A. I. Yusipovich

Laser interference microscopy in erythrocyte study

With the laser interference microscopy (LIM) technique, one can measure phase height of cells—a variable proportional to the cell thickness and the difference in the refractive indices of the cell and the surrounding medium. This makes functional optical cell imaging possible, and estimation of shape, thickness, and area of erythrocytes feasible. In this paper, we studied changes in erythrocyte shape and volume with osmolarity and pH. Obtained from the LIM technique, erythrocyte phase heights and area values, as well as the hematocrit-measured erythrocyte volume, were used to estimate changes in the refractive index with osmolarity and pH. A comparison between the estimated refractive index with the refractive index, calculated in the assumption that it can only depend on the hemoglobin concentration in the cell, indicates that these two estimates are identical in the range of osmolarity (250–1000 mOsm) and pH (4.5–10.0) values. Thus, refractive index changes result exclusively from the changes in hemoglo...

Combined Raman and atomic force microscopy study of hemoglobin distribution inside erythrocytes and nanoparticle localization on the erythrocyte surface

The letter describes a combined technique of atomic force microscopy (AFM) and micro-Raman spectroscopy (mRS) to estimate the distribution of cytosolic hemoglobin (Hb) and nanoparticles (NPs) inside and on the erythrocyte surface, respectively. We have shown that cytosolic hemoglobin is distributed uniformly inside the cell while NPs absorb on the cell surface irregularly, forming nanoaggregates. The obtained data provide new insight into the surface enhanced Raman spectroscopy of living cells.

Laser interference microscopy of amphibian erythrocytes: impact of cell volume and refractive index

This study examined the action of anisosmotic media on the volume of nucleated erythrocytes isolated from Rana temporaria. Elevation of medium osmolarity from 100 to 345 mOsm resulted in attenuation of mean cell volume by more than 3‐fold, estimated by hematocrit measurement. By contrast to this ‘classic’ erythrocyte volume evaluation technique, we did not observe any significant cell volume modulation by examining the 3D reconstruction of erythrocyte interference images obtained by laser interference microscopy. Comparative analysis of mean cell volume, phase height and cell area appraised by laser interference microscopy showed that the lack of visible alterations of phase image geometry was caused by sharp elevation of the average refractive index of the cytoplasm in shrunken cells. Thus, our results show for the first time that laser interference microscopy in combination with a direct method for cell volume measurement may be employed for estimation of the refractory index of intracellular milieu and for assessment of changes of physical chemical properties of the cytoplasm evoked by diverse stimuli including osmotic stress.

Non-invasive study of nerve fibres using laser interference microscopy

This paper presents the results of a laser interference microscopy study of the morphology and dynamical properties of myelinated nerve fibres. We describe the principles of operation of the phase-modulated laser interference microscope and show how this novel technique allows us to obtain information non-invasively about the internal structure of different regions of a nerve fibre. We also analyse the temporal variations in the internal optical properties in order to detect the rhythmic activity in the nerve fibre at different time scales and to shed light on the underlying biological processes. We observe pronounced frequencies in the dynamics of the optical properties and suggest that the oscillatory modes have similar origin in different regions, but different strengths and mutual modulation properties.

Evaluation of erythrocyte shape and status by laser interference microscopy

The area, thickness, and volume of erythrocytes of different types (discocytes, stomatocytes, and echinocytes) from normal subjects and coronary patients were studied by laser interference microscopy. Increase of pH value leading to the stomatocyte-discocyte-echinocyte transformations resulted in a slight decrease of cell volume. In coronary patients, erythrocyte had larger area and volume and exhibited increased aggregation capacity compared to erythrocytes from controls. The results recommend laser interference microscopy as an adequate method for erythrocyte evaluation in laboratory diagnostic measurements.

Visualization of the extracellular polymeric matrix of Chromobacterium violaceum biofilms by microscopic methods

517 We have previously presented results that indicated direct correlation between the ability to form a fully functional matrix and the sensitivity of microbial bio films to thermal, hyperosmotic, and acidic shock [1]. In particular, we compared the sensitivity to these fac tors in the pigment free (producing no violacein) strain Chromobacterium violaceum WT, which pos sesses a normal quorum sensing system and matrix forming ability, and in the mutant strain C. violaceum CV026, with impaired synthesis of N acyl homoserine lactones (AHL), components of the regulatory quo rum sensing system, and with a decreased capacity for matrix formation. Violacein synthesis by strain CV026 is induced by N hexanoyl homoserine lactone (C6 AHL), as well as by other AHLs with acyl side chains containing from 4 to 8 carbon atoms. This strain is therefore used as a biosensor for AHLs [2]. In the previously reported experiments [1], biofilm matrix production was quantified according to the standard technique [3] with minor modifications: the matrix was stained with 1,9 dimethylmethylene blue (DMMB) specific to the matrix components, DMMB was extracted with 96% ethanol, and optical density of the extract was measured at 540 nm [1]. Earlier, such dyes as Congo red (staining only cellulose and cellu lose like glycans) [4] and Alcian blue (staining acidic polysaccharides) [5] were proposed to visualize the biofilm matrix. However, the results of the cited work [3], as well as our data (see below) show that DMMB has the highest selectivity toward the matrix compo nents and practically does not stain matrix free bacte rial cells.

Changes in the State of Hemoglobin in Patients with Coronary Heart Disease and Patients with Circulatory Failure

Morphology of erythrocytes and conformation of hemoglobin-derived hematoporphyrin were studied in patients with coronary heart disease (CHD) and patients with circulatory failure using laser interference microscopy and Raman spectroscopy. Correlation was revealed (r=0.81) between hemoglobin oxygen saturation and oxyhemoglobin fraction in erythrocytes evaluated by Raman spectroscopy. Patients with CHD and patients with circulatory failure showed reduced oxygen-releasing capacity of hemoglobin and hemoglobin content and increased oxygen-binding capacity of hemoglobin, and hemoglobin affinity for oxygen. Significant differences from the control were observed only in patients with circulatory failure. It was found that hemoglobin content, hematocrit, and the shape of erythrocytes during CHD and circulatory failure did not differ from the control, whereas the area of erythrocytes was increased.

Studies of the blood antioxidant system and oxygen-transporting properties of human erythrocytes during 105-day isolation

Effects of strict 105-day isolation on the blood antioxidant status, processes in erythrocyte membranes, and oxygen-binding properties of hemoglobin were studied in six male volunteers (25 to 40 years old) in ground-based simulation of a mission to Mars (Mars-105 experiment). The study was performed with venous blood samples and red blood cells isolated from them, which were collected during the baseline data collection period, on days 35, 70, and 105 of the experiment, and on days 7 and 14–15 after its completion. Biochemical (determination of enzyme activity and thin-layer chromatography) and biophysical (laser interference microscopy and Raman spectroscopy) methods showed changes in the relative content of lipid and phospholipid fractions, suggesting an increase in the membrane microviscosity and the content of TBA-RP (active lipid peroxidation products interacting with thiobarbituric acid). A significant increase in the activity of glucose-6-phosphate dehydrogenase and superoxide dismutase and a reduction in the catalase activity was found, which indicates both reparative processes in red blood cells and imbalance between the amount of generated reactive oxygen species and antioxidant protection mechanisms in cells. The hemoglobin affinity for oxygen and the blood level of oxyhemoglobin also increased. It is assumed that the adaptation of the body to stresses experienced during and after the experiment can disturb the balance between the antioxidant defense systems. The latter, in turn, leads to peroxidation of membrane phospholipids, alteration in their content, increase in membrane microviscosity, and eventual disturbance of the gas-exchange function of red blood cells.

Use of nanoparticles for studying the conformations of submembrane hemoglobin

The advantages and specific features of integrated application of atomic force microscopy, laser interference microscopy, and Raman microscopy in the study of erythrocytes are discussed. For successful application of Raman microscopy in the surface-enhanced mode, use was made of silver colloids. The dependence of the enhancement of Raman signals on silver nanoparticle size is demonstrated. The use of developed methods in clinical diagnostics is discussed.

Role of the state of erythrocyte cytoplasm in the change of hemoglobin affinity for oxygen

The dimensions and refractive properties of erythrocytes and the affinity of their hemoglobin for oxygen have been assessed using laser interference microscopy and Raman spectroscopy. Lowering the pH and addition of 2,3-diphosphoglycerate cause comparable changes in the cell shape and the state of cytoplasm, as well as in the oxygen affinity of hemoporphyrin, but the mechanisms of these effects appear to be different. It is shown that acidification mainly decreases the hemoporphyrin oxygen-binding capacity, whereas 2,3-diphosphoglycerate increases the ability of hemoporphyrin to release oxygen.