Biopolymers are actively used in tissue engineering for the production of biomedical products with desired and controlled properties. Particular attention is paid to the creation of an artificial biomimetic matrix - a framework for targeted regeneration, effective cell growth and their integration with the surrounding tissue. The purpose of the work is to identify the possibilities of interference microscopy for studying the structural, functional and biological properties of biopolymer matrices based on fibroin and chitosan. The analysis of the thickness, relief features, and structure of biopolymer matrices was carried out using methods of superresolving interference and atomic force microscopy. We used a MIM-340 laser modulation interference microscope (Shvabe, Russia) with following technical characteristics: resolution in the lateral plane - 13 nm; vertical resolution - 0.1 nm; the range of measurement of linear vertical dimensions - 0.01–0.10 μm, in the lateral plane (for a 20x lens) - 0.1–8.0 μm; field of view of the measuring channel in the lateral plane of not less than 20 microns. Atomic force microscopy was performed on the basis of the NtegraPrima micro-cantilever system (NT-MDT, Russia) in semi-contact mode. We visualized and quantified the relief and heterogeneity of the internal structure of biopolymer matrices in the obtained interferograms. The effect of the addition of fibroin and jenipine on the thickness, roughness, and structural uniformity of the composite matrix coating has been studied. To assess the biocompatibility, adhesive and immunogenic properties of chitosan and fibroin-containing polymers, populations of living cells (lymphocytes, neutrophils and mesenchymal stem cells) were used. High informativeness of the QPI in the study of the morpho-functional properties of films and nanofiber materials was established. Based on the results of a comparative analysis of new biopolymer composites, the most promising samples for creating medical devices with characteristics as close to physiological as possible are determined.
In the last years, there has been an increasing interest to the elaboration of new biocompatible and biodegradable medical polymers meant to contact the living body milieu. Amine-based biopolymer films with intrinsic biological activity have a significant potential for the synthesis of contemporaneous materials intended for surgery and tissue engineering. Our investigation is aimed to perform a non-invasive assessment of the structural characteristics and biological properties of biodegradable polymeric composites with anti-inflammatory activity, by means of ultra-high resolution laser interferometric microscopy.
Various samples of biodegradable polymers were studied with a phase-modulation laser interferometric microscope MIM-340 (Yekaterinburg, Russia) at a wavelength of 532 nm and magnification of x 20, with superficial plane resolution of up to 15 nm, vertical resolution of 0,1 nm and possibility to control the relief depth of up to 600 nm.
We have performed an in vitro non-invasive assessment of the impact of the structure, composition and modification conditions of the obtained biopolymer composites on the viability, adhesive properties and functional activity of the living blood cells (neutrophils, lymphocytes, and platelets). We propose a number of densitometry criteria to identify the most promising biopolymer samples for the development of medical products with characteristics maximally resembling the physiological ones.
Modulation interference microscopy is one of the promising technologies for early personalized cancer diagnostics that allow for assessment of the real-time changes of subcellular microstructures with 2D and 3D reconstructions of the images and multifactorial data analysis. Our investigation is aimed to assess the heterogeneity of the CTCs population in patients with breast cancer using modulation interference microscopy (Quantitative Phase Imaging (QPI) technology). Morpho-functional changes in living CD326 (EpCam)+ and Annexin V+ cell were measured with a laser modulation interference microscope MIM (PA UOMZ, Russia): height accuracy 0,1 nm, coordinate accuracy 10 nm, image area 1280x1024 pixels, optical magnification 1000, acquisition time 0,3 sec. The complex algorithm included the definition of optic and geometrical characteristics of living cells and , statistical analysis of data and creation of medical documents. We evaluated the functional cellular conditions based on the phase-interference features of their nuclear structures including nucleoli organizer areas which reflect metabolic and proliferative activity of cells and also serve the markers of their malignant transformation.
Revealing and characterization of circulating tumor cells (CTCs) is one of the most actively investigated field of oncology. It was established by the past time studies that some of these cells were in condition of apoptosis and so couldn’t initiate formation of a clinically significant metastasis. The novel opportunities associated with development of computer technologies and interferential microscopes facilitate the solution of many medico-biological problems. One of them is a method of determination of CTCs functional condition based on the phase-interferential characteristics of their nuclear structures which reflect cellular metabolic and proliferative activity and are the markers of their malignant transformation.
The analysis was carried out of the morphodensitometric biomarkers of the metastatic activity of circulating tumorous cells including conformational alterations of their nuclear structures. The reference collection of phase images of metastatic cells was created as well as the data of the morphodensitometric parameters of their metabolic activity was received.
A new information was received about heterogeneity of the circulating tumorous cells with different metastatic activity which allows the broadening of fundamental scientific knowledge concerning cancerogenesis, individualization of the observation and treatment of patients, improvement of the quality of treatment, and optimization of its cost.
The development of robust non-invasive laboratory screening methods for early diagnosis on the out-patient basis seems quite relevant for practical medicine. It is known, that platelet is an original biosensor, a detector of early changes in hemostasis condition. The aim of this study was to assess a potential of the quantitative phase imaging (QPI) technique for real time evaluation the influence of low-molecular weight and unfractionated heparin on platelets in patients with the end-stage of chronic renal failure, who were treated with program hemodialysis (PHD).
The main group consisted of 21 patients who were administered a low-molecular weight heparin for hypocoagulation during the procedure of hemodialysis. The control group (15 patients) received unfractionated heparin. Morphodensitometric state of living platelets we evaluated by QPI using computer phase-interference microscope MIM (Moscow, Russia). We analyzed the optical–geometrical parameters and the morphological features of living platelets which reflected the degree of their activation at the beginning of PHD (before administration of heparin), in 15 minutes after it and at the end of the procedure. The results allow us to conclude that the use of low-molecular weight heparin provides better ratio of efficacy/safety and causes a reduction of the platelet activation during the hemodialysis procedure. Practical implementation of QPI for clinical monitoring of platelets makes it possible to obtain important information on hemostasis cell. It opens new opportunities to assess the efficacy of treatment, as well as for early diagnosis of complications for disease.
Quantitative phase imaging (QPI) can be considered as a potential tool to extract important information on the refractive index of the cellular and subcellular structures. Interphase chromatin is an original biosensor, a detector of early changes in morphofunctional cell condition. The authors presented a technology of densitometric segmentation based on the quantitative phase microscopy and computed analysis of the changes in the optic density of interphase chromatin as a biosensor. The purpose of this work to evaluate the possibility of quantitative phase imaging for visualizing the nuclear proteins conformation and chromatin decondensation degree. Modification of chromatin structure, compactness of its package, and so on, are indicative of cell condition alteration and may be projected on the organism as a whole not only for the early preclinical diagnostics but also for assessment of prognosis in crisis conditions.
It is well known that platelets play a central role in hemostasis and thrombosis, they also mediate tumor cell growth, dissemination and angiogenesis. The purpose of the present experiment was to evaluate living platelet size, function and morphology simultaneously in unactivated and activated states using Phase-Interference Microscope “Cytoscan” (Moscow, Russia). We enrolled 30 healthy volunteers, who had no past history of aeteriosclerosis-related disorders, such as coronary heart disease, cerebrovascular disease, hypertention, diabetes or hyperlipidemia and 30 patients with oropharynx cancer. We observed the optic-geometrical parameters of each isolated living cell and the distribution of platelets by sizes have been analysed to detect the dynamics of cell population heterogeneity. Simultaneously we identified 4 platelet forms that have different morphological features and different parameters of size distribution. We noticed that morphological platelet types correlate with morphometric platelet parameters. The data of polymorphisms of platelet reactivity in tumor progression can be used to improve patient outcomes in the cancer prevention and treatment. Moreover morphometric and functional platelet parameters can serve criteria of the efficiency of the radio- and chemotherapy carried out. In conclusion the computer phase-interference microscope provides rapid and effective analysis of living platelet morphology and function at the same time. The use of the computer phase-interference microscope could be an easy and fast method to check the state of platelets in patients with changed platelet activation and to follow a possible pharmacological therapy to reduce this phenomenon.
KEYWORDS: Fluctuations and noise, Phase imaging, Diagnostics, Microscopy, Blood, Proteins, Real time imaging, Phase interferometry, Medical research, Interferometry
The objective of the present study is to increase the quality of the early diagnosis using cytological differential-diagnostic criteria for reactive changes in the nuclear structures of the immunocompetent cells. The morphofunctional status of living cells were estimated in the real time using new technologic platform of the hardware-software complex for phase cell imaging. The level of functional activity for lymphocyte subpopulations was determined on the base of modification of nuclear structures and decreasing of nuclear phase thickness. The dynamics of nuclear parameters was used as the quantitative measuring for cell activating level and increasing of proliferative potential.
Combined analysis of optic-geometrical characteristics makes it possible to comprehensively evaluate the morphological and functional state of the cytological object, which can not be done during visual observation. The technique is discussed for real-time monitoring of the functional status of platelets using computer-aided phase microscope (CPM) 'Cytoscan'. High accuracy and sensitivity of CPM with respect to determination of local temporal phase make it possible to register the dynamic processes in the voluntarily chosen points and sections of micro-objects, to obtain the Fourier's spectra and other characteristics suitable for statistical analysis. Human platelets were prepared from venous blood of healthy donors and pregnant women by standard methods, suspended in culture medium 199 and treated by different doses of 6% Infukoll HES. Nonfixating and nonstaining cells were studied with CPM: height accuracy 0.5 nm, magnification 1000, acquisition time 4 - 30 s. In our experiments we used time resolution about 0.03 s and 30 x lens with numerical aperture 0.65. During investigations of temporal processes a certain section was chosen in the topogram of cell image and local values for the phase of scattered wave in each of the points of the chosen cell's profile were measured. On the basis of the results of automated phase image analysis of optic-geometrical characteristics of living cells, the new quantitative express-method for evaluating of the functional status of human platelets was developed and tested. The structural changes of cells were visualized in alteration of 3-D images, phase profiles, in the decrease of mean cell phase diameters, heights, volumes, in disturbance of histograms of phase heights distribution by cell image points. New data on the behavior of platelets treated by Infukoll in vitro and in vivo were obtained. Analysis of intracellular dynamics was allowed to characterize the cell's regions of maximal activity, but the intensity of processes taking place in these regions in accordance with frequencies as well. The difference between the calculated values of frequencies (from 0.1 to 17 H) is associated with the influence of Infukoll on the metabolism and functional activity of living platelets.
In recent years we have been working out a new method of treatment of steroid-resistant asthma -- extracorporeal He-Ne-laser blood irradiation. The procedures gave good clinical effect and allowed reduction of steroid dose in a majority of patients. The monocytes were investigated by luminol-dependent chemiluminescence and the method of living cell microcopy. Extracorporeal He-Ne laser blood irradiation normalized both monocytes chemiluminescence and cell oscillation in asthmatic patients significantly earlier than in cases of ordinary treatment, although the mechanisms of action of He-Ne-laser irradiation upon blood steroid-resistant asthmatics were not fully determined.
Living cells investigation and cell response to external factors are of great interest for practical medicine and biology. The main advantages of computer aided phase microscope (CPM) Cytoscan which permits us to observe the cell surface and internal structure consists in superresolution and the possibility of the dynamic processes registering. We attempt to characterize some aspects of the morphofunctional status of human lymphocytes determining the dynamics of the selected points in normal or pathological cells. To evaluate the lymphocyte homeostasis donors and persons of autoimmune diseases were analyzed and the changes of optical and geometrical cell parameters registered by CPM Cytoscan. The dynamic process registration allowed us to perform the real-time quantitative analysis of the living lymphocyte activity in norm and pathology.
The main advantages of the computer-aided phase microscope (CPM) Airyscan which permits the user to observe the cell surface and internal structure consists in superresolution and the possibility of the dynamic processes registering. Immunocompetent cells in process of activation with different agents were studied. Several criterions for evaluation of the functional status of immunocompetent cells based on the shape changes were suggested on the basis of real-time immunology reaction monitoring. The perspectives of the proposed method for immunology testing are discussed.
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