Modulation Interference Microscopy Research Articles

Analysis of micro-relief of biofilm of yeast Candida albicans of basic plastics by the method of laser modulation interference microscopy.

The development of mycotic colonization of the base surface with further biodegradation of acrylic plastics is currently of undoubted interest. The oral cavity is a favorable ecological niche for colonization by fungi and their subsequent possible invasion into the epithelium of the oral mucosa. The method of modulation interference laser microscopy is of considerable interest to researchers in medicine in the context of obtaining the necessary information about the morphological characteristics of microbial cells and the microbiome community as a whole during the colonization of a certain ecological niche in the human body. Purpose of the study: to analyze the microrelief of the biofilm of yeast-like fungi of the species Candida albicans of base plastics of the hot type of polymerization using the method of laser modulation interference microscopy. An experimental study was carried out in order to study biofilms of yeast-like fungi of the genus Candida on samples of basic plastics, an image of a biofilm of yeast-like fungi of the species Candida albicans was obtained on the surface of a plastic of a hot type of polymerization (polymethyl methacrylate) in the visualization of the phase portrait, a description of its horizontal and vertical bioprofile. As a result of the research, the heterogeneous structure of the biofilm was determined, due to the different density and accumulation of cells along the surface, the characteristics of the surface were established in accordance with the roughness criteria. The microrelief parameters on a separately arbitrarily selected section line allow one to determine the characteristics of the biofilm in the required area and make it possible to judge the nature of its formation in a certain biological niche.

МЕТОДОЛОГИЧЕСКИЕ АСПЕКТЫ ИССЛЕДОВАНИЙ СОСТАВА БИОПЛЕНОК in vitro, ex vivo, in vivo

The article presents the general patterns of formation of a three-dimensional multilayer heterogeneous structure biofilms in vitro, ex vivo, in vivo. It was found, that the mass fraction of cells of biofilms was 86,8-91,4%, of the matrix – 8,9-13,2%, which is due to the structure of the cell wall of microorganisms synthesizing the extracellular matrix. The advantages of transmission and scanning electron microscopy are the visualization of L-shapes and the quantitative assessment of the diameter, perimeter, height, area, volume and chemical composition of cells and the extracellular matrix of microorganisms. Laser modulation interference microscopy made it possible to study biofilms in real time without fixing and staining microorganisms; phase-contrast microscopy – intrapopulation and interpopulation interactions depending on changes in the pH of the environment, oxygen content and other chemical and physical factors. Fluorescent microscopy is a promising method for detection of non-culturable viable cells of microorganisms. The use a modified Gram stain, the use of solution of «Crocus sativus» made it possible to differentiate gram-negative and gram-positive microorganisms and to assess the degree of repair of tissue. The developed new methodological approach made it possible to scientifically substantiate and experimentally confirm the effectiveness of the use of nutrient media containing components for the repair of the cell wall of microorganisms.

Comparison of modulation interference microscopy, DNA spectrometry, DNA cytometry, and flow cytofluorimetry in the assessment of phytohemagglutinin-induced activity of human blood lymphocytes

Rationale: The study of the structural particulars and functional state of immune cells and primarily lymphocytes is of great importance for both fundamental and clinical medicine. It requires the development of simple and reliable analytic methods that would allow for fast and effective real-time assessment of cell activity.Aim: To evaluate the effectiveness of the interference microscopy compared to DNA spectrometry, DNA cytometry, and flow cytometry with an internalized fluorescent label CFSE (carboxyfluorescein succinimidyl ester) in the assessment of PHA-induced proliferation of human blood lymphocytes.Materials and methods: Phytohemagglutinin (PHA)-induced proliferative activity of blood lymphocytes from 10 healthy volunteers was studied with various methodological strategies. Blast transformation of lymphocytes was induced by their incubation for 5 days with PHA 5 μg/mL. The cell proliferative activity was assessed as follows: 1) by DNA spectrometry at 260/280 nm using Tecan Infinite 200 Pro with a specialized NanoQuant Plate™; 2) by cytophotometry followed by cell distribution analysis assessing deoxyribonucleic acid (DNA) content after staining with Felgen's dye with an imaging system based on an Olympus BX41 light microscope with a ProgRes CF camera; 3) by flow cytometry using an internalized fluorescent label CFSE; the analysis was performed with a BD FACS Calibur flow cytometer; 4) by measurement of the lymphocyte interference profile with a modulation interference microscope MIM-340 (Schwabe, Russia). The functional activity of the nucleus (FAN) was determined and used as a criterion for assessment of the lymphocyte functional state.Results: Incubation of lymphocytes with PHA led to an increase in the linear size by 22.2±2.8%, a decrease in phase height by 46.3±4.7% (p=0.019), and an increase in FAN by 75.9±9.4%, vs control (p=0.046). As measured by isolated DNA spectroscopy, PHA stimulation of lymphocytes was associated with an increase in the amount of DNA by 55% vs baseline (409.8±22.3 ng/μL and 264.3±25.0 ng/μL, respectively, p=0.049). Felgen's reaction revealed that the proportion of nuclei containing more than 2n DNA was 2% in the control cells and 14.8% in the PHA-activated lymphocytes, with a difference between the groups of 12.8%. CFSE staining with subsequent incubation and assessment by flow cytofluorimetry demonstrated an increase in the percentage of proliferating cells from 1.68±0.9% in the control to 55.56±5.6% (p=0.00068) in the mitogen-stimulated sample.Conclusion: Modulation interference microscopy does not require the sample preparation and demonstrated comparable and even higher effectiveness compared to conventional methods for assessment of lymphocyte activity. At the same time, it allows for evaluation of the lymphocyte functional state in real time in the process of cultivation. This opens ample opportunities for evaluation immune cells for research and diagnostic purposes.

Use of modulation interference microscopy in applied immunology

Objective: to determine the osmotic resistance of red blood cells using modulation interference microscopy technologies in the light microscopy mode of biological objects to identify the dynamics and determine the possibilities for continuing apitherapy in patients with autoimmune diseases. Methods. Methodological approaches to the use of modulation interference microscopy and computed tomography for the tasks of diagnostic medicine and applied immunology are described. The technology of vital computer dynamic phase metering, special methods for sample preparation of cytoobjects, as well as a computer-aided automated analysis of cytological images were used; recognition algorithms, measurement and identification of micro-objects; methods of statistical data processing. Results. Using the domestic innovative laser microscope MIM340, the osmotic resistance of erythrocytes was estimated using the method of modulation interference microscopy to identify the dynamics and determine the possibilities for continuing apitherapy in patients with rheumatoid arthritis and multiple sclerosis. Using computer methods of cytodiagnostics, new aspects of the functional morphology of living cells were revealed, clinical and morphological parallels were established. It was possible to evaluate the diagnostic and prognostic value of vital cell morphometry in various pathological processes and assess the effectiveness of therapeutic measures. A data bank of graphic images of red blood cells and blood lymphocytes of patients with diseases of the immune system has been created. Findings. The study of the structural features and functional usefulness of circulating blood cells is of great importance in addressing the pathogenesis, diagnosis, assessment of the severity of various pathological conditions and the effectiveness of the therapy. We believe that the study of living cytoobjects using the new method of coherent phase microscopy will provide the most objective data and increase the information content of the analysis, which is undoubtedly an urgent and promising task. The immediate plans include the refinement of mathematical, algorithmic, and software to support decision-making in computer-aided analysis of images of the epidermis and the surface of the dermis in neoplastic processes - malignant skin diseases. It is also necessary to create algorithmic and software tools for computerizing studies of cell models for quantitative and qualitative assessment of the selective accumulation of xenobiotics by laser microscopy.

Use of modulation interference microscopy in applied immunology

Use of modulation interference microscopy in applied immunology

Production and application of selenium nanoparticles to prevent ionizing radiation-induced oxidative stress

The technology of obtaining selenium nanoparticles using laser ablation is presented. Selenium nanoparticles in water and aqueous solutions form stable colloids. For the obtained nanoparticles, the evolution in size and in mass has been established; optical properties have been characterized. The nanoparticles were studied using a transmission electron microscope, a modulation-interference microscope and a Bruker X-ray diffractometer. It has been previously shown that selenium containing preparations can prevent oxidative stress caused by ionizing radiation. In this work, it was shown that the nanoparticles obtained by us are also able to prevent oxidative stress caused by ionizing radiation and protect animals from radiation-induced death. In laboratory mice, it was established that selenium nanoparticles at a concentration of up to 10 mg / kg do not cause acute toxic effects. It is shown that the most effective concentration of selenium nanoparticles is a concentration of 5 mg / kg. The optimal administration time is 5 hours before exposure to ionizing radiation. Also set the dose reduction factor, it was 1.2.

Evaluation of nonlinear dynamics of the cell structure damage as a promising method for personalized cancer diagnostics

Background:Modulation interference microscopy is one of the promising technologies for early personalized cancer diagnostics, while it gives the possibility to obtain real-time images of living functioning cells at a nanometer resolution, as well as to assess spatio-temporal changes of their intracellular structures.Aim:To identify the markers of malignant transformation of cells based on a multiple scale spatio-temporal of the data obtained by lase interferometry duringin situregistration of changes in intracellular structures.Materials and methods:The study was performed with the breast adenocarcinoma MCF-7 cell line and human kidney epithelial cells HEK 293 provided by the N.N. Blokhin National Medical Research Centre of Oncology (Moscow, Russia). Changes in fluctuation of the living cell phase thickness were measured with a laser modulation interference microscope MIM-340 (PA UOMZ, Russia). The multifractal detrended fluctuation analysis (MF-DFA) was used to assess the results obtained.Results:By comparison of the scaling exponential spectra and corresponding spectra of the singular fluctuations in the minimal and maximal diameters of the phase pictures of HEK 293 cells and MCF-7 cells, it was shown that the cancer cell dynamics has definite monofractal properties: a linear dependency of the scaling exponentsτfrom the actualqparameter and “dotted” singularity spectrum (the spectral width does not exceed 0.1). The dynamics of healthy cell demonstrated multifractal properties, such as a non-linear dependencyτ(q)and a bell shaped singularity spectrum (the spectral width over 0.1).Conclusion:We have proposed a new approach to assess the intracellular structural damage during oncogenesis. The signs of monofractality are objective criteria of malignization. The results obtained seems promising as early diagnostic markers of cancer transformation (damage) of cells and determination of tumor cell sensitivity / resistance to anti-tumor agents in an individual patient.

The use of the interference microscopy to study structural characteristics of cultured dermal fibroblasts

Rationale: The use of electron, nuclear power and confocal microscopy for the screening of biologically active compounds, medical products and express diagnostics of some diseases at the cell level is associated with laborand time-consuming sample preparation, which cannot exclude potential measurement errors and artifacts. The modulation interference microscopy does not have these disadvantages; it allows for non-invasive studies of cell structures, imaging with nanometer resolution and analysis of the optical properties of an object.Aim: To assess the potential of the interference microscopy in the evaluation of morphofunctional characteristics of in vitro mitomycin conditioned cultured cell nuclei (dermal fbroblasts taken as a model).Materials and methods: Native culture of human dermal fbroblasts of the 6th passage, grown on glass with mirror coating in the cell culture laboratory of the Institute of Experimental Medicine and Biotechnology of Samara State Medical University (Russia), was examined with a modulation interference microscope MIM-340 (JSC PA UOMZ, Russia). Changes over time in the structural characteristics of dermal fbroblast nuclei conditioned with mitomycin were evaluated. The control group included fbroblasts cultured in the same conditions on glass with mirror coating without mitomycin. Imaging with MIM-340 was done at three hours, one and four days after adding the cytostatic. The control group was assessed at the same time points.Results: We have shown that the cell culture grown on dielectric glasses does not differ in its morphofunctional characteristics from the culture grown on culture plastics. This proves the possibility to study the adhesive native culture using interference microscopy. We have found that the cells respond to a single mitomycin 0.04% exposure with a change to a globular shape and a sharp increase in the nuclear phase thickness (217.8 vs. 142.18 nm in the control group, p ≤ 0.05). Thereafter, the morphofunctional characteristics of the cells are restored, which is confrmed by the changes over time in the culture density, cell shape and size, and the phase thickness of the nucleus.Conclusion: The results obtained make it possible to recommend the method of modulation interference microscopy for evaluation of toxicity and biocompatibility of drugs, medical products and physical factors for diagnosis and treatment.

Translational studies of electrophoretic mobility and phase picture of erythrocytes with consideration of development of stress response during a pathological process

Rationale:Modern cell diagnostic methods are in high demand during the development of new approaches in personalized medicine. Coherent phase interferometry and cell microelectrophoresis are among such methods that are being actively introduced into the diagnostic process in medical institutions.Aim:To substantiate the potential use of biophysical and morphodensitometrical erythrocytes parameters as criteria of treatment efcacy and course of adaptation process in patients with gastrointestinal tract disorders.Materials and methods:The study included 25 patients aged from 40 to 54 years (11 males and 14 females), among them 9 (36%) with gastric peptic ulcer, 3 (12%) with duodenal ulcer, 8 (32%) with acute gastritis, and 5 (20%) with acute pancreatitis. Biophysical and morphological particulars of peripheral blood erythrocytes were assessed before and after treatment using cell diagnostic techniques, such as microelectrophoresis and laser modulation interference microscopy. Also, we evaluated changes over time in routine clinical laboratory tests, such as red and white blood cell counts, hemoglobin levels, and erythrocyte sedimentation rate (ESR), and differential leukocyte counts. The control group included 10 healthy donors aged from 36 to 52 years.In vitroexperiments were performed to assess the erythrocyte electrophoretic mobility (EEPM) and morphology of erythrocytes treated with epinephrine or cortisol.Results:After the treatment, the patients demonstrated a decrease in their leukocyte counts (by 27%), a 2-fold increase in monocyte counts and an ESR decrease (by 10%), compared to the corresponding baseline values before treatment (p < 0.05 for all comparisons). EEPM increased by 12% (1.37 vs. 1.22 mcm × cm/V × s, p < 0.05). The erythrocyte pool of the patients before treatment, had a decreased proportion of discocytes, compared to that in the control group (85.2 vs. 95.4%, р < 0.05), increased proportions of echinocytes, stomatocytes and degenerative forms (11, 2.8 and 1%, respectively, р < 0.05). After the treatment, the discocytes counts increased virtually up to their physiological normal range (91.3%). However, the surface of the discoid cells remained heterogeneous with multiple microspicules; this resulted in changes of electrokinetic and morphological properties of erythrocyte response to stress reaction occurring in the body. The impact of the stress effectors was confrmed inin vitroexperiments assessing the effects of epinephrine (1 × 10-9 g/mL) and cortisol (5 × 10-7 g/mL) on erythrocytes. At 120 minutes of the experiment, epinephrine decreased EEPM (1.14 vs. 1.24 mcm × cm/V × s at baseline, р < 0.05) and increased cell sphericity. On the contrary, cortisol increased EEPM (1.72 vs. 1.36 mcm × cm/V × s, р < 0.05), with non-signifcant echinocytic transformation.Conclusion:Biophysical and morphodensitometric parameters of red blood cells obtained with the use of current express methods of cell microelectrophoresis and coherent interference microscopy help to objectivize the intensity of stress response during a pathological process and activation of adaptation mechanisms during the treatment.

Nanoscale surface characterization using laser interference microscopy

Nanoscale surface characterization is one of the most significant parts of modern materials development and application. The modern microscopes are expensive and complicated tools, and its use for industrial tasks is limited due to laborious sample preparation, measurement procedures, and low operation speed. The laser modulation interference microscopy method (MIM) for real-time quantitative and qualitative analysis of glass, metals, ceramics, and various coatings has a spatial resolution of 0.1 nm for vertical and up to 100 nm for lateral. It is proposed as an alternative to traditional scanning electron microscopy (SEM) and atomic force microscopy (AFM) methods. It is demonstrated that in the cases of roughness metrology for super smooth ( Ra >1 nm) surfaces the application of a laser interference microscopy techniques is more optimal than conventional SEM and AFM. The comparison of semiconductor test structure for lateral dimensions measurements obtained with SEM and AFM and white light interferometer also demonstrates the advantages of MIM technique.

Laser Modulation Interference Microscopy as a Means of Controlling the Form and Roughness of Optical Surfaces

The use of the technology of modulation interference microscopy as an effective means of quality control of precision optical surfaces with roughness on the order of 1 nm and flatness up to 10 nm at an aperture up to 100 mm is considered.

Laser Interference Microscopy for Nanobiotechnologies

Interference microscopy is widely used in different areas of science for comprehensive study of optical and geometric properties of various microand nanoobjects. The fact that phase imaging makes it possible to overcome the classical resolution threshold has been well known since the 1950s (1). However, only recently the progress in electronics and computer technology has allowed the capacities of phase microscopy to be used to the full extent. Presently, many models of interference micro� scopes are available on the market. The specifications of the interference microscopes are consistent with the area of application. Optical profilometers based on whitelight interferometers are most commonly used. Optical pro� filometers are used in semiconductor technology for quality control. In recent years, interference microscopy finds con� tinuously increasing application for studying biological objects. The advantages of interference microscopy are high spatial resolution, noninvasive monitoring, and lack of special requirements for the medium in which the measurement is taking place (vacuum or chemical dyes are not required). Interference microscopy, which is a strong alternative to conventional optic and confocal microscopy, is based on modern methods of interfero� gram processing. These methods include: coherence phase microscopy (2), optical coherence microtomogra� phy (3), coherence correlation interferometry (4), and digital holographic microscopy (5). The main problem of interference microscopy is insufficient correlation between spatial resolution and efficiency. The method of coherence phase microscopy provides lateral resolution up to 100 nm, but a frame 128 × 128 pixels is recorded within 14 sec, which is too long to test the fast processes in biological objects. On the other hand, the method of digital holographic microscopy allows the images to be obtained at a rate 200 frame/sec, but the spatial resolution of the method is below 200 nm, which is inappropriate for visualization of cell organelles or intracellular structures. The goal of this work was to describe a new modifi� cation of modulation interference microscope (MIM) for biomedical research. The MIM detects the distribution of optical and material parameters of the microscopic object of interest (refraction coefficient, reflection coefficient, anisotropy, and polarization) with high spatial and tem� poral resolution.

Modulation interference microscope as a tool for measuring the linear dimensions of nanostructures

A new version of a modulation interference microscope with long-path coordinate table resting on aeromagnetic guides that enables travel of the microscope with deviation from linearity of at most 0.1 μm on path lengths up to 300 mm has been developed. The metrological aspects of the use of the microscope for measurement of the linear dimensions of nanostructures are considered. Results of measurements of the basic parameters of the topology of integrated circuits are presented.

Metrologic platform with a modulation interference microscope

This paper presents the results of the development of a unique metrologic platform intended for the study of the nanodefects of an object and the measurement of its surface flatness on large areas. The design of the platform is based on the simultaneous use of modulation interference microscopy and noncontact aeromagnetic guides. The optical layout and an algorithm for obtaining panoramic images are considered. It is shown to be promising to use the metrologic platform in the optics and semiconductor industries.

Laser-based modulation-interference microscopy of optical surfaces

A new version of the modulation interference microscope with a long-path coordinate stage on aeromagnetic guides has been developed that can displace the microscope with nonrectilinearity no greater than 0.1 µm on a path length of up to 300 mm. The given modification of the microscope can be used to investigate the surface of large optical items having dimensions up to 300 × 300 × 100 mm with a resolution of 0.1 nm along the vertical and 10–100 nm in the sample plane. The design and operating principles of the main modules of the microscope are considered. Examples are used to show that the microscope thus developed has possibilities and advantages when optical surfaces are being investigated.

Investigating the optical properties of nanostructures by modulation interference microscopy

A modulation interference microscope has been developed with record-breaking lateral resolution (10–100 nm) and high image-production rate (up to 250 frames per second). The optical layout of the device, the operating principles, and the problems of superhigh optical resolution in phase images are described. Possible ways of developing a method for modulation interference microscopy are discussed, along with areas in which the method can be used to study nanostructured materials, the topology of integrated microcircuits, and the morphology of biological objects.

Long‐living nanobubbles of dissolved gas in aqueous solutions of salts and erythrocyte suspensions

Results of experiments combining laser modulation interference microscopy and Mueller matrix scatterometry show that macroscopic scatterers of light are present in liquids free of external solid impurities. Experimental data on distilled water and aqueous NaCl solutions of various concentrations as well as physiological saline solution are reported. The experimental data can be interpreted by using a model of micron-scale clusters composed of polydisperse air nanobubbles having effective radii of 70-100 nm. Their concentration increases with the growth of ionic content. We hypothesize that under certain conditions those clusters of nanobubbles can affect the erythrocyte structure.

A high-speed modulation interference microscope for biomedical studies

A new modulation interference microscope has been developed with high speed in obtaining phase images (time required for a complete frame with resolution 1280 × 1024 pixels constitutes 0.3 sec). An optical scheme is considered with principles of operation and fundamental problems for ultrahigh optical resolution, together with results from measurement of morphology and the dynamics of nerve fibers. There is a discussion of development prospects for the modulation interference microscope and the possible use of it for biomedical research.

Determination of the microstructure of gas bubbles in highly purified water by measuring the elements of the laser radiation scattering matrix

Modulation interference microscopy and measurements of the elements of the light scattering matrix showed that doubly distilled water purified from solid impurities contains macroscopic scatterers in the form of micron clusters formed by polydisperse air bubbles with the effective radius 70–90 nm. The fractal dimension of clusters lies within 2.4–2.8 and their concentration is ∼106 cm-3.

Nanoscale structure of dissolved air bubbles in water as studied by measuring the elements of the scattering matrix

Results of experiments with laser modulation interference microscopy and the Mueller-matrix scatterometry show that macroscopic scatterers of light waves are present in doubly distilled water free of external solid impurities. The experimental data can be interpreted using a computational model of micron-scale clusters composed of polydisperse air bubbles having effective radii of 70-90 nm. The fractal dimension of such clusters was evaluated as 2.4-2.8 and their concentration appeared to be approximately 10(6) cm(-3).