Methods of Cytology

The morphology, ultrastructure and function of cell organelles is currently using the following basic methods: light and electronic, dark-field, phase contrast, polarizing, fluorescent microscopy used to study the structure, ultrastructure of fixed cells, and differential centrifugation, allowing you to select individual organelles and analyze their cytochemical, biochemical, biophysical, and other methods.

Light microscopy. The principle of the method is that the light beam passing through the object enters the lens system of the lens and generates a primary image, which increases with the lens in the eyepiece. The main optical part of the microscope, determining its main features, lens.

Electronic microscopy. In principle, the electron microscope is arranged as the light, only the light beam takes the electron beam and focuses the beam lenses, and electromagnets. However, for electron beam wavelength much shorter than the wavelengths of visible light that provides better resolution electron microscope in comparison with the light microscope. Permission from modern electron microscopes 0.2 to 1 nm.

Dark-field microscopy. Its essence is that like the dust in the beam of light (Tyndall effect) in the cell with the side light glow smallest particles (less than 0.2 μm), the reflected light which enters the lens of the microscope. This method has been successfully applied in the study of living cells.

The method of phase - contrast microscopy is based on the fact that certain parts transparent, in General, cells, although a little, but still differ from each other in density and refraction. Passing through them, the light changes its phase, but the phase change of the light wave our eyes can't follow, as it is sensitive only to changes in light intensity.

Using a polarized microscope study objects with so-called isotropy, i.e., an ordered orientation of submicroscopic particles (fibers spindle, myofibrils, and others). Such a microscope before the condenser is placed a polarizer which transmits light waves at a certain speed polarization. After preparation and the lens is placed in the analyzer, which can transmit light from this plane of polarization. When between crossed prisms will be the object having double refraction, i.e., the ability to polarize light, it becomes visible as a glowing against a dark field. Using a polarizing microscope can be seen, for example, in the oriented arrangement of the micelles in the cell wall of plants.

When studying living cells are widely used fluorescent dyes and method of fluorescent microscopy. Its essence lies in the fact that a number of substances have the ability to glow (fluoresce, luminesce) in the absorption of light energy. Fluorescence spectrum is always biased towards larger wavelengths with respect to vozbujdayuschego fluorescence radiation. This principle allows us to consider fluorescent objects in the area of short waves. In such microscopes apply filters, giving light in the blue-violet region. There are ultraviolet fluorescence microscopes.

Native fluorescence have some pigments (chlorophyll, bacterial pigments), vitamins (A, B2), hormones. You can use the method of fluorescence microscopy, adding living cells fluorescing substances that selectively bind to specific structures, causing them to secondary luminescence.

To study the cells of organs and tissues of animals used method of cell cultures. A simple variant of this method is that in the chamber filled with a nutrient medium, put a small piece of living tissue. After some time on the periphery of this piece begins the division and growth of cells. In another case, cut a piece of cloth lightly treated with a solution of the enzyme trypsin or hellatone of versene that leads to its dissociation, to complete separation of the cells from each other. Then such a suspension of washed cells is placed in a container with a nutrient medium, where they sink to the bottom, attached to the glass and begin to multiply, forming a first colony, and then a continuous cellular layer. So grow a single-layer cell culture, very convenient for in vivo observations. When cultivated outside the body in addition to changing environment it is important to maintain and required temperature, and sterility. In the culture can be grown and plant cells.

Observation of living cells is usually captured in the form of photographs, made using a special fotonation to the microscope. The living cell can be removed and the film. By applying such accelerated or delayed filming (time-lapse shooting), you can detail to see the flow of important processes such as cell division, phagocytosis, throughout the cytoplasm, the beating of cilia and so on Despite the importance and sufficient simplicity vital observations, most of the information about the structure and properties of cells received on the fixed material.

Task fixation is to kill the cell, stop the activity of intracellular enzymes, to prevent the breakdown of cellular components, as well as to avoid the loss of structures and substances that prevent the emergence of structures that are absent in the cell. As the clamps used aldehydes and their mixtures with other substances, alcohol, mercuric chloride, cityregional osmium, etc. After fixing the objects are subjected to additional treatment to staining. For painting use a variety of natural (hematoxylin and Carmine and others) and mainly synthetic dyes. But for staining cells in the part of the authorities need to get their slices. Cuts up to 5 -10 µm get on the microtome.

There are a number of specific colorful techniques aimed at identifying specific chemicals received navaneethakrishnan or cytochemical. This is actually the cytochemical reactions. The main requirements for such reactions, the following: specificity, binding dye, invariance, localization substances. The quantity of the finished product cytochemical reactions can be determined using cytophotometry. To identify proteins specific sequences of nucleotides in DNA or to identify the localization of the RNA-DNA hybrid molecules using the method of immunofluorescence assay.

To clarify the localization of sites of synthesis of biopolymers, to determine the transfer of substances in the cell, to monitor migration or properties of individual cells widely used method of autoradiography - registration of substances labeled isotopes. For example, using this method when using labeled precursors RNA was shown that all RNA is synthesized only in the interphase nucleus, and the presence of cytoplasmic RNA is the result of migration of the synthesized molecules from the nucleus.

In Cytology apply various analytical and preparative methods of biochemistry. In the latter case can be obtained in the form of separate fractions of various cellular components and to study their chemistry, ultrastructure and properties. At the present time in the form of pure fractions get almost any cellular organelles and structures.

One of the main methods for the isolation of cell structures is differential (separation) centrifugation. The principle of its application is that the time for sedimentation of particles in the homogenate depends on their size and density: the larger the particle is, or what she is heavier, the faster it will settle to the bottom of the tube. To speed up the sedimentation process using acceleration generated by the centrifuge. When repeated fractional centrifugation mixed padmakshi you can obtain a pure fraction. In cases of more subtle division of fractions using centrifugation in density gradient of sucrose, which allows a good separation between the components, even slightly differ from each other in weight. The fractions obtained before they are analyzed by biochemical methods, should be checked for purity using an electronic microscope.

Test questions:

1. Levels of organization of living matter

2. The cell theory of organization of organisms

3. Research methods in cytology

4. Objectives and subject of Cytology

5. Apparatus light microscope

6. The device of an electron microscope

7. Safety at work cytological

8. Requirements for the preparation of biological material for cytology

9. Fixing agents whose mechanism of action

10. cytochemistry, material requirements and opportunities

11. Quantitative analysis (morphometry), the requirements and opportunities

12. Artifacts in cytology, the way objectification results