Cell research methods

Microscopic methods

Special devices are used to study the structure of biological objects: microtomes (for preparing ultra-thin sections), hand or stand magnifiers, as well as light and electron microscopes.

Light microscopy uses visible light or ultraviolet radiation. Depending on the type of object and tasks, different modifications of the method are used:

• Phase-contrast microscopy. Significantly increases image contrast, which is especially important for studying transparent and unstained structures.
• Polarization microscopy. Relies on the phenomenon of double refraction (anisotropy). In such a microscope, anisotropic structures appear as brightly glowing elements against an absolutely dark background.
• Interference microscopy. The light beam is split: one part passes through the cell, the other past it. The transmitted beam receives a phase shift, and the final image is built on the density difference of cell regions (the denser, the higher the contrast).
• Dark-field microscopy. The image is created by light reflection from the studied object. The smallest particles appear as glowing points on a dark background because direct light does not enter the objective.
• Ultraviolet microscopy. Based on the ability of cellular components to specifically absorb rays of a certain wavelength. Allows the identification of structures without prior staining.
• Fluorescence microscopy. Used for living cells in ultraviolet light. Either the intrinsic glow of substances or the glow after adding dyes (fluorophores) is evaluated. Helps visualize fats, vitamins, and nucleic acids.

Electron microscopy achieves the highest resolution through the use of an electron beam instead of ordinary light.

• Transmission electron microscopy. The image appears on a fluorescent screen when the electron beam is scattered. The higher the scattering capacity of a particular area (it holds back more electrons), the darker it looks.
• Scanning electron microscopy. The electron beam passes over the surface, making it possible to obtain an image of the three-dimensional surface of the cell.

Study of chemical composition

In addition to structure, it is important for researchers to understand the chemical composition and distribution of substances inside the cell. A separate group of methods is used for this:

1. Cytochemical methods. Rely on the specific binding of certain dyes to specific chemical components of the cell.
2. Autoradiography. Substances labeled with radionuclides (radioactive isotopes) are introduced into the cell, after which devices record their movement and accumulation.
3. Immunohistochemical methods. Use the antigen-antibody reaction to precisely determine antigens in cells and detect tissue antibodies.
4. Cytospectrophotometry. Allows quantitative assessment of substance content. The method is based on measuring the absorption of light rays of a certain wavelength.
5. X-ray crystallography. A highly specialized method used to decode the spatial configuration of complex molecules, primarily proteins and nucleic acids.

Physiological and vital studies

To understand how a cell functions in real time, intravital (vital) methods are used:

• Intravital staining. Living cells are treated with dyes at very low concentrations. This avoids the toxic effect and death of the object.
• Tissue culture method. Living cells (for example, tissue culture cells) are placed in an artificial nutrient medium where they can autonomously grow and multiply outside the organism.
• Cytophysiological method. Implies the study of physiological processes in isolated cells — for example, in muscle cells, nerve trunks, or ganglia.
• Vital study of cells. Observation of living free-living protozoa or blood cells.
• Microsurgery. Performing delicate operations on the cell itself and its organelles using special micromanipulators.

Fractionation method

For a detailed study of the composition of individual cellular components, differential ultracentrifugation is used. This method is based on the separation of organelles and intracellular inclusions according to their density. The tissue is placed in a special device — an ultracentrifuge. Due to very rapid rotation, organelles precipitate, forming distinct layers in accordance with their density. Subsequently, these layers are carefully separated and subjected to individual study.

Briefly: answers to topical questions

Class and exam questions

What else to study on this topic

• Mechanisms of specific dye binding in cytochemistry
• The principle of microtome operation and stages of histological section preparation
• Features of the antigen-antibody reaction in the immunohistochemical method
• Properties of radioactive isotopes used in autoradiography
• Selection of nutrient media for the tissue culture method

Other topics in the section "Basics of Cytology"

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