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Research Laboratory staff Publications
The main methods, used in laboratory.

1. Atomic force microscopy.
Atomic force microscope (AFM) is a scanning probe microscope of high resolution. It is used to determine the surface topography with a resolution of tens of angstroms up to atomic resolution. Its advantage is a possibility to study surfaces of non-conductive structures, particularly, the biological membranes and other biological objects. Operating principle of AFM is based on the use of forces of the atomic interactions between the atoms of substances. Repulsive forces acts at short distances between two atoms (about one angstrom, 1 A0 = 10-8 cm), and attractive forces acts at large distances. In scanning AFM these bodies are the investigative surface of biological object and cantilever tip moving over it. As the result we can construct volumetric relief of the sample surface in the real time regime. The resolution capability of this method is approximately 0.1-1 nm horizontally and 0.01 nm vertically.

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The general view, the idea of AFM, the cantilever construction, erythrocyte in 3D format and its profile are presented on the fig. 1. Scanning methodic allows to distinguish any fragment of the scan interested for investigator for its detailed analysis. This procedure is shown on the fig.2.

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2. Calibrated electroporation.

Electroporation is the process of formation of through irreversible pores in the membranes under the action of the external impulse electrical field. Electroporation appears if the transmembrane potential φ ≥ φcr (φ critical - potential of the membrane breakdown). The value of φcr is determined by the number of active centers of the membrane in normal state. Under the action of exogenic physico-chemical agents the number of active centers increases and the critical value of breakdown potential decreases. As the result of the membranes breakdown osmotic hemolysis occurs. The rate of the hemolysis depends on the value of membrane breakdown potential under given conditions:
n(t) = n0 exp (- βt) + nr

β is the constant of the hemolysis rate. The curve n (t) is the kinetic curve of hemolysis. It is possible to estimate the average number of defects in membranes by the kinetic curve of hemolysis and the constant of hemolysis rate. The more the external factor causes damages, the more is the rate constant. The main idea of the method is shown on the fig.3, equipment on the fig. 4, scheme of the experiment on the fig.5.

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The main results.
For the analysis of red blood cells structure in nanoscale it is necessary to distinguish informative indicators, which form objective criteria of estimation. For this purpose the spatial Fourier transform is used for obtaining of surfaces of three orders in accordance with different spectral windows. Each spectral window corresponds to the physiological structure level of the membrane. This method of surface decomposition is shown on Fig. 6.

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Different substances and actions cause different reaction of membrane structures. Thus, for example, fixative gluteraldehyde, impulse electrical field, relaxant cause different change of surface orders under their action on cells membranes. This is shown on Fig. 7.

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Under the action of erythrocyte membrane modificators, which cause aggregation of proteins band 3, defects (non- through pores) appear on membrane surface. Their sizes and configuration depends on modificator concentration. The defects appeared on erythrocyte membrane under action of Zn ions are shown on Fig.8.

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The use of perfluorocarbon emulsion is perspective for correction of such defects. The effect of correction of membrane defects by perfluorocarbon is shown on Fig. 9.

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Perfluorocarbon is also highly effective after membrane damage by ionizing radiation. The corrective action of perfluorocarbon after the influence of shortwave ultraviolet radiation is shown on Fig.10.

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The result of action of heavy nuclei B 11 on red blood cells membrane, produced by an elementary particle accelerator in JINR, Dubna, is shown on Fig. 11.

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The series of experiments was carried out in the study “Influence of perfluorocarbon on membrane damages after reinfusion” by the scheme hypotension - reinfusion - correction by perfluorocarbon. 186 cells were scanned, 720 images of 3 orders were mathematically processed. It was shown that the first 5 minutes of hypotension cause the greatest stress of erythrocyte membranes structures. The first order – flickerihg – was maximally changedAfter 60 minutes flickering was decreased, but the second and the third orders were increased. Thus, the structure of spectrin matrix was disordered and proteins were aggregated. The effective method of membrane defects correction by perfluorocarbon is shown in these experiments. Subsequent injection of emulsion gives better result than perfluorocarbon injection before hypotension (Fig. 12,13).

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