[Investigation of lymphocytes activation using a method of coherent phase microscopy].

Investigation of initial stages of T-lymphocytes activation was performed by coherent phase microscopy (CPM) based on the measurements of phase thickness and diameter of the cell and its organelles. Lymphocytes where activated in two ways: by addition of 2 10-6 g/ml phytohemagglutinin (PHA) and with He-Ne laser (lambda = 633 nm, 10-3 W) radiation. It was shown that phase thickness is proportional to refractivity (difference between refractive indices of an object and the surrounding medium) and geometrical thickness. Phase thickness decreased during activation independently of the nature of the stimuli. Phase thickness in healthy donors was 220 + 60 nm; decreased to 110 + 30 nm 1 h after addition of PHA. The same values were achieved 30 min after stimulation with the He-Ne laser. Characteristic changes in phase thickness in the nucleus and nucleolus were observed after lymphocytes were stimulated with the laser.

[Coherent phase microscopy: a new method of identification of intracellular structures based on their optical and morphometric parameters].

A new method for the identification of intracellular structures of a living cell and obtaining the quantitative parameters characterizing these structures by means of coherent phase microscopy is proposed. The method is based on the analysis of the histogram of a cell phase image and its decomposition by phase height levels. In the spherical and cylindrical approximation of the cell, the method makes it possible to separate the contributions of phase-contrast intracellular structures to the integral refractive index of the whole cell. The calculation of refractive indices of intracellular structures is illustrated on a two-component model of a spherical cell. The possibility of determining the refractive indices of cellular organelles was shown by an example of cyanobacterium Anabaena variabilis ATCC 29413 cells and Cancer mammae breast cancer cells. Three most contrast cellular structures in the phase images of the cells were identified, and their refractive indices were determined. For Anabaena variabilis cells, these structures were the cell wall ( = 1.39), tylakoids ( = 1.47), and the nucleoid ( = 1.58); for breast cancer cells, these were the cytoplasm ( = 1.34), the nucleus ( = 1.36), and the nucleolus ( = 1.44). Using this method is possible to identify phase-contrast intracellular structures and calculate their refractive indices. This enables one to follow morphofunctional changes of not only the whole cell but also its individual organelles.

[Cooperative processes in mitochondria: registration by dyanmic phase microscopy]. / Kooperativnye protsessy v mitokhondriiakh: registratsiia metodom dinamicheski fazovoi mikroskopii.

The spectra of phase fluctuations in mitochondria were measured by the method of dynamic phase microscopy. Contrasting components were revealed whose intensity markedly changed at distances of 100-300 nm. Similar frequency components were observed in the spectra of ATP-stimulated fluctuations in liposomes with the incorporated ATPase. The values of the frequency and intensity of contrasting components in spectra of liposomes, mitochondria, and cells are presented. The possibility of determining the position of active enzyme complexes from their characteristic frequencies is discussed.

[Registration by dynamic phase microscopy of characteristic frequencies of phase height fluctuations of the myelin nerve fiber at rest and under stimulation]. / Registratsiia metodom dinamicheskoi fazovoi mikroskopii kharakternykh chastot fluktuatsii fazovoi vysoty uchastkov mielinovogo nervnogo volokna v pokoe i pri stimuliatsii.

The method of dynamic phase microscopy was used to study the dynamics of changes in the structure of paranodal and nodal regions of a myelin nerve fiber of brown frog Rana temporaria at rest and under stimulation. Regular structural changes with frequencies of 5.3 and 10.8 Hz in the nodal region of the myelin nerve fiber were detected. A rhythmic excitation leads to additional changes in the structure of the nodal region with a new frequency of 5.6 Hz. It is likely that the regular changes in the nodal region of the myelin nerve induced by rhythmic excitation are due to slow changes in the axolemma (changes in the mode of lateral diffusion of membrane phospholipids), induced by developing trace changes in the membrane potential of the axolemma. The fact that these changes do not occur in the paranodal region of the fibre may indicate either the localization of regular structural changes in the axolemma or the difficulties that arise during the registration of the useful signal in the vicinity of myelin by this method.

[Quantitative characteristics of polarization images]. / Kolichestvennye kharakteristiki poliarizatsionnykh izobrazhenii.

Paper shows that quantitative data on the anisotropic structures in the form of three-dimensional images having a high spatial resolution could be practically obtained and presents the results of examining a number of anisotropic objects using the experimental model of a computerized polarizing microscope.

Reduction of phase thickness is a characteristic reaction of the nucleoli to toxicity as shown by coherent phase microscopy.

Optical parameters of human cell nucleoli (HCT116 colorectal cancer cells) in depolymerization of microtubules and depletion of intracellular ATP pool were studied by coherent phase microscopy. These influences were associated with a rapid (recorded within the first minutes) reduction of the phase thickness of the nucleoli. These changes are similar to the nucleolar response to direct inhibitors of transcription. Hence, quantitative parameters of coherent phase microscopy describe common reaction of the nucleolus to stress; reduction of optical thickness of the nucleolus is a component of this reaction.

[Research on the early stages of spore germination in Bacillus subtilis using dynamic phase microscopy].

The changes in the state of Bacillus subtilis spores that occur during germination were analyzed using dynamic phase microscopy (DPM). DPM is based on monitoring and analyzing the interference image of a specimen in a coherent laser beam. The optical path difference (the phase thickness of the specimen, PT) depends on the geometrical height of the specimen and its refractive index. We demonstrated that the maximum PT value is a convenient criterion of the physiological state of the organism involved: PT is > or = 80 nm, 40-50 nm, and < or = 0 in dormant, developing (initiated), and heat-killed spores, respectively. We established that (i) heating a spore suspension to 40 degrees C results in a reversible twofold decrease (from 80 to 40 nm) in their PT under conditions that do not promote the development of the bacteria; this decrease is irreversible under growth-promoting conditions; (ii) the PT values of germinating spores oscillate with a considerable fluctuation amplitude (up to 7 nm), in contrast to the limited fluctuation amplitude (within 1 nm) in dormant spores; (iii) activated spores were heterogenous with respect to the PT pattern: a majority of the spores exhibited a usual spatial profile (with a maximum thickness in the center), whereas a minor fraction of them were characterized by an erythrocyte-like profile with a concave center; this implies that the central zone of the spore was more rapidly hydrated (with a decrease in refractive index) than the peripheral zone.

Study of optical parameters of the nucleoli under the effect of transcription inhibitors by coherent phase microscopy.

The use of coherent phase microscopy for online quantitative registration of nucleolar reaction to transcription inhibition is validated. Reduction of phase thickness of the nucleoli was detected during the first minutes of the experiment; 30 min after addition of the drug rarefaction zones predominated and areas of condensation were seen. These changes reflect the dynamics of disorders in the nucleolar ultrastructure during transcription inhibition.

A study of demyelination of nerve fibers using dynamic phase contrast microscopy.

Dynamic phase microscopy was used for evaluation of changes in myelinated axon segment in the paranodal region of nerve fibers during demyelination. Normally paranodal myelin sheath is characterized by regular oscillations of the optical path difference with frequences of 4.2 and 6.7 Hz. Demyelination decreased the amplitude and conduction velocity in nerve fibers and shifted the characteristic frequencies of optical path difference oscillations to 2.8, 3.2, and 11 Hz. These shifts of optical path difference frequencies probably resulted from disturbances in the state of charged phospholipids and a decrease in the level of bound Ca(2+)during demyelination of nerve fiber.