescent microscope.
Recently, a combination of light microscopy (especially phase-contrast) with electronic-computer image processing has begun to be widely used to study living cells or their components. It uses video recording electronic processing image, which, as it were, "removes" background levels and highlights, contrasts the observed structures. This technique allows you to see such structures on a television screen.
as microtubules, the size of which (20 nm) is much smaller than the resolving power of a light microscope. The use of such systems not only replaces time-lapse filming, instead of which video recording is used, but also allows computer processing of the image: information about the density of structures, their parameters, number and three-dimensional organization. In combination with fluorescence microscopy, these methods open up great prospects in the study of living cells.
Conventional methods of light microscopy are difficult to use to reproduce a three-dimensional picture of the object under study due to the small depth of field of the microscope. Cells are usually viewed as optical sections at a given depth of focus. In order to obtain a complete three-dimensional reconstruction of the object, a special confocal scanning light microscope is used. With the help of this device, a series of successive optical sections are obtained, taken from different depths and images of which are accumulated in a computer, and according to a special
The program reconstructs a three-dimensional, three-dimensional image of an object. Typically, objects stained with fluorochromes are used.
Study of fixed cells
Despite the importance and simplicity of vital observations, most of the information about the structure and properties of cells has been obtained using fixed material. If the cell is damaged, it begins to undergo a series of changes, and after the death of the cell, autolytic enzymes are activated in it, which leads to gross changes in the cellular structure. Therefore, the tasks of fixation are to kill the cell, stop the activity of intracellular enzymes, prevent the decay of cellular components, as well as avoid the loss of structures and substances, and prevent the appearance of structures that are absent in a living cell (artifact structures). Unfortunately, no chemical fixative has yet been found that satisfies all these requirements.
Aldehydes and their mixtures with other substances are often used for fixation. Alcohols are also used as fixatives.
causing irreversible denaturation of proteins, precipitation of nucleic acids and polysaccharides. Sublimate fixatives and fixatives with picric acid have a precipitating effect. Fixatives containing osmium tetroxide (OsO4) preserve lipids well.
After fixing, objects can be subjected to additional processing in the future. One of the main such treatments is cell staining. It was the additional staining of the cells that made it possible to reveal a lot of details in them.
Slides with fixed smears of unicellular organisms or with tissue culture cells can be directly placed in stains. But in order to stain cells in the composition of organs, it is necessary to obtain their sections. Such sections and individual cells are studied.
To do this, after fixation, pieces of organs are dehydrated in alcohols of increasing concentration, alcohol is replaced by xylene, and xylene is replaced by paraffin. Thus, the fixed tissue, bypassing air drying, is encapsulated in a solid mass of paraffin, which can be cut.
Sections up to 5-10 microns thick are obtained on a special device - a microtome. Such sections are glued to a glass slide: paraffin is dissolved in xylene, xylene is removed with alcohols, which are replaced by water. Sections can now be stained with aqueous dye solutions. To make permanent preparations, stained sections are again dehydrated and embedded in Canadian balsam under a coverslip; these preparations can be stored for a long time.
Various natural and mainly synthetic dyes are used for staining fixed tissues and cells. Natural dyes (hematoxylin, carmine, etc.) are used in combination with mordants (oxides of various metals), with which they form complex compounds (varnishes).
Synthetic dyes are divided into acidic and basic. Basic paints are salts of coloring bases containing amino groups in their composition, which determine their alkalinity. These dyes form salt bonds with
acid groups in cell structures. Consequently, areas of cells rich in acidic groups will bind to basic dyes, will be, as they are called, basophilic. Acid dyes contain hydroxyl groups, or SO2OH groups. Cell structures with basic (alkaline) properties bind to acidic dyes and are called acido- or oxyphilic. There are many mixtures of such dyes that can simultaneously stain different parts of cells in different colors and thereby increase the contrast of cellular and extracellular components. Thus, using various dyes, researchers not only achieve a clear morphological picture of the cell, but also obtain some information about the chemistry of a particular structure.
A number of colorful techniques aimed at identifying specific chemicals are called histochemical and cytochemical. There are a lot of methods of cytochemical analysis.
There are a number of specific colorful techniques, directly
detecting certain substances. These are actually histochemical (cytochemical) reactions. The main requirements for such reactions are as follows: the specificity of dye binding, the immutability of the localization of the substance.
An example of this kind of cytochemical reactions can be a widely used reaction for DNA, the Feulgen reaction (Fig. 8). Its essence is that after specific acid hydrolysis only on DNA, as a result of the cleavage of purines on deoxyribose, aldehyde groups are formed. These groups can react with a specific indicator, Schiff's reagent (discolored fuchsin base), giving red staining at DNA localization sites. The binding of the dye in this case is strictly quantitative, which allows not only to detect and indicate the places where there is DNA, but also to measure its amount. Using the same principle of identifying aldehyde groups, one can see the location of polysaccharides in cells after their hydrolysis with periodic acid (the so-called PAS reaction).
It is also possible to specifically determine the localization of proteins by reactions to individual amino acids (tyrosine, tryptophan, arginine, etc.). Lipids and fats are detected in cells with special dyes (Sudan black), which dissolve well and accumulate in fatty inclusions.
A whole group of cytochemical reactions is associated with the detection of enzymes. The general principle of these reactions is that it is not the protein enzymes themselves that are visible under a microscope, but the places of their localization, which are detected by the products of their specific enzymatic activity.
Quantity final product cytochemical reaction can be determined using the method of cytophotometry. It is based on determining the amount of chemicals by their absorption of light of a certain wavelength. It was found that the intensity of absorption of the rays is proportional to the concentration of the substance at the same thickness of the object. Therefore, by estimating the degree of absorption of light by a given substance, one can find out its amount. For this type of research,
devices - microscopes-cytophotometers; they have a sensitive photometer behind the lens, which registers the intensity of the passed through the object luminous flux. Knowing the area or volume of the measured structure and the absorption value, it is possible to determine both the concentration of a given substance and its absolute content. The method of cytophotometry is widely used in determining the amount of DNA per cell after the Feulgen reaction. In this case, it is not the DNA itself that is photometered, but the content of red-colored fuchsin, the amount of which is directly proportional to the content of DNA. By comparing the obtained absorbance values with standards, it is possible to obtain exact values of the amount of DNA, expressed in grams. This method allows measuring the amount of DNA up to 10-12 - 10-14 g, while microchemical methods have a sensitivity of no more than 10-6 g. With the help of cytophotometry, the DNA content in cells is determined much more accurately than conventional biochemical methods.
Not only light-absorbing
objects and substances, but also radiating (luminous). Thus, methods of quantitative fluorometry have been developed, which make it possible to determine the content of substances with which fluorochromes bind by the degree of luminescence.
To identify specific proteins, immunochemical reactions using fluorescent antibodies are used. This immunofluorescence method has very high specificity and sensitivity. It can be used to identify not only proteins, but also individual nucleotide sequences in DNA or to determine the localization of RNA-DNA hybrid molecules. To do this, first, specific sera containing antibodies are obtained for a protein (for example, tubulin). Purified antibodies are chemically coupled to fluorochromes. Such preparations are poured onto objects and, using a fluorescent microscope, the locations of the desired proteins in the cell are found by the fluorescence of the fluorochrome. However, in order for fluorochrome-labeled antibodies to enter the cell, a plasma membrane is required.
make it permeable. This is usually achieved by cell fixation and partial extraction of lipids from the membranes. To study cytoskeletal proteins using this method, one resorts to the dissolution of cell membranes with various detergents.
To determine the localization of the sites of biopolymer synthesis, to determine the pathways for the transfer of substances in the cell, to monitor the migration or properties of individual cells, the method of autoradiography is widely used - the registration of substances labeled with isotopes (Fig. 9). The principle of this method is very simple, it repeats the method of Becquerel, who discovered radioactive decay. During radioautographic examination, cells are introduced into the medium with a precursor of one of the macromolecular compounds (for example, an amino acid or nucleotide), one of the atoms of which is replaced by a radioactive isotope. For example, instead of 12C, a 14C atom is introduced, instead of hydrogen, tritium 3H, etc. In the process of synthesis, the labeled precursor molecule will also be included in the biopolymer. Register her place
in a cage it is possible by means of a photographic emulsion. If the cells in the layer or on the cut are covered with a photographic emulsion, then after some time, as a result of the decay of the isotope, particles flying randomly in different directions will fall into the zone of the sensitive photolayer and activate silver bromide grains in it. The longer the exposure time, i.e. contact of such a labeled cell with a photographic emulsion, the more AgBr grains will be exposed. After exposure, it is necessary to develop the preparation; in this case, silver is reduced only in the illuminated granules; when the preparation is fixed, unexposed AgBr granules dissolve. As a result, from the mass of granules that covered the object, only those that were activated by? radiation will remain. Looking through the microscope such preparations, on top of which a layer of photographic emulsion is applied, the researcher finds the localization of silver grains, which are located opposite the places where the labeled substance is contained (Fig. 9).
This method has limitations: its accuracy will depend
on the AgBr grain size and on the particle energy. The larger the grain size, the less accurate is the location of the isotope. And the higher the energy of the particle and the longer its path, the farther from the place of decay the activation of AgBr grains will occur. Therefore, special fine-grained photographic emulsions (0.2-0.3 μm) and isotopes with low energy?-particles, mainly the hydrogen isotope, tritium (3H) are used for the autoradiography method. Any precursors of biological macromolecules can be labeled with tritium: nucleotides, amino acids, sugars, fatty acids. Labeled hormones, antibiotics, inhibitors, etc. are also used for radioautographic studies. Water-soluble compounds cannot be studied by autoradiography, since they may be lost during the treatment of cells with aqueous solutions (fixation, development, etc.). Another limitation of the method is a rather high concentration of these substances, since at a low concentration of a radioactive substance, the exposure time increases, while
the danger of the appearance of a background of illuminated AgBr granules due to cosmic radiation.
The autoradiography method is one of the main methods that allows one to study the dynamics of synthetic processes, to compare their intensity in different cells on the same preparation. For example, using this method, using labeled RNA precursors, it was shown that all RNA is synthesized only in the interphase nucleus, and the presence of cytoplasmic RNA is the result of the migration of synthesized molecules from the nucleus.
The method of autoradiography is also used to determine the location of certain types of nucleic acids or individual nucleotide sequences in the composition of cell nuclei or chromosomes - the method of molecular hybridization. To do this, a solution with a labeled nucleic acid (for example, with ribosomal RNA) or with its fragment (for example, with satellite DNA) is applied to a preparation pre-treated so as to denature DNA (break hydrogen bonds in native DNA) in the composition
chromosomes or nuclei, which is achieved by alkaline or temperature treatment of the sample. In the process of DNA renaturation, a molecular hybrid is formed between the labeled nucleic acid from the solution and its complementary DNA region in the preparation. The place of such hybridization is determined radioautographically. This method of molecular hybridization of nucleic acids makes it possible to localize places with a given nucleotide sequence on a chromosome or even the location of certain genes with great accuracy.
The method of molecular hybridization of nucleic acids is also used when staining them with fluorochromes. For example, if the isolated nucleolar DNA responsible for the synthesis of ribosomal RNA is first bound to some fluorochrome, then after DNA renaturation on preparations with this fluorescent ribosomal DNA, it can be seen that fluorescence will be observed only in the nucleoli of interphase cells or only in zones nucleolar organizers of mitotic chromosomes. In this way
it is possible to localize any DNA sequences in cells and even the location in the nuclei of individual chromosomes. This technique is called the FISH method (fluorescent in situ hybridization).
electron microscopy
Considering the characteristics of a light microscope, one can be convinced that the only way to increase the resolution of an optical system is to use an illumination source that emits waves with the shortest wavelength. Such a source can be a hot filament, which in an electric field ejects a stream of electrons, the latter can be focused by passing through a magnetic field. This served as the basis for the creation of an electron microscope, in which a resolution of 1 A (0.1 nm) has already been achieved. According to the design principle, an electron microscope is very similar to an optical microscope: it has an illumination source (electron gun cathode), a condenser system (condenser magnetic lens), an objective (objective magnetic lens), an eyepiece (projection magnetic lenses), but instead of
In the retina of the eye, electrons fall on a luminescent screen or on a photographic plate (Fig. 7).
The main part of such a microscope is a hollow cylinder (microscope column), from which air is pumped out so that there is no interaction of electrons with gas molecules and oxidation of the tungsten filament in the cathode of the electron gun. A high voltage (from 50 to 200-5000 kV) is applied between the cathode and the anode, which causes the electrons to accelerate. There is a hole in the center of the anode, passing through which the electrons form a beam that goes down the microscope column. The lenses of an electron microscope are electromagnets whose field can change the path of electrons (as glass lenses change the path of photons). In a condenser lens, an electron beam is fixed and hits an object with which the electrons interact, are deflected, scattered, absorbed, or pass through unchanged. The electrons passing through the object are focused by an objective lens, which forms
enlarged primary image of the object. Just like in a light microscope, the objective lens determines its main indicators. The primary image is enlarged by a projection lens and projected onto a screen covered with a luminescent layer that glows when electrons hit it. Instead of a luminous screen, the image can be placed on a photographic plate and a picture can be obtained.
The voltage used to accelerate electrons in most transmission (transmission) electron microscopes reaches 50-150 kV. At a voltage of 50 kV, an electron has a wavelength of 0.05 A, and in this case, theoretically, a resolution of 0.025 A (d ~ 0.5?) could be obtained. However, in modern designs of electron microscopes, a resolution of about 1 A is achieved due to insufficient voltage stability, lens current stability, inhomogeneity of the metal of magnetic lenses and other imperfections of the device (theoretically, it is possible to further increase the resolution of an electron microscope
100 times). But the resolution achieved is also enormous (recall that O-N value bonds in a water molecule is 0.99 A): it is now already 106 times higher than the resolving power of the eye!
On the screens and photographic plates of electron microscopes, you can get magnification up to 50,000 times, then another 10 times magnification can be obtained with photo printing, so that the final magnification, at which resolution is maximized, can reach 106 times (for example, if 1 mm is increased by 106 times, then it will reach a length of 1 km).
At present, an electron microscopic image from a fluorescent screen is transmitted directly to a computer using a digital television camera, where it can be processed on the monitor screen in various ways (change the magnification, image contrast, apply densitometry, plani- and morphometry of individual components). Using the printer, you can get prints of the received images.
The maximum resolution of an electron microscope (EM) is realized
now only in the study of metals or crystal lattices. So far, it has not been possible to obtain such a resolution on biological objects due to the low contrast of the object. Biological objects for study in EM are placed on copper grids coated with thin films - substrates (formvar, collodion, carbon), consisting mainly of carbon. Biological objects also mainly contain carbon and, therefore, will differ little in density from the background, will have little contrast. It is shown that the minimum thickness of a biological object with a density of about 1 g/cm3, detected at an accelerating voltage in an electron microscope of 50 kV, is 50 A. Viruses located on the supporting film will be visible in this case in the form of structureless spots, and nucleic acid molecules (DNA thickness is 20 A) are not visible at all due to low contrast. The contrast of biological objects can be increased by using heavy metals or their salts.
Contrasting corpuscular objects
Corpuscular objects can be called particles of viruses, phages, isolated cellular components (ribosomes, membranes, vacuoles, etc.), macromolecules.
One of the widely used methods of contrasting biological objects is metal shading. In this case, thermal evaporation of the metal is carried out in special vacuum installations. In this case, the metal atoms scatter from the place of evaporation along straight trajectories. When they meet an object, they are deposited on it in the form of a layer; its thickness will be greater in places perpendicular to the direction of flight of metal particles. In areas where the object shields the particle beam, "shadows" will appear. Thus, the deposited part of the object has a higher density than the deposited substrate (background), and therefore the object will be visible. This method is widely used not only for contrasting viruses, ribosomes, but also for fairly thin molecules of nucleic acids. The disadvantage of this method is that it leads to an increase in the size of the object by the thickness
deposited layer, which at best reaches 10-15 A. Another disadvantage of it is that it provides information only about appearance and volume of particles. For contrasting shading, platinum, palladium, their alloys, and uranium are used.
In case of negative contrasting of objects with solutions of salts of heavy metals, ammonium molybdate, uranyl acetate, phosphotungstic acid (PTA) are used (Fig. 10). If aqueous solutions of such substances are mixed with biological objects and then applied to substrate films and dried, then the objects (for example, viruses or protein complexes) will appear to be immersed in a thin layer of a high-density amorphous substance. In an electron microscope, they look like light objects on a dark background (like a photo negative). The advantage of the method is that dissolved salts can penetrate deep into the object and reveal its additional details. Negative contrasting is widely used in the study of viruses and membrane enzyme complexes. Filamentous nucleic acid molecules by this method
are poorly identified due to their small thickness.
Heavy metal salts can be used in so-called positive staining. In this case, the contrast agent binds to the structure and increases its electron density. Often, solutions of uranyl acetate in alcohol or acetone are used to positively stain nucleic acids. Uranyl acetate, contrasting nucleic acids, well stains the central cavities of spherical viruses, significantly increases the contrast of ribosomes and allows you to see thin strands of isolated nucleic acids.
Ultramicrotomy
When studying objects in an electron microscope, another complication arises - this is their thickness. The fact is that when an electron beam passes through an object, some of the electrons are absorbed, which leads to heating of the object and to its deformation. Therefore, it is necessary to have thin objects (not higher than 0.1 microns). Another limitation is that even if we consider non-changing objects of large thickness (about
0.5-1 µm), which is possible in principle (for example, in a megavolt electron microscope, see below), then the projections of structures located at different levels along the thickness of the object will be superimposed on the final image. Thus, to study in transmission microscopes internal structure whole cells are bad and inconvenient. The way out of this situation is similar to what was found for light microscopy - to make sections of very small thickness, ultrathin sections (0.05-0.10 microns).
The procedure for their manufacture is in principle similar to that used in light microscopy. Cells and tissues for this are first fixed. Buffer solutions of glutaraldehyde or osmium tetroxide are used as fixatives. The most commonly used double fixation: first glutaraldehyde, and then osmium, which, like a heavy metal, contrasts cellular structures. Then, after dehydration, the fabrics are impregnated with epoxy resins or other plastics in a liquid, monomeric
form. During the polymerization of such plastics, the object impregnated with them is enclosed in solid blocks, which can already be cut into thin sections. Two problems arise here: where to get the ideal knives, the flaws of which would not affect the study of cells at almost molecular levels, and how to make sections as thin as hundredths of a micron. The first problem was solved in this way: it turned out that glass chips have an ideally sharp and notch-free cutting surface (Fig. 11). But glass knives are very short-lived, they are used only once. Diamond knives are used: these are small diamonds sharpened in a special way, they serve for several years.
The problem of making an ultra-thin section was also solved, it would seem, simply - this is the thermal supply of the object. A block with an object enclosed in plastic is mounted on a metal rod, which heats up and thereby moves the object forward by a certain amount in a known time. And if this thermal supply is coordinated with rhythmic cycles
cutting, it is possible to obtain a series of slices of a given thickness. This is achieved using special devices - ultramicrotomes. There are designs of ultramicrotomes, where the object is fed mechanically.
The area of the resulting ultrathin sections is usually very small (0.1-1 mm2), so all operations during ultramicrotomy are under microscopic control. Sections mounted on grids with a substrate must be additionally contrasted - “stained” with the help of salts of heavy metals. In this case, lead and uranium salts are also used, which, by binding to intracellular structures on the cut, positively contrast them.
The technique of making ultrathin sections has opened up enormous opportunities for the application of electron microscopy in literally all areas of biology and medicine.
This method allows the use of cytochemical techniques at the level of electron microscopy: in this case, it is necessary that the reaction products be electron dense, reject
would be electrons. In addition, the reactions should not lead to the appearance of artifact patterns already at the ultrastructural level. The number of cytochemical methods in electron microscopy is not yet large, but this direction is being intensively developed.
In electron microscopic studies, it was possible to apply the methods of autoradiography. In this case, ultra-fine-grained emulsions are used (the size of the granules is about 0.02-0.06 µm). The disadvantage of this method is the very long exposure time, in some cases reaching several months.
Increasing use is being made of methods for preparing ultrathin sections without fixation and for embedding cells in solid plastics. These are cryoultramicrotomy methods, i.e. obtaining sections from frozen tissues, instantly cooled to the temperature of liquid nitrogen (-196 ° C). In this case, almost instantaneous inhibition of all metabolic processes occurs, and water from the liquid phase passes into a solid, but not crystalline,
its molecular structure is disordered (glassy state). Such solid blocks at liquid nitrogen temperature can be cut into ultra-thin sections (the knife is also cooled). The resulting sections are used to detect the activity of enzymes in them, to carry out immunochemical reactions on them, for enzymatic digestion, etc.
For immunochemical studies, antibodies are used that are associated with particles of colloidal gold, the localization of which on the preparations indicates the location of the desired antigen.
The study of sections obtained on cryoultratomes showed that the general structure and composition of cellular components in this case differ little from what is seen when using chemical fixation and conventional methods for obtaining ultrathin sections. Consequently, those structures that have the same composition with different methods of processing the material, apparently close to their lifetime structure, are not artifact.
This is supported by data on the study of cells using other methods of electron microscopy.
The freeze–cleave method is used to study the structure of various membrane components of a cell. It consists in the fact that the object is first quickly frozen with liquid nitrogen, and then transferred to a special vacuum unit at the same temperature. There, the frozen object is mechanically cleaved off with a chilled knife. In this case, the internal zones of frozen cells are exposed. In a vacuum, part of the water that has passed into a vitreous form is sublimated (“etching”), and the cleavage surface is sequentially covered with a thin layer of evaporated carbon, and then metal. Thus, a replica of its cleavage is obtained from a material frozen and preserving its intravital structure (Fig. 12). Then, already at room temperature, the tissue or cells are dissolved in acids, but the replica film remains intact, it is studied under an electron microscope. This method has two advantages: they study replicas from chips
native samples; study the relief of the surface of cell membranes, which is unattainable by other methods. It turned out that in this case, too, the overall organization of the cell and its components is similar to what we see during chemical fixation or cryotomy. This method made it possible to see that globules of integral proteins are located both on the surface and in the thickness of cell membranes, and that the membranes are not uniform in their structure.
The method of obtaining replicas from the microrelief of a sample is widely used in the study of fibrillar components of a cell. So, when studying the cytoskeleton of tissue culture cells or blood cells, the cells are treated with detergents in order to dissolve all membranes. This leads to the fact that all components are washed out of the cell, except for the fibrillar protein components of the cytoskeleton and nuclear materials. Such preparations are then fixed, dehydrated and dried in a special way. Following this, dry preparations are sprayed with carbon and counterstained by spraying.
heavy metals, after which such a replica is removed from the glass on which the cells grew, and viewed in a transmission electron microscope.
Other special methods of electron microscopy of biological objects
Recently, methods of high-voltage (or rather, ultra-high-voltage) microscopy have begun to be used. Devices with an accelerating voltage of 1-3 million volts have been designed. These are very expensive devices, which hinders their widespread use. The advantage of this class of electron microscopes is not that they can obtain a higher resolution (at a shorter electron wavelength), but that at high electron energies, which are less absorbed by the object, it is possible to view samples of large thickness (1-10 µm). The additional use of stereoscopic imaging makes it possible to obtain information on the three-dimensional organization of intracellular structures with their high resolution (about 0.5 nm).
The method of scanning (raster) electron microscopy allows
study the three-dimensional picture of the cell surface. In scanning electron microscopy, a thin beam of electrons (probe) runs over the surface of an object and the information obtained is transmitted to a cathode ray tube. The image can be obtained in reflected or secondary electrons. With this method, a fixed and specially dried object is covered with a thin layer of evaporated metal (most often gold), reflected from which electrons enter a receiving device that transmits a signal to a cathode ray tube. Due to the huge depth of focus of the scanning microscope, which is much greater than that of the transmission microscope, an almost three-dimensional image of the surface under study is obtained. The resolution of this type of instrument is somewhat lower than that of transmission electron microscopes, but instruments with a resolution of 3–5 nm are already being produced (Fig. 13).
Using scanning electron microscopy, one can obtain information about the chemical composition in certain areas.
cells. Thus, the method of X-ray spectral microanalysis is based on the identification and quantitative assessment of the content of chemical elements according to the spectra of characteristic X-ray radiation arising from the interaction of primary electrons with atoms of an object. To obtain such information, of course, objects should not be covered with a layer of metal, as in the usual method of scanning electron microscopy. Moreover, the object must be prepared so that there is no loss or additional introduction of elements. For this, quickly frozen and vacuum-dried objects are used.
Fractionation of cells
In cytology, various methods of biochemistry, both analytical and preparative, are widely used. In the latter case, various components can be obtained in the form of separate fractions and studied. chemical composition, ultrastructure and properties. So, at present, almost any cell organelles and structures are obtained in the form of pure fractions: nuclei, nucleoli, chromatin,
nuclear membranes, plasma membrane, endoplasmic reticulum vacuoles, its ribosomes, hyaloplasmic ribosomes, Golgi apparatus, mitochondria, their membranes, plastids, peroxisomes, microtubules, etc., etc. Recently, pure fractions of centrioles and nuclear pores have been obtained.
Obtaining cell fractions begins with the general destruction of the cell, with its homogenization. Then fractions can already be isolated from the homogenates. One of the main ways to isolate cellular structures is differential (separation) centrifugation. The principle of its application is that the time for particles to settle in the homogenate depends on their size and density: the larger the particle or the heavier it is, the faster it will settle to the bottom of the test tube. To speed up this settling process, the accelerations created by the centrifuge are used. During centrifugation, nuclei and undestroyed cells will settle first and at small (1-3 thousand g) accelerations, at 15-30 thousand g large particles, macrosomes consisting of mitochondria, small plastids,
peroxisomes, lysosomes, etc., at 50 thousand g, microsomes, fragments of the vacuolar system of the cell, will settle. By repeated fractional centrifugation of these mixed sub-fractions, pure fractions can be obtained. So, when separating the macrosomal subfraction, mitochondria, lysosomes, and peroxisomes are obtained separately. When separating microsomes, it is possible to obtain a fraction of the membranes of the Golgi apparatus, fragments of the plasma membrane, vacuoles, and granular reticulum. In cases of finer separation of fractions, sucrose density gradient centrifugation is used, which makes it possible to separate components well, even slightly differing from each other in specific gravity.
The obtained fractions, before they are analyzed by biochemical methods, must be checked for purity using an electron microscope.
Obtaining individual cellular components makes it possible to study their biochemistry and functional features. So you can create a cell-free system for ribosomes, which will
to synthesize a protein according to the messenger RNA specified by the experimenter, isolated mitochondria under selected conditions can carry out ATP synthesis RNA synthesis can occur on the isolated chromatin with the participation of the corresponding enzymes, etc.
Recently, cell-free systems have been used to recreate cellular supramolecular structures. So, using yolks purified from granules, extracts of the cytoplasm of amphibian eggs or eggs sea urchins, it is possible to obtain nuclei with a nuclear envelope from foreign DNA introduced into this cell-free system (for example, bacteriophage DNA). Such DNA binds to histone proteins, which are in excess in such an extract, chromatin (deoxyribonucleoprotein) is formed, which is covered with a double membrane membrane, which even carries nuclear pores. Such model systems help to study subtle, intimate processes, such as the transport of macromolecules from the cytoplasm to the nucleus and vice versa. In cytoplasmic extracts of amphibian and echinoderm eggs
such nuclei can periodically divide by mitosis. These models have made an enormous contribution to the deciphering of the nature of cell cycle regulation.
A great contribution to the biology of the cell is made by the methods of cell engineering. It was found that various living cells can merge with each other if their plasma membranes are treated in special ways. So you can merge a chicken erythrocyte and a human lymphocyte. In this case, a binuclear cell is obtained, a heterokaryon, in which the nucleus of a chicken erythrocyte is activated (Fig. 14). If a heterokaryon is formed from closely related cells (for example, mice and hamsters), then when they enter mitosis, the chromosomes can unite into one metaphase plate. After separation of such a cell, a true hybrid cell will be obtained. Other techniques make it possible to construct cells from nuclei and cytoplasm of different origins (Fig. 15). Thus, by destroying the actin component of the cytoskeleton and subjecting the cells to centrifugation, the cell can be divided into two parts: the nucleus
with a narrow rim of the cytoplasm - karyoplast and on the rest of the cytoplasm - cytoplast. Then, using different karyoplasts and cytoplasts, you can create different combinations of reconstructed cells.
Cell engineering methods are widely used not only in experimental biology, but also for biotechnological purposes. For example, in the production of monoclonal antibodies, cell hybrids are used between lymphocytes from immunized animals and rapidly proliferating myeloma cells. The resulting primary dikaryons form true hybrid cells that intensively proliferate at the expense of the genome of tumor myeloma cells and simultaneously secrete a large number of antibodies, due to the work of the genome of immunized lymphocytes. This technique allows you to get a large number of hybridoma cells that produce large quantities necessary antibodies.
It is not necessary to describe all the methods and techniques used in cytology to study the structure, chemistry and
functions of cells or their components. This overview enough to show the richness of the arsenal of methods in cytology, allowing to give an accurate analysis, starting from the form, general view and cell size, ending with the molecular composition of its individual parts.
The properties of bulk glass to enlarge the image have been familiar to people for a very long time. The oldest lens found by archaeologists in Iraq near the city of Nimrud dates back to the 8th century BC. The inventors of this useful device have remained unknown. It is also unclear who first used it to create a microscope. There is reliable information that the famous scientists of the 16th-17th centuries used combinations of two lenses for their devices - Galileo Galilei, Girolamo Fracastoro, Christian Huygens. History is silent whether these devices were invented before them or not. But it was in that era that optics began to be used for the first time to study the microworld.
Researchers quickly realized that when using several lenses at once, their magnification factors of objects do not add up, but multiply each other. And this gives a significant effect, allowing you to consider the objects of the microworld. The problem was that the first lenses were imperfect and rather rough. Therefore, the image was obtained with defects that increased along with the object of study. To solve this problem, microscopes were developed with a single powerful lens, one of which allowed Antony van Leeuwenhoek to see the plant cell. Only a century and a half later, multi-piece microscopes with several lenses gained wide popularity among scientists. And with the advent of electricity, illumination began to be used, which greatly facilitated the process of observation. This is how a device appeared, similar in principle to a modern light microscope.
Principle of operation
A light microscope uses one of the inherent properties of a beam of light - refraction. The illumination rays are reflected in the mirror, diverge from the object and go in a parallel beam inside the tube, in which the lenses are placed. With the help of lenses, the rays are refracted, i.e. change the angle of their incidence in such a way that they are concentrated on the retina. In this way, the object of observation is enlarged and its previously imperceptible details appear.
Magnification ratios
The eyepiece of a microscope is the lens through which the eye of the observer looks directly. Typically, lenses with a tenfold magnification are used for these purposes. Below, in the tube, there is a number of lenses, each of which has its own magnification - 4, 10, 40 or 100. Since the magnifications are multiplied, then, depending on the chosen lens in combination with a tenfold eyepiece, you can achieve a magnification from 40 to 1000, respectively .
Typically, observation begins with the choice of a quadruple lens, which gives the smallest magnification of 40 times. What for? The fact is that for a detailed consideration of any object, you must first find this object. It is inconvenient to carry out such a search at too high a magnification. Therefore, when studying a microscopic object, as a rule, one starts from the smallest magnification to the largest. A low magnification lens allows you to focus much faster than a high magnification lens.
Useful and useless magnification
The increase is both useful and useless. What is the difference between one and the other? The fact is that the possibilities of any light microscope have a limit. It is theoretically possible to increase the magnification of the device to infinity by using a plurality of lenses.
But in practice there comes a limit, after which a further increase does not make visible the new details of the object. Up to this limit, the increase is considered useful, and after - useless.
Resolution
It makes no sense to enlarge the image to infinity because the resolution of the device is finite. This ability is the distance between two close lines, allowing you to see them separately. For a light microscope, this distance reaches a maximum of 0.2 µm. It is this factor, and not the finite values of the multiplicity, that limit the scope of light microscopy. Smaller objects are accessible to electron and other more modern microscopes.
The lens is a metal cylinder (tube) in which several lenses are mounted. Its increase is indicated by numbers.
Two or three lenses are used for the eyepiece. The purpose of the diaphragm located between them is to focus the field of view. The lower lens focuses the rays emanating from the object, and the observation itself takes place with the help of the upper one.
The lighting device uses a mirror or an electric illuminator. An important detail is the presence of a condenser, which includes two or three lenses. Rising or lowering on a bracket with a special screw, it can concentrate or scatter the light falling on the object. The diameter of the light flux is changed by a special diaphragm controlled by a lever. The degree of illumination of the object regulates the ring, which has a frosted glass or light filter.
Components mechanical system microscope:
- Stand.
- Box with micrometer accessories.
- Tube.
- Tube holder.
- Coarse screw.
- Bracket and condenser displacement screw.
- Revolver.
- Subject table.
The object of observation is placed on the object table. Micrometer mechanisms are designed for small movements of the tube holder with the tube, so that the distance between the lens and the object is optimal for observation. For a more significant displacement, coarse adjustment screws are used. The function of the revolver is a quick change of lenses. This is an extremely convenient device that the first microscopes did not have, so the testers of the past were forced to spend an extremely long time and effort on this procedure. The bracket that holds the condenser is also able to be raised and lowered with a screw.
Usually microscopic biological objects are considered in a light microscope. It was with his help that a living cell was discovered. Today, using a light microscope, you can examine a number of cell organelles that play important role in the functioning of a living organism.
It is this microscope that is used in the teaching of a school biology course.
In particular, with this device you can see:
- The core, which is its main component.
- The wall that forms the surface cellular apparatus, including the membrane.
- Chloroplasts contain chlorophyll, which is important for the plant cell, with the help of which hydrocarbons are taken from water and carbon dioxide.
- Mitochondrial structures and the Golgi complex important for cellular metabolism.
various types of cilia, flagella, vacuoles and photosensitive organelles.
Latest Advances - Most Powerful Microscopes
In 2006, a research group led by the German scientist Stefan Hel and the Argentinean Mariano Bossi completed the development of an optical (light) microscope, which became a real breakthrough in research technologies using high-precision optics. The invention, which was called a nanoscope, allows you to observe objects smaller than 10 nm. At the same time, their high-quality images in three-dimensional format are obtained. This is probably not the limit - studies in different countries aimed at improving the resolving power of the light microscope are ongoing.
To study cells, many methods have been developed and applied, the capabilities of which determine the level of our knowledge in this area. Advances in the study of cell biology, including the most outstanding achievements recent years usually associated with the use of new methods. Therefore, for a more complete understanding of cell biology, it is necessary to have at least some understanding of the relevant methods of cell research.
Light microscopy
The oldest and, at the same time, the most common method of studying cells is microscopy. We can say that the beginning of the study of the cell was laid by the invention of the light optical microscope.
The naked human eye has a resolution of about 1/10 mm. This means that if you look at two lines that are less than 0.1 mm apart, they merge into one. To distinguish structures located more closely, optical instruments are used, for example, a microscope.
But the possibilities of the light microscope are not unlimited. The resolution limit of a light microscope is set by the wavelength of light, that is, an optical microscope can only be used to study such structures, the minimum dimensions of which are comparable to the wavelength of light radiation. The best light microscope has a resolution of about 0.2 µm (or 200 nm), which is about 500 times better than the human eye. It is theoretically impossible to build a high resolution light microscope.
Many cell components are similar in their optical density and, without special processing, are practically invisible in a conventional light microscope. In order to make them visible, various dyes with a certain selectivity are used.
AT early XIX in. There was a need for dyes for dyeing textile fabrics, which in turn caused the accelerated development of organic chemistry. It turned out that some of these dyes also stain biological tissues and, quite unexpectedly, often preferentially bind to certain components of the cell. The use of such selective dyes makes it possible to study the internal structure of the cell more subtly. Here are just a few examples:
Hematoxylin dye stains some components of the nucleus in blue or purple;
· after treatment successively with phloroglucinol and then with hydrochloric acid, lignified cell membranes become cherry-red;
Sudan III dye stains corky cell membranes pink;
A weak solution of iodine in potassium iodide turns starch grains blue.
For microscopic studies, most tissues are fixed before staining. After fixation, the cells become permeable to dyes, and the cell structure is stabilized. One of the most common fixatives in botany is ethyl alcohol.
Fixation and staining are not the only procedures used to prepare preparations. Most tissues are too thick to be immediately observed at high resolution. Therefore, thin sections are made on a microtome. This appliance uses the principle of a bread slicer. Slightly thicker sections are made for plant tissues than for animals, since plant cells are usually larger. The thickness of plant tissue sections for light microscopy is about 10 µm - 20 µm. Some fabrics are too soft to cut straight away. Therefore, after fixing, they are poured into molten paraffin or a special resin, which impregnate the entire fabric. After cooling, a solid block is formed, which is then cut on a microtome. True, for plant tissues, filling is used much less frequently than for animals. This is due to the fact that plant cells have strong cell walls that make up the framework of the tissue. Lignified shells are especially durable.
However, filling can disrupt the structure of the cell, so another method is used, where this danger is reduced? fast freezing. Here you can do without fixing and pouring. Frozen tissue is cut on a special microtome (cryotome).
Frozen sections prepared in this way have a clear advantage, as they better preserve the features of the natural structure. However, they are more difficult to cook, and the presence of ice crystals still breaks some details.
Microscopists have always been concerned about the possibility of loss and distortion of certain components of the cell during fixation and staining. Therefore, the results obtained are verified by other methods.
It seemed very tempting to examine living cells under a microscope, but in such a way that the details of their structure were more clearly manifested. This possibility is provided by special optical systems: phase-contrast and interference microscopes. It is well known that light waves, like water waves, can interfere with each other, increasing or decreasing the amplitude of the resulting waves. In a conventional microscope, as light waves pass through the individual components of a cell, they change their phase, although the human eye does not detect these differences. But due to interference, waves can be transformed, and then different components of the cell can be distinguished from each other under a microscope without resorting to staining. These microscopes use 2 beams of light waves that interact (superimpose) on each other, increasing or decreasing the amplitude of the waves entering the eye from different components of the cell.
Confocal microscope and images made with it: anther cracking, xylem vessels, chloroplasts in stigma cells.
Light microscopy
One of the main methods of cytology today remains microscopy, designed to study the structure of the cell, it is widely used in fundamental and applied research. The invention of the microscope is associated with names Galileo Galilei(Italian) and the Jansen brothers (voice) in 1609-1611. The term "microscope" was proposed by Faber (German) in 1625.
At the moment, there are two main types of microscopy - light and electron. The differences between them are in the principle of considering the object. In the first case, the object is considered in the flow of the visible part of electromagnetic radiation (wavelength = 400-750 nm), in the second case - in the electron flow. These two methods have different resolution. Resolution or resolution limit is the minimum distance between two points at which they can be seen separately. The resolution limit of a microscope is set by the wavelength of the radiation flux in which the object is studied. Therefore, radiation of a given wavelength can be used to study only such structures, the minimum dimensions of which are comparable with the wavelength of the radiation itself. The resolution limit of light microscopy was achieved by microscope designers at the end of the 19th century, and amounted to 0.2 microns. This means that two objects, if they are separated by a distance of less than 0.2 microns, will look like one, even if we greatly enlarge the image, for example, by projecting it onto a screen. Therefore, with the help of a light microscope, it is not possible to examine two centrioles in the cell center, they look like one point (it must be said that in modern commercially produced microscopes, the maximum resolution is not realized). Due to the limited resolution of the light microscope, it can be used to study a limited number of intracellular structures, including: the nucleus, plastids, large vacuoles, and the plant cell membrane. The smallest objects that are clearly visible in a light microscope are bacteria and mitochondria, the size of which is about 500 nm (0.5 μm), smaller objects are not clearly visible, increasing the accuracy of lens processing cannot overcome this limitation, which is set by the wave nature of light.
The resolution depends not only on the wavelength of the light source, but also on the refractive index of the medium through which the object is observed, as well as on the angle of inclination at which the light rays enter the lens. The standard set of microscope objectives are: low magnification objectives (x8) with aperture A=0.2 and high magnification objectives (x20) with A=0.40 and - (x40) with A=0.65. These lenses are called "dry", since the object is viewed through the air (refractive index n = 1). But most microscopes are equipped, in addition, with special immersion objectives, which require a special immersion medium (n=1). Such a medium can be water, the x40 VI lens has an aperture of 0.75. Oil immersion is the most common (n=1.51), at x90 the objective aperture value is A=1.25. In the case of immersion, the resolving power of the light microscope is improved. However, high-resolution lenses have disadvantages: shallow depth of field and low contrast.
The most common method of light microscopy is the bright field method, in which the light rays of the illuminator pass through the object and enter the lens. In this way, fixed and stained cells are studied. The discovery of basic cellular structures is associated with the development and application of a set of dyes that selectively stain cell components and provide contrast for their observation. There is a wide variety of dyes. Some of them are extracted from plants and animals, so far there are no synthetic analogues. For example, the widely used hematoxylin is an extract of a tropical log tree, carmine is a pigment in the fat body of some aphids. These are all so-called nuclear dyes that stain structures containing nucleic acids. The use of a non-nuclear dye, silver nitrate, allowed Camillo Golgi in 1898 to observe and describe what was later called the Golgi apparatus.
Staining of a living cell is possible only in rare cases, so other methods are used to study them. In contrast to the bright field method, when observing objects using the dark field method, the illuminator rays do not enter the lens and the image is created only by scattered rays coming from the object. In this case, against a dark background, one can see luminous particles, which are smaller in size than the resolution of the lens, although it is difficult to determine the size and shape of the particles. Transmitted light dark field microscopy is used to study transparent objects normally invisible in a bright field, and especially to view living cells. Living and dying cells look completely different against a dark background. The protoplast of dying cells glows brighter, there is no explanation for this fact. This method was invented by Zsigmondy (Austrian) in 1912. In a light microscope, objects that change the amplitude of the illumination rays can be distinguished, but living cells are transparent to visible light and the rays passing through the cell practically do not change their amplitude. The human eye is not able to perceive the phase shift of rays without changing the amplitude. Therefore, specifically for the study of living cells, the methods of phase-contrast (invented by Zernike (Dol.) in 1934) and interference microscopy (invented by Lebedev in 1932) are used. In such systems, the passage of light through a living cell is accompanied by a change in the phase of the light wave. Light is delayed as it passes through thick areas of the cell, such as the nucleus. There is a recombination of two sets of waves that create an image of cellular structures.
To study objects with birefringence (starch grains, plant fibers, crystals), polarizing microscopy is used, the foundations of which were laid by Ebner in 1882. This method uses a special polarizer device that converts multidirectional light waves and they acquire one direction.
In fluorescence microscopy, an object is viewed in the light emitted by itself. The first luminescent microscope was designed by Keller and Sindentokf in 1908. This method is based on the ability of a number of substances to glow when illuminated with short-wavelength rays (violet or ultraviolet). Often fluorescence microscopy is used to detect specific proteins, antibodies, Koons was the first to use fluorochromes to bind to antibodies, and this reaction is named after him. In cytoembryological studies, this method is used to study structures containing the carbohydrate callose. This method uses a special optical system with a mercury lamp connected to a light microscope.
Recently, the possibilities of light microscopy have increased significantly due to the use of sensitive video systems. The image created by a light microscope is processed in a video camera. It is cleared of "noise", converted into digital signals and sent to a computer, where it undergoes additional processing to extract hidden information. Computed interference microscopy makes it possible to achieve strong contrast and analyze transparent objects and living cells.
electron microscopy
Long continuous efforts to improve research methods brought the desired results at the end of the Second World War. It was then, thanks to an amazing combination of circumstances, almost at the same time, that scientists were enriched by a number of powerful new tools and methods of research. In morphology, the electron microscope has become such a tool. Created back in the 30s of the 20th century, it had sufficient resolution to penetrate into the cell, up to nanometer-sized structures. At the same time, the electron beam had a weak penetrating power, and this required the preparation of very thin material samples and high vacuum. Such stringent requirements created serious difficulties, but surprisingly short term succeeded in developing methods for preparing tissue samples and constructing devices for obtaining thin sections from them. The quality of the objects steadily improved, and by the early 1960s, many of the previously unknown cellular structures had been described.
So, the resolution of an electron microscope is much higher than that of a light microscope. Theoretically, at a voltage of 100,000 V, its resolution is 0.002 nm, but due to the correction of electronic lenses, it decreases and in reality it is 0.1 nm for modern electron microscopes. Significant difficulties in observing biological objects further reduce the normal resolution, which does not exceed 2 nm. However, this is 100 times greater than that of a light microscope, which is why electron microscopy is called ultramicroscopic.
The general scheme of a transmission electron microscope resembles that of a light microscope. It is significantly larger than the light one and is, as it were, inverted. The electron microscope uses a cathode filament that emits electrons (an electron gun) as a radiation source. Electrons are emitted from the top of a cylindrical column about two meters high. So that there are no obstacles for the movement of electrons, this happens in a vacuum, the electrons are accelerated by the anode and penetrate through a tiny hole into the bottom of the column with a narrow electron beam. The electron beam is focused by ring magnets along the column, which act like the glass lenses of a light microscope. The sample is placed in the path of the electron beam. At the moment of its passage through the sample, part of the electrons is scattered in accordance with the density of the substance, the rest of the electrons are focused, forming an image on a photographic plate or on a screen.
The first electron microscope was created by Siemens in 1939. He made it possible to see many amazing structures in the cell. But for this it was necessary to invent completely new methods for preparing preparations, which began to be used since 1952. Fixation of cells in this case is carried out with glutaraldehyde, which covalently binds proteins, and then with osmic acid, which stabilizes protein and lipid layers. The sample is dehydrated and impregnated with resins that form a solid block after polymerization. Sections for electron microscopy should be approximately 1:200 of the thickness of a single cell. To make such sections, an ultramicrotome (1953) was created, which uses glass or diamond knives. The resulting sections are placed on a special copper mesh. The image in an electron microscope depends on the scattering of electrons, which is determined by the atomic number of the substance. Biological objects consist mainly of carbon, oxygen and hydrogen, which have a low atomic number. To enhance the contrast, they are impregnated with heavy metals such as osmium, uranium, and lead. Thin sections with transmission electron microscopy do not allow one to judge the three-dimensional structure of the cell; this disadvantage can be compensated for by a series of sections, according to which the cell is reconstructed. This is a long process.
There is also a direct method for studying the three-dimensional structure of biological objects - scanning electron microscopy - it was created in 1965. In this case, electrons scattered or emitted by the surface of an object are used to obtain an image, which must be fixed, dried and covered with a film of heavy metal. This method is applicable only to the study of surfaces and its resolution is low - about 10 nm.
Electron microscope
Transmission, probe and scanning electron microscopes. Electron microscopic image of the surface of the anther and pollen grain
Chemical methods of cell research
The classical light microscope has a low resolution, which does not allow studying the details of the structure of a cell with a size of less than 0.25 microns. The second stage of the study of the cell dates back to the time when microscopists were working on improving their instruments. At the same time - the end of the 18th century. - French scientist Antoine de Lavoisier and Englishman Joseph Priestley create a new science - chemistry. Unlike morphology, which progresses from the complex to the simple, chemistry progresses from the simple to the complex. Chemistry began with the identification of elements, atoms, and then moved along the path of studying some of their simplest combinations - molecules.
The synthesis of the biological molecule urea, first carried out in 1828 by the German scientist Friedrich Wöhler, helped to cross the border between inorganic and organic chemistry and allow penetration into the living world of chemistry. This was the beginning of the application of the chemical approach to the study of the cell. In the next hundred years, amino acids, sugars, fats, purines, pyrimidines and other small molecules were discovered, purified, structurally studied and obtained synthetically. Scientists managed to get an idea about the metabolism of these substances in the body and the ways of formation of the main biological molecules from them: proteins, polysaccharides and nucleic acids. But again, formidable obstacles arose in the way of progress: in the face of the complexities of the structural complexity of these large molecules, classical chemistry turned out to be powerless. For a long time, cells were studied mainly by observing them. But with the development of the experimental method in natural sciences they began to resort to it in the study of living organisms. This was facilitated by powerful biomedical research carried out in the second half of the 19th century. At the beginning of the 20th century American Ross Garrison and Frenchman Alexis Carrel found that animal cells can be cultured in a test tube, similar to how they do with single-celled organisms. Thus, they demonstrated the ability of cells to live independently and created a cultivation method that is now one of the most relevant.
But all these methods, essentially revolutionary, were still indirect, the cage remained a closed black box. A huge gap remained unexplored between the smallest particle distinguishable in a light microscope and the largest molecule accessible to chemical research. In this unknown space, important concepts and concepts were hidden, the functions of the described cellular structures, their relationship with known biomolecules remained unknown - without all this, the life of the cell remained unsolved.
In turn, biochemistry has also been enriched by a number of fundamentally new instruments and methods. Of particular interest was chromatography, based on a very simple phenomenon - the formation of a border or halo around the spot (what we see when we try to remove the spot with a special solution). This phenomenon is based on differences in the speed of movement of different colors in a flow of a spreading liquid. At the beginning of the 20th century, the Russian physiologist and biochemist Mikhail Semenovich Tsvet was the first to use this phenomenon. By passing the extract from the leaves through a vertical tube filled with adsorbent powder, he was able to separate the main leaf pigments - green and orange - and get them in the form of separate colored bands or rings along the tube. He called his method chromatography (Greek khroma - color, graphein - write). Tsvet died relatively young and the potential of his method remained unused until the early 1940s. Now there are many variants of chromatography - applicable to all substances that can be identified chemically.
Close to chromatography is gel electrophoresis, in which not a solvent stream, but electromotive force promotes the movement and separation of electrically charged components. These methods revolutionized the field of chemical analysis. Now, trace amounts of a mixture of almost any composition can be analyzed.
The second method, which radically changed chemical research living cells, was the method of isotope labeling. Isotopes are varieties of the same chemical element, differing in atomic mass. Some isotopes exist in nature, many can be obtained artificially through the process nuclear reactions. Isotopes are used to specifically label certain molecules, such molecules can be distinguished from their relatives without disturbing the overall structure. This method is used in the analysis of biosynthetic processes that could not be studied in any other way. For example, with the production of labeled amino acids, it became possible to study their combination into proteins in a living organism or under experimental conditions, even despite the infinitely small amount of newly formed protein, due to its radioactivity. This method became widespread with the creation of atomic reactors and the production of a wide range of radioisotopes. Without the method of labeled atoms, the achievements of cellular and molecular biology would not have been possible.
Thus, both morphology and biochemistry, enriched by new methods, were constantly improved, the gap between their knowledge became smaller and disappeared altogether when it became possible to divide the cell into parts in such a way that each part could be independently studied.
The methods used for such fractionation are mainly based on centrifugation. This method makes use of the differences in physical properties, in particular the size and density, of certain components of the cell to separate them from each other. This made it possible to study a large part of the cell and combine morphological and biochemical knowledge.
However, one part of the cell - its most important central part, the nucleus - remained largely inaccessible until another event occurred. And it began with an attempt to analyze, using genetics, the characteristics of some simple viruses that infect bacteria and are called bacteriophages or bacteria eaters. This study turned out to be the right approach to solving the problem of genetic organization, which, even in the simplest non-cellular organisms, was unusually complex. For a long time, the new discipline known today as molecular biology was limited to the study of viruses and bacteria, but then it literally broke into the eukaryotic cell, allowing the study of the regulation of the cell's vital activity.
Detailed biochemical analysis is needed to study the molecular basis of cell organization. It requires a significant number of cells of a certain type, so it is impossible to use pieces of tissue, because they contain cells of different types. At the first stage of work, pieces of tissue are turned into a suspension. This can be done by destroying the intercellular substance and intercellular connections. To do this, the tissue is treated with proteolytic enzymes that destroy proteins (trypsin, collagenase). Calcium plays an important role in the connection of cells, their adhesion, therefore chelating substances that bind calcium are also used. Then the tissues are subjected to mild mechanical destruction and divided into individual cells. The second stage is the separation of the suspension into separate fractions. To do this, centrifugation is used, with the help of which large cells are separated from small ones, and lungs from heavy ones, or antibodies are used, and the ability of cells with different strengths to attach to glass or plastic. The third stage is the introduction of isolated cells into culture. The first experiments were carried out in 1907 by Harrison, who cultivated the spinal cord of amphibians in a plasma clot. Culture media have a rather complex composition. The standard medium was developed in the early 70s, it contains a set of 13 amino acids, 8 vitamins, mineral salts. In addition, glucose, penicillin, streptomycin, horse or calf serum may be included in the medium. As Hayflick and Moorhead showed in 1961, most mammalian cells die in culture after a certain number of divisions. Human skin cells divide 50-100 times in culture. However, mutant cells sometimes appear in culture, which can multiply indefinitely, forming a cell line. In 1952, a transplantable cell line was isolated from cervical cancer known as the HeLa line. Such lines are stored at a temperature of -70 C; after thawing, they retain the ability to divide. The plant cell culture method was developed by 1964. Using it, it was possible to grow in vitro a whole carrot plant from root cells.
