Microscopic research methods are methods of studying a variety of objects using special equipment. It allows us to consider the structure of substances and organisms, the magnitude of which is beyond the limits of the resolution of the human eye. In the article, we will briefly analyze microscopic research methods.
General information
Modern methods of microscopic examination are used in their practice by different specialists. Among them are virologists, cytologists, hematologists, morphologists and others. The main methods of microscopic examination have been known for a long time. First of all, this is a light method of viewing objects. In recent years, other technologies have been actively introduced into practice. Thus, the phase-contrast, luminescent, interference, polarization, infrared, ultraviolet, stereoscopic methods of research have gained popularity. All of them are based on various properties. Sveta. In addition, electron microscopic research methods are widely used. These methods allow you to display objects using a directed stream of charged particles. It should be noted that such methods of study are used not only in biology and medicine. The microscopic method of studying metals and alloys in industry is quite popular. Such a study allows us to evaluate the behavior of joints, develop technologies to minimize the likelihood of failure and increase strength.
Light ways: characteristics
Such microscopic methods for studying microorganisms and other objects are based on different resolutions of the equipment. Important factors in this case are the direction of the beam, the features of the object itself. The latter, in particular, may be transparent or opaque. In accordance with the properties of the object, the physical properties of the light flux change - brightness and color, due to the amplitude and wavelength, plane, phase and direction of wave propagation. Various microscopic research methods are based on the use of these characteristics.
Specifics
To study by light methods, objects are usually painted. This allows you to identify and describe certain of their properties. This requires that the tissues be fixed, since staining will reveal certain structures only in killed cells. In living cells, the dye is isolated as a vacuole in the cytoplasm. It does not paint structures. But with the help of a light microscope, living objects can also be examined. For this, a vital method of study is used. In such cases, a dark-field condenser is used. It is built into a light microscope.
Studying unpainted objects
It is carried out using phase-contrast microscopy. This method is based on the diffraction of the beam in accordance with the features of the object. In the process of exposure, a change in the phase and wavelength is noted. There is a translucent plate in the microscope objective. Living or fixed, but not colored objects, due to their transparency, almost do not change the color and amplitude of the beam passing through them, provoking only a shift in the wave phase. But at the same time, having passed through the object, the light flux deviates from the plate. As a result, between the rays passed through the object and entering the light background, a difference in wave length appears. At a certain value, a visual effect occurs - a dark object will be clearly visible against a light background, or vice versa (in accordance with the features of the phase plate). To obtain it, the difference must be at least 1/4 of the wavelength.
Anoptral method
It is a kind of phase-contrast method. The anoptral method involves the use of a lens with special plates that change only the color and brightness of the background light. This significantly expands the possibilities of studying unpainted living objects. A phase-contrast microscopic research method is used in microbiology, parasitology in the study of plant and animal cells,the simplest organisms. In hematology, this method is used to calculate and determine the differentiation of blood and bone marrow elements.
Interference techniques
These microscopic research methods generally solve the same problems as phase-contrast ones. However, in the latter case, specialists can only observe the contours of objects. Interference microscopic research methods allow you to study their parts, to perform a quantitative assessment of the elements. This is possible due to the bifurcation of the light beam. One flow passes through the particle of the object, and the other passes by. In the eyepiece of a microscope, they converge and interfere. The resulting phase difference can be determined by the mass of different cellular structures. By successively measuring it with given refractive indices, it is possible to determine the thickness of non-fixed tissues and living objects, the protein content in them, the concentration of dry matter and water, etc. In accordance with the data obtained, specialists are able to indirectly evaluate membrane permeability, enzyme activity, and cellular metabolism.
Polarization
It is carried out using Nicol prisms or filmy polaroids. They are placed between the drug and the light source. The polarization microscopic research method in microbiology makes it possible to study objects with inhomogeneous properties. In isotropic structures, the speed of light propagation does not depend on the chosen plane. In this case, in anisotropic systems, the velocity changes in accordance withdirectivity of light along the transverse or longitudinal axis of the object. If the magnitude of refraction along the structure is greater than along the transverse one, double positive refraction is created. This is characteristic of many biological objects that have a strict molecular orientation. They are all anisotropic. This category, in particular, includes myofibrils, neurofibrils, cilia in the ciliated epithelium, collagen fibers and others.
Polarization value
Comparison of the nature of the ray refraction and the anisotropy index of the object makes it possible to evaluate the molecular organization of the structure. The polarization method acts as one of the histological methods of analysis, is used in cytology, etc. Not only colored objects can be studied in the light. The polarization method makes it possible to study unstained and unfixed - native - preparations of tissue sections.
Luminescent tricks
They are based on the properties of some objects to give a glow in the blue-violet part of the spectrum or in UV rays. Many substances, such as proteins, some vitamins, coenzymes, drugs, are endowed with primary (intrinsic) luminescence. Other objects begin to glow when fluorochromes, special dyes, are added. These additives selectively or diffusely spread to individual cellular structures or chemical compounds. This property formed the basis for the use of luminescence microscopy for histochemical andcytological studies.
Usage areas
Using immuno-fluorescence, experts detect viral antigens and determine their concentration, identify viruses, antibodies and antigens, hormones, various metabolic products, and so on. In this regard, in the diagnosis of herpes, mumps, viral hepatitis, influenza and other infections, luminescent methods for examining materials are used. The microscopic immunofluorescence method makes it possible to recognize malignant tumors, determine ischemic areas in the heart in the early stages of a heart attack, etc.
Using ultraviolet light
It is based on the ability of a number of substances included in living cells, microorganisms or fixed, but uncolored, visible-light-transparent tissues to absorb UV rays of a certain wavelength. This is typical, in particular, for macromolecular compounds. These include proteins, aromatic acids (methylalanine, tryptophan, tyrosine, etc.), nucleic acids, pyramidal and purine bases, and so on. Ultraviolet microscopy makes it possible to clarify the localization and amount of these compounds. When studying living objects, specialists can observe changes in their life processes.
Extra
Infrared microscopy is used to study objects that are opaque to light and UV rays by absorbing themflow structures, the wavelength of which is 750-1200 nm. To apply this method, there is no need to preliminarily expose the preparations to chemical treatment. As a rule, the infrared method is used in anthropology, zoology and other biological fields. As for medicine, this method is used mainly in ophthalmology and neuromorphology. The study of volumetric objects is carried out using stereoscopic microscopy. The design of the equipment allows you to perform observation with the left and right eyes at different angles. Opaque objects are examined at a relatively low magnification (no more than 120 times). Stereoscopic methods are used in microsurgery, pathomorphology, and forensic medicine.
Electron microscopy
It is used to study the structure of cells and tissues at the macromolecular and subcellular levels. Electron microscopy has made it possible to make a qualitative leap in the field of research. This method is widely used in biochemistry, oncology, virology, morphology, immunology, genetics and other industries. A significant increase in the resolution of the equipment is provided by the flow of electrons that pass in a vacuum through electromagnetic fields. The latter, in turn, are created by special lenses. Electrons have the ability to pass through the structures of an object or be reflected from them with deviations at different angles. As a result, a display is created on the instrument's luminescent screen. With transmission microscopy, a planar image is obtained, with scanning, respectively, a volumetric one.
Necessary conditions
It is worth noting that before undergoing electron microscopic examination, the object undergoes special preparation. In particular, physical or chemical fixation of tissues and organisms is used. Sectional and biopsy material, in addition, is dehydrated, embedded in epoxy resins, cut with diamond or glass knives into ultrathin sections. Then they are contrasted and studied. In a scanning microscope, the surfaces of objects are examined. To do this, they are sprayed with special substances in a vacuum chamber.
