Laser Interference Microscopy Research Articles

МОРФОЛОГИЧЕСКИЕ И ФУНКЦИОНАЛЬНЫЕ ПОКАЗАТЕЛИ СПЕРМАТОЗОИДОВ БЫКОВ ПРИ КРИОКОНСЕРВАЦИИ

The purpose of the study is to analyze the influence of the freezing and thawing cycle on the morphofunctional parameters of bull sperm. The study was conducted at Nizhegorodskoe LLC on breeding work in the Kstov municipal District of the Nizhny Novgorod Region, on the basis of the Department of Physiology and Anatomy of the Institute of Biology and Biomedicine of the Nizhny Novgorod State University named after N.I. Lobachevsky and the Department of Physiology, Animal Biochemistry and Obstetrics of Nizhny Novgorod State Technical University. The object of the study is the sperm production of black-and-white bulls. Semen was diluted with sterile BioXcell medium. Native diluted sperm and sperm after deep freezing were studied. Motility and the average speed of sperm movement were analyzed on a sperm analyzer SA-500 from Biola (Russia). To assess the ultrastructure of spermatozoa, a Hitachi SU8220 electron microscope (Japan) was used. Using a laser interference microscope MIM-340 (Russia, Yekaterinburg), the morphology of spermatozoa was studied in real time without fixation or staining. After cryopreservation of the sperm, there was a decrease in the biological usefulness of sperm, which is confirmed by a decrease in motility by 13.77% (p ≤ 0.05) and the average speed of sperm movement by 12.95% (p ≤ 0.05). Using electron and laser interference microscopy, it was shown that the chromatin of thawed spermatozoa was insufficiently condensed and contained fibrils. After cryopreservation, the position of the acrosome was changed in 6.23% (p ≤ 0.05) of sperm, and the shape of the acrosome was changed in 18.64% (p ≤ 0.05) of cells. After freezing and thawing, 7.01% of sperm showed a decrease in head length, 17.90% - a decrease in body length, and 9.53% - a decrease in sperm tail length. The use of modern methods for assessing sperm viability is of great importance for understanding the processes occurring during sperm cryopreservation and allows us to develop ways to improve the quality of sperm du¬ring freezing and thawing.

State-of-the-Art Materials Used in MEMS Micromirror Arrays for Photonic Applications

This work provides an overview on micromirror arrays based on different material systems such as dielectrics, element silicon, compound semiconductors, metals, and novel 2D materials. The goal is to work out the particular strength of each material system to enable optimum performance for various applications. In particular, this review is intended to draw attention to the fact that MEMS micro-mirrors can be successful in many other material systems besides silicon. In particular, the review is intended to draw attention to two material systems that have so far been used less for MEMS micromirror arrays, that have been less researched, and of which fewer applications have been reported to date: metallic heterostructures and 2D materials. However, the main focus is on metallic MEMS micromirror arrays on glass substrates for applications like personalized light steering in buildings via active windows, energy management, active laser safety goggles, interference microscopy, and endoscopy. Finally, the different micromirror arrays are compared with respect to fabrication challenges, switching speed, number of mirrors, mirror dimensions, array sizes, miniaturization potential for individual mirrors, reliability, lifetime, and hinge methodology.

Assessment of the biofilm surface of clinical strains of yeast-like fungi on dentures using scanning electron microscopy and laser interference microscopy

Assessment of the biofilm surface of clinical strains of yeast-like fungi on dentures using scanning electron microscopy and laser interference microscopy

Effects of lipoperoxidation and mitochondrial state on milk yield of dairy cows under technological stress

Evaluation of the physiological state of cattle is crucial in creating healthy, high-performing dairy cattle herds. Technological stress is one of the most critical factors determining the biological potential of higher-yielding cows. This work aimed to assess the effect of technological stress on various oxidative parameters and mitochondrial states in dairy cows' blood, milk yield and milk composition. The study was conducted on the black-and-white breed of healthy herds. Regrouping, changing service personnel, and carrying out veterinary and sanitary manipulations were considered technological stress factors. The concentration of cortisol in the blood serum was studied by the immunological method. The concentrations of malonic dialdehyde (MDA), diene conjugates (D.C.), Schiff bases (S.B.), reduced glutathione and catalase activity were measured spectrophotometrically. The mitochondrial state was estimated by laser interference microscopy. While the milk yield, protein and lipid composition of cow milk were studied using an ultrasound analyzer. The researched indicators were analyzed before and for 30 days after the effect of technological stress. Results of the study suggested that technological stress caused an increase in oxidative processes, along with a reduction of antioxidant activity of blood and milk at the initial stages of registration (1-7 days). The concentration of glutathione remained reduced for 30 days after technological stress. A decrease in mitochondrial refractoriness and disintegration accompanied these processes. The milk yield indicator decreased was not restored to the values of intact animals by 30 days after technological stress. Further, the protein and lipid composition also reduced. Thus, a decrease in the quantity and quality of milk under technological stress may be mediated by the development of oxidative stress, which the refractoriness and disintegration of mitochondria might trigger.

Real-time observation of the precursor film for low viscosity liquids spreading on solid substrates by a customized differential laser interference microscopy.

While static wettability is well treated with Young's equation via its static contact angle, theoretical analyses for wetting dynamics are not yet reaching consensus due to a singularity of the spreading forces worked at the vapor/liquid/solid contact line. One plausible explanation to overcome the singularity problem is that there is a so-called precursor film spreading outside the apparent contact line. After its first finding in 1919, many researchers have attempted to visualize its shape. However, because its length and thickness are as small as micrometer and nanometer-order, respectively, its visualization stillremains a challenging issue especially for low-viscosity liquids. In the present study, we developed a differential laser interference microscope, which has a thickness resolution of approximately 2nm at the best, and applied it to the wetting front of 10 cSt of silicone oil spreading on a silicon wafer with an almost constant spreading velocity. As a result, the precursor film of 14µm long and 108nm thick was clearly visualized. While the macro contact line has a finite advancing contact angle of 4.0°, the gradient of the precursor film surface gradually decreased and converged to ~ 0.1° at the micro-contact angle. The shape of the precursor film was independent of the time after the dropping for the range of 600s ± 10%, which is consistent to theoretical estimation. The present study demonstrated that our interferometer simultaneously achieved nanometer thickness resolutions, micrometer in-plane spatial resolution, and at least a millisecond temporal resolution with a simple optical setup.

Возможности прижизненной визуализации клеток крови при стрессе для оценки состояния организма

Biological specimens are the most difficult to study as almost exclusively light elements are considered, a little visibility yielded and structures are sparsely distinguishable. In addition cell structures are easily damaged on any stage of microscopic exploration. The optimal solution is to study their phase changes using laser interference microscopy. The method is based on the analysis of refractive index of intracellular medium of cells with subsequent computer analysis and processing of phase images followed by three-dimensional reconstruction of the object. The obtained images are digitally recorded, and any point of view can be introduced for viewing. This work uses laser interference microscopy followed by 3D reconstruction of cell images to study blood cells: neutrophils and erythrocytes by 3D reconstruction of neutrophils have demonstrated changes of their functional activity under technological stress, while 3D reconstruction of erythrocytes revealed a violation of the normal discoidal cell shape. Interpretation of the obtained data indicates a restructuring of the cellular component of nonspecific resistance and deterioration of the oxygen transport function of erythrocytes under technological stress. Visualization of interference patterns of blood cells through 3D image processing allows obtaining n a vital analysis of cells, the technique that is not available for other methods of microscopy, in particular light microscopy, which is traditionally used as a prognostic method of the state of the body. This direction represents the most important condition for further development of works in the field of cell diagnostics. The obtained results prove the applicability of 3D reconstruction of cell images for bioscanning of technological stress.

Изменение конформации и распределения гемоглобина при изменении объема эритроцита

The intracellular space of red blood cells (RBC) includes 40% hemoglobin (Hb) molecules and 60% being water molecules. When the intracellular volume changes, which is possible when erythrocyte cells pass through the blood stream, the possibility of redistribution of intracellular components of the cell due to its volume changes is assumed. By methods of optical spectroscopy (non-invasive methods of infrared spectroscopy, Raman-spectroscopy, laser interference microscopy) changes of morphology, conformation and redistribution of Hb have been revealed in the human erythrocyte due to an increase in the ratio of [Na+]in and [K+]in when Na+/K+-ATPase is blocked in the cell. The decrease of activity of Na+/K+-ATPase by a ouabaine (3 mM) has been found to lead not only to an increase in [Na+]in cell but also to an increase in positive charge on the cytoplasmic surface of the RBC membrane. In these conditions, changes in the conformation of both the heme and globine parts of the cytoplasmic Hb have been identified. It has been shown that cell depolarization, together with cell volume change, leads to a decrease in packing density of Hb molecule, which may be related to sorption of intracellular Na+ (or Ca2+) c Hb, as well as increasing the amount of water molecules in the cell and redistributin Hb in the cell. These processes can lead to a change in the conformation of Hb, as well as to a redistribution and alteration of the conformation of the cytoplasmic Hb.

Morphofunctional Characteristics of Erythrocytes and Blood Erythropoietin Level in Patients as Predictors of Severe Course of COVID-19.

Morphological and functional characteristics of erythrocytes, hemoglobin, and erythropoietin level in the venous blood were evaluated by laser interference microscopy, Raman spectroscopy with a short-focus extreme aperture lens monochromator, and by ELISA, respectively, in 30 patients with verified moderate COVID-19 at the time of hospitalization and 30 healthy volunteers. The patients whose course of COVID-19 has worsened to critical by day 5 had already had lower (p<0.001) indicators at the time of hospitalization such as the area and thickness of erythrocytes, the hemoglobin distribution and packing density, hemoglobin conformation index (I1355/I1550)/(I1375/I1580) reflecting its oxygen affinity, and blood erythropoietin content. Our findings suggest that these characteristics of erythrocytes, hemoglobin, and erythropoietin can serve as potential predictors of COVID-19 aggravation in hospitalized patients.

Research into Morphofunctional Characteristics of Erythrocytes in COVID-19 Patients.

In this paper, the erythrocytes of healthy donors and people with a confirmed diagnosis of COVID-19 were tested by Raman spectroscopy and laser interference microscopy. We argue that erythrocytes (red blood cells) in COVID-19 patients have an irregular shape, and their morphometric characteristics are impaired in comparison to healthy red blood cells. Raman spectroscopy also allows us to detect a decreased oxygen transport function of these erythrocytes. The combination of these methods—Raman spectroscopy and laser interference microscopy—is a highly effective method for the diagnosis of SARS-CoV-2.

Conditions of Erythrocytes during the Post-Traumatic Period of Cranial Cerebral Injury in Rats under the Effect of Cytoflavin: Assay by Laser-Interference Microscopy

Conditions of Erythrocytes during the Post-Traumatic Period of Cranial Cerebral Injury in Rats under the Effect of Cytoflavin: Assay by Laser-Interference Microscopy

Analysis of morphometric and fractal properties of phase-contrast images of native and apoptotically changed MCF-7 cells

This work was aimed at the determination of the morphometric and fractal characteristics of native and apoptotic MCF-7 cells on the basis of the analysis of phase contrast images obtained using laser interference microscopy. MCF-7 cells were cultured under standard conditions. The cell phase images were obtained using a laser interference microscope (model MIM-340) at Perm Federal Research Center of the Ural Branch of the Russian Academy of Sciences. In the work, 30 native MCF-7 cells and 30 MCF-7 cells apoptotically changed under the action of doxorubicin were studied. The phase images obtained were treated to highlight the cell contour so as to subsequently determine the morphometric parameters and fractal dimension of the cell phase image contour. The maximal and minimal diameters, perimeter and volume of the cell phase images were chosen as geometric characteristics of the images. The fractal dimension of the phase image contour was calculated as a scale-invariant parameter indicating the indented character of the cell contour. The comparative analysis of the morphology of native and apoptotic MCF-7 cells enabled differentiating the cells under study using the two-sample non-parametric Wilcoxon–Mann–Whitney test.

Assessing the Functional State of Red Blood Cells by Using the Laser Interference Microscopy

Assessing the Functional State of Red Blood Cells by Using the Laser Interference Microscopy

Label-Free Single Cell Viability Assay Using Laser Interference Microscopy.

Laser interference microscopy (LIM) is a promising label-free method for single-cell research applicable to cell viability assessment in the studies of mammalian cells. This paper describes the development of a sensitive and reproducible method for assessing cell viability using LIM. The method, based on associated signal processing techniques, has been developed as a result of real-time investigation in phase thickness fluctuations of viable and non-viable MCF-7 cells, reflecting the presence and absence of their metabolic activity. As evinced by the values of the variable vc, this variable determines the viability of a cell only in the attached state (vc exceeds 20 nm2 for viable attached cells). The critical value of the power spectrum slope βc of the phase thickness fluctuations equals 1.00 for attached MCF-7 cells and 0.71 for suspended cells. The slope of the phase fluctuations' power spectrum for MCF-7 cells was determined to exceed the threshold value of βc for a living cell, otherwise the cell is dead. The results evince the power spectrum slope as the most appropriate indicator of cell viability, while the integrated evaluation criterion (vc and βc values) can be used to assay the viability of attached cells.

Diagnostics possibilities of erythrocytes analysis by the method of laser interference microscopy.

The morphofunctional state of erythrocytes significantly changes by the onset and progression of various diseases, as well as in critical conditions, injuries, accompanied by severe tissue damage. This leads to a violation of microcirculation and has an effect on hemorheology in general. In connection with this, there is a need for the development of adequate methods for assessing the functional activity of erythrocytes. The aim of the work was to study the possibilities of laser interference microscopy in the analysis of the functional state of erythrocytes. The results allowed us to quantify the state of the phase height and phase diameter of erythrocytes. Analysis of the surface nanostructure gave a qualitative assessment of the functional activity of cells. The following patterns were revealed: the action of adrenaline causes the appearance of echinocytes with an increase in phase height and decrease phase diameter and the appearance of spicules on the surface of cells. The action of cortisol leads to a decrease in the phase height and the phase diameter with the appearance of numerous loosening of the structure of the cell surface. Moreover, the traditional method of analyzing the distribution of erythrocytes in a population by morphology (discocytes, stomatocytes, and degeneratively altered cell forms) did not reveal statistically significant differences. Thus, the results indicate that the interference microscopy method allows a qualitative assessment of the morphofunctional activity of native cells. This increases the information content of the analysis and leads to the objectification of data on the functional capabilities of erythrocytes.

Analysis of the relief of MCF-7 tumor cells during apoptisis on the basis of phase-contrast images obtained by laser interference microscopy

Data on the surface topography of the cell makes it possible to predict its behavior and is an indicator to assess whether it is in a state of apoptosis, that is, in a state of death due to the activation of intracellular reactions; this state of a cell occurs both during normal development and as a result of a pathological process. A universal method for recording the state of the relief of the cell and its other mechanobiological parameters has not been developed, and therefore laser interference microscopy is promising for this purpose. The aim of this work is to evaluate the state of the surface of tumor cells during the apoptotic changes in breast cancer cells by analyzing the phase contrast (phase) images obtained by laser interference microscopy. The object of the study is the cells of the MCF-7 line (epithelial-like adenocarcinoma of the human mammary gland) in a native state and in a state of induced apoptosis under the action of doxorubicin. The analysis of the cell surface involves interpretation of data on the optical thickness of cells and their morphology through the construction of isolines. Based on the analysis of phase images of cells, their general morphometric parameters were determined. Visualization of optical data and their interpretation was performed using the ImageJ-Fiji software. According to the Wilcoxnon-Mann-Whitney test, the samples under study are significantly different in maximum diameter, perimeter, and sphericity coefficient. Based on the analysis of isolinear images, a diagram of the phase state of cells in a native state and in a state of apoptosis was constructed. According to the diagram, the cross-sectional area of apoptotic cells is generally much smaller than the cross-sectional area of native cells at the same height levels. The characteristics of cells within the framework of the proposed phase diagram are consistent with the morphological cell apoptosis signs, which are recorded using classical microscopic methods. The construction of the diagrams of the state of cells according to the parameters of their relief holds promise for assessing the cellular behavior, predicting it, as well as for analyzing the effect of drugs and therapeutic methods on pathological cells.

DNA Transformation, Cell Epigenetic Landscape and Open Complex Dynamics in Cancer Development

Statistical thermodynamics allowed the formulation of mesoscopic approach of DNA transformation in course of the excitation of collective distortion modes (denaturation bubbles) associated with hydrogen bond breaking between the base pairs. Intermediate (non-continual limit) of DNA modeling (the Peyrard-Bishop model) is combined with the field description (generalized Ginzburg-Landau approach) to analyze the dynamics of collective open complex modes associated with mesodefects in the DNA ensemble. Collective modes dynamics describes different scenario of gene expression according to statistically predicted form of out-of-equilibrium potential (epigenetic landscape) reflecting specific type criticality of “soft matter” with mesodefects (open complexes) – the structural-scaling transition. Principal difference of thermodynamics of non-continual and continual models is thermalization conditions related to thermal fluctuations responsible for the DNA breathing (localized excitation with breather dynamics) and structural-scaling parameter responsible for spinodal decomposition of out-of-equilibrium potential metastability due to generation of open complex collective modes. Open complex collective modes have the nature of self-similar solutions (breathers, auto-solitary and blow-up modes) of open complex evolution equation accounting qualitative different types of potential metastabilities. Sub-sets of collective modes represent the phase variables of attractors associated with different scenario of expression dynamics, which allows the interpretation of multistability of the epigenetic landscape and the Huang diagram of gene expression. It was shown different epigenetic pathway in attractors phase space corresponding to normal and cancer expression scenario. These scenarios were supported by laser interference microscopy of living normal and cancer cells illustrating multi- and monofractal dynamics.

VITAL MORPHOMETY OF RBC IN THEIR INTERACTION WITH STRESS HORMONES

The possibility to use the laser interference microscopy in order to assess the RBCs morphology in normal state and in interaction with cortisol and adrenalin as stress hormones was studied. It was established that the laser interference microscopy represented an important instrument permitting to receive the information about physiological state of the RBCs as well as of the organism in the whole in extreme condition. New aspects of cortisol and adrenalin impact on the RBC morphology are defined

Nanostructured Particles of Wood: A Promising Substrate for the Production of Lignocellulolytic Enzymes by the Wood-Degrading Fungus Lentinus tigrinus

The use of wood nanoparticles (NPs) to increase the efficiency of the synthesis of lignolytic and cellulolytic enzymes using the Lentinus tigrinus basidiomycete is proved. It is shown by the laser diffraction technique that the particle size of the main fractions decreases with an increase in the time of dispersion to 40 min and NPs are detected (the particle size of birch is smaller than that of pine). Using laser interference microscopy and dynamic light scattering (DLS), it is demonstrated that the mean NP size of birch wood after 30 min of dispersion corresponds to 160.9 nm, while after 40 min this size is 95.3 nm. During the cultivation of the L. tigrinus fungus on coarse dispersed particles and NPs of wood, it is shown that the maximum lignolytic and cellulolytic activities are observed on the substrate with birch NPs after six days of growth, 528.44 units/mg in the case of laccase, 61.2 units/mg in the case of peroxidase, 4383 units/mg in the case of Mn-peroxidase, and 69.2 units/mg in the case of endoglucanase. A significant enhancement of the synthesis of ligninlytic and cellulolytic enzymes upon cultivation of the L. tigrinus basidiomycete with the NPs is presumably caused by the increase in the total surface area of the substrate for fungus enzymes. In addition, wood NPs can act as inductors of the biosynthesis of enzymes of the lignocellulolytic complex.

Сравнительная характеристика липидного состава и морфофункциональных показателей ядерных и безъядерных эритроцитов в условиях гипоксии

A comparative study of the properties of nuclear and non-nuclear erythrocytes in experimental hypoxia. The phospholipid and fatty acid composition of individual phospholipids, the content of free fatty acids in the composition of nuclear-free and nuclear red blood cells were determined by thin-layer chromatography. The method of laser interference microscopy was used to study the morphometric characteristics of nuclear and nuclear-free red blood cells and the distribution of hemoglobin. Conformational changes in the hemoporphyrin of nuclear and nuclear-free red blood cells under hypoxia were studied by Raman spectroscopy. It has been shown that under conditions of hypoxia, changes in the phospholipid and fatty acid composition occur in the membranes of both nuclear and non-nuclear erythrocytes, leading to the accumulation of lysoforms of phospholipids, diacylglycerol and free fatty acids, as well as to an increase in the degree of saturation of fatty acids in phospholipids. These changes indicate structural changes in the membrane of nuclear-free and nuclear red blood cells and, as a result of this, functional changes both on the part of the red blood cell in general and the main oxygen-transporting protein of red blood cells - hemoglobin.

Heat damage of cytoskeleton in erythrocytes increases membrane roughness and cell rigidity.

The intensity of erythrocyte membrane fluctuations was studied by laser interference microscopy (LIM), which provide information about mechanical properties of the erythrocyte membrane. Atomic force microscopy (AFM) was used to study erythrocyte surface relief; it is related to the cytoskeleton structure of erythrocyte membrane. Intact human erythrocytes and erythrocytes with a destroyed cytoskeleton were used. According to the obtained results, cytoskeleton damage induced by heating up to 50 °С results in a reduced intensity of cell membrane fluctuations compared to non-treated cells (20.6 ± 10.2 vs. 30.5 ± 5.5 nm, correspondingly), while the roughness of the membrane increases (4.5 ± 1.5 vs. 3.4 ± 0.5 nm, correspondingly).