Microscopic research methods are methods of studying a variety of objects using special equipment. It allows you to consider the structure of substances and organisms, the magnitude of which is beyond the boundaries of the resolving power of the human gaze. The article will conduct a brief analysis of 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, it is a light method of viewing objects. In recent years, other technologies have been actively introduced into practice. So, phase-contrast, luminescent, interference, polarization, infrared, ultraviolet, stereoscopic research methods have gained popularity. All of them are based on the diverse properties of light. In addition, electron microscopy methods are widely used. These methods allow you to display objects using a directed flow of charged particles. It is worth noting that such methods of study are used not only in biology and medicine. The microscopic method for studying metals and alloys in industry is quite popular. Such a study allows us to evaluate the behavior of compounds, develop technologies to minimize the likelihood of fracture and increase strength.
Lighting methods: characteristic
Such microscopic methods for the study of microorganisms and other objects are based on different resolution of the equipment. An important factor in this is the directivity 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. Using these characteristics, various microscopic research methods are built.
Specificity
For study by light methods, objects are usually stained. This allows you to identify and describe their particular properties. In this case, it is necessary that the tissues are fixed, since the stain will reveal certain structures exclusively in dead cells. In living elements, the dye is isolated in the form of a vacuole in the cytoplasm. She does not paint over the structure. But with the help of a light microscope, living objects can also be examined. For this, a vital way of studying is used. In such cases, a dark-field capacitor is used. It is built into a light microscope.
Exploring unpainted objects
It is carried out using phase contrast microscopy. This method is based on beam diffraction in accordance with the characteristics of the object. In the process of exposure, a change in phase and wavelength is noted. A translucent plate is present 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 wave phase shift. But at the same time, passing through the object, the light flux deviates from the plate. As a result, between the rays transmitted through the object and entering the light background, a wavelength difference appears. At a certain value, a visual effect arises - a dark object will be clearly visible on 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 type of phase-contrast method. Anoptral method involves the use of a lens with special plates that change only the color and brightness of the background light. This greatly expands the possibilities of studying unpainted living objects. The phase-contrast microscopic method of research is used in microbiology , parasitology in the study of plant and animal cells, simple organisms. In hematology, this method is used to calculate and determine the differentiation of blood elements and bone marrow.
Interference techniques
These microscopic research methods generally solve the same problems as phase-contrast ones. However, in the latter case, specialists can observe only the contours of objects. Interferential microscopic research methods allow you to study parts of them, to quantify the elements. This is possible due to the bifurcation of the light beam. One stream passes through the particle of the object, and the other by. In the eyepiece of the microscope, they converge and interfere. The resulting phase difference can be determined by the mass of different cellular structures. By sequentially measuring it with given refractive indices, it is possible to establish the thickness of unfixed 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 Nicolas prisms or filmy polaroids. They are placed between the drug and the light source. The polarizing microscopic method of research in microbiology allows you to study objects with heterogeneous properties. In isotropic structures, the speed of light propagation does not depend on the chosen plane. Moreover, in anisotropic systems, the speed changes in accordance with the directivity of the light along the transverse or longitudinal axis of the object. If the refractive index along the structure is greater than along the transverse, a double positive refraction is created. This is characteristic of many biological objects in which a strict molecular orientation is detected. They are all anisotropic. This category, in particular, includes myofibrils, neurofibrils, cilia in the ciliated epithelium, collagen fibers, and others.
Polarization value
A comparison of the nature of radiation refraction and the anisotropy index of an 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. In the light, it is possible to study not only colored objects. The polarization method makes it possible to examine unpainted and non-fixed - native - tissue section preparations.
Fluorescent 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 are added - special dyes. These additives selectively or diffusely spread to individual cellular structures or chemical compounds. This property formed the basis for the use of luminescent microscopy in histochemical and cytological studies.
Areas of use
Using immunofluorescence, specialists detect viral antigens and establish their concentration, identify viruses, anti bodies 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 are used to study materials. Microscopic immunofluorescence method allows to recognize malignant tumors, determine ischemic areas in the heart in the early stages of a heart attack, etc.
Use of ultraviolet light
It is based on the ability of a number of substances included in living cells, microorganisms or fixed, but unpainted, transparent tissues with visible light to absorb UV rays of a certain wavelength. This is characteristic, in particular, for macromolecular compounds. These include proteins, aromatic acids (methylalanine, tryptophan, tyrosine, etc.), nucleic acids, pyramidine and purine bases, and so on. Ultraviolet microscopy allows you to clarify the location and quantity of these compounds. When studying living objects, specialists can observe changes in their life processes.
Additionally
Infrared microscopy is used to study objects that are opaque to light and UV rays by absorbing them with flux structures whose wavelength is 750-1200 nm. To apply this method, there is no need to pre-chemize the preparations. As a rule, the infrared method is used in anthropology, zoology and other biological industries. 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 observation by the left and right eye at various angles. Opaque objects are examined at a relatively small magnification (not 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 a quantum leap in research. This method is widely used in biochemistry, oncology, virology, morphology, immunology, genetics and other industries. A significant increase in the resolving power of the equipment is provided by the flow of electrons that pass through the electromagnetic fields in a vacuum. The latter, in turn, are created with special lenses. Electrons have the ability to pass through the structure of an object or be reflected from them with deviations at different angles. As a result, a display is created on the luminescent screen of the device. With transmission microscopy, a planar image is obtained, with scanning, respectively, three-dimensional.
The necessary conditions
It is worth noting that before undergoing electron microscopic examination, the object undergoes special training. 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 ultrafine sections with diamond or glass knives. Then they are contrasted and studied. A scanning microscope examines the surfaces of objects. To do this, they spray special substances in a vacuum chamber.