Lichen sclerosus (LS) is the most common vulvar dermatosis, which is characterized by damage to the connective tissue of the dermis. The affected area in lichen sclerosus is characterized by a sequential change in the main components of the connective tissue - collagen and elastin fibers. The affected area is polymorphic and remains poorly defined from a histological point of view. Among histopathologists, there are no unequivocal opinions on changes in the connective tissue of the dermis in LS. However, an assessment of the degree of dermis damage is important for the timely diagnosis of the condition and adequate treatment. Nonlinear microscopy includes second-harmonic generation (SHG) and twophoton autofluorescence (TPEF). SHG allows to selectively examine the signal from heterotypic collagen fibers of the dermis that contain type 1 collagen. TPEF allows to identify elastic fibers of the connective tissue matrix. It has been demonstrated that nonlinear microscopy allows visualizing the changes in the microstructure of collagen and elastin fibers. Three histological patterns were revealed as a result of the analysis of the nonlinear optical microscopy of the classical VLS. These histological patterns cannot be distinguished using histological stains and indicate a polymorphism of connective tissue changes. Nonlinear microscopy makes it possible to assess the changes in tissue structure, which is important for the histological interpretation of changes in the dermis and to clarify histological classification system in the future.
3D-models based on patient-specific induced pluripotent stem cells are widely used for biomedical research. Thus, non-invasive and continuous comprehensive description of these of 3D-models using FLIM and multiphoton microscopy requires.
KEYWORDS: Fluorescence lifetime imaging, Oxygen, 3D modeling, Luminescence, Biological research, 3D displays, Tissues, Stem cells, Microscopy, Medical research
3D models based on cells differentiated from patient-specific induced pluripotent stem cells (iPSCs) are widely used to identify disease phenotypes, to accurately analyze dysfunctions at the level of human tissues and organs, to screen new drugs, which makes them more promising tool for biomedical research tasks than monolayer cultures, which is associated with their proximity to in vivo. The metabolic activity with oxygenation level of cells, assessed by optical imaging methods, can be used as markers of cell viability, proliferative activity and the degree of differentiation in 3D culture conditions. In this paper we used fluorescence and phosphorescence lifetime imaging microscopies (FLIM and PLIM) to study the metabolic status and the oxygenation level of derived from iPSCs neural stem cells (NSC) cultured in 3D condition. An analysis of the fluorescence intensities and FLIM data showed that NSCs in monolayer and at the periphery of large spheroids have more glycolytic phenotypes, NSCs in the center of large spheroids and NSCs grouped into small spheroids have more oxidative state. For determination of the relative oxygen level in spheroids PLIM of BPTDM stained neurospheres was carried out. As it was supposed, oxygen transport in the spheroid depended on it size. In neurospheres with an average size 600 μm O2 distribution is radial, with the lowest concentration in the center. Thus, the metabolic status and oxygenation level of the NSC in the spheroid composition was assessed in a life-time and noninvasive manner.
The aim of our work was to investigate energy metabolism of patient’s tumor ex vivo using fluorescence lifetime imaging (FLIM). Tumor metabolism was examined by detecting the fluorescence lifetime of metabolic cofactor NAD(P)H. In glioma tumor samples was shown a different energy metabolism with white matter. Colorectal tumors displayed marked intertumor heterogeneity and shift towards oxidative energy metabolism. Thereby, the possibility of assessing the energy metabolism in postoperative tumor samples of patients was shown.
Successful gastrointestinal surgery is based on the precise knowledge of the morphological, functional and metabolic state of the bowel wall at a specific time point. Current trends include the development of real-time, minimally invasive, label-free and rapid techniques for tissue assessment in combination with algorithms of data processing. The aim of the study was to evaluate the performance of trans-serous multimodal optical coherence tomography (MM OCT) and FLIM macro-imaging in detecting changes in microstructure, blood circulation and metabolism of intestinal wall caused by acute arterial ischemia in experiment. The study was supported by the Russian Science Foundation, project No. 19-75-10096.
Breast cancer is a genetically heterogeneous disease characterized by various biomolecular and morphological features that affect the diagnosis, prognosis, and treatment response. In this study we combined cross-polarization optical coherence tomography (CP OCT) and multiphoton tomography (MPT), based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) to visualize tumor stroma and tumor cells in specimens of a human breast tissue. The data obtained by both techniques were compared with histopathology. The CP OCT and MPT images were quantitatively assessed to distinguish a breast normal tissue from a cancer as well as between a low and a high grade of cancer. Quantitative assessment of the CP OCT image included the calculation of signal attenuation coefficients separately for co- and cross- polarization channels and the formation the color-coded en-face distribution maps of these coefficients. The attenuation coefficient in cross- polarization channel showed better difference between breast cancer of low and high grades and distinguish them from normal tissue. The SHG images were processed using texture analysis techniques to quantify the density of collagen fibers in normal tissue and tumor. Thus, both imaging techniques have great potential to distinguish nontumorous and tumorous human breast tissue of varying degrees of malignancy and could provide identifying breast cancer margins for in surgery.
Non-invasive optical techniques on the basis of quenching of phosphorescence by molecular oxygen represent a powerful tool to perform mapping of oxygen content in biological systems. The development of phosphorescent oxygensensitive probes suitable for biological applications, especially in vivo, remains a challenging task. In this paper we applied for biological investigations one novel and one previously published Ir(III)-based complexes. This complexes demonstrate low cytotoxicity at the concentrations ≤70 μM and good uptake by cultured cancer cells. Both compounds were primarily located within the cytoplasm. Using TCSPC-based phosphorescence lifetime macro-imaging in vivo, we found that IR-2 complex shows detectable phosphorescence in tumor tissue in mice upon local injection. These results illustrate the high potential of organometallic complexes under study for mapping of oxygen level in cells and tissues.
Colorectal cancer is one of the most commonly diagnosed and poorly responding to chemotherapy types of cancer, which emphasizes the importance of personalized approach to treatment selection. Short-term primary cell cultures established from patients’ tumors represent a valuable model for testing drug response. In this study, we developed protocols for generation of the short-term primary cell cultures from colorectal cancer tissue and assessment their chemosensitivity using MTT test. Additionally, we showed the possibility of metabolic analysis of patient-derived cancer cells using fluorescence lifetime imaging (FLIM) of autofluorescent cofactor NAD(P)H. Since FLIM of NAD(P)H demonstrates the potential to detect early responses to cancer treatment, we assume that this method, alone or in combination with MTT assay, can be used for choosing the optimal chemotherapy for patients.
Bladder cancer, occurring at different times after radiation therapy for tumors of the pelvic organs (cervical cancer, uterus cancer, prostate cancer) is a specific problem of oncourology. The main visual manifestations of the adverse events of radiation therapy in early time after irradiation are pronounced edema and hyperemia of the bladder mucosa, in the case of severe complications – hemorrhages. In years after radiation exposure atrophic changes of the mucous membrane, telangiectasia and reduction of the capacity of the bladder occur; late complications of a severe degree manifest as ulcers and fistulas. The main clinical symptoms of bladder cancer are hematuria and dysuria. The cystoscopy still remains the “gold standard” for its diagnosis, but this method appears to have significant limitations if a tumor arises in bladder tissue suffering from radiation exposure. In this case, the clinical symptoms and the cystoscopic picture can be regarded as manifestations of the side effects of RT, especially in case of grade 3 and 4 of complication. Optical methods may play a key role for distinguishing between a metachronous bladder cancer and severe complications of radiation therapy. Optical coherence tomography (OCT) has a spatial resolution corresponding to several micrometers and makes it possible to assess the structure of biological tissues at a depth of up to 2 mm. Besides, polarization-sensitive methods OCT (CP OCT) allow evaluating the state of the connective tissue matrix which loses orderliness and organized structure of the arrangement of collagen fibers and bundles in malignant tumors. The study presents the case reports of bladder cancer which arose against the radiation-induced changes of bladder tissue after previous irradiation for cervical cancer and were diagnosed by optical coherence tomography.
Cirrhosis is defined as the histological development of regenerative nodules surrounded by fibrous bands in response to chronic liver injury that leads to portal hypertension and end stage liver disease. Conventional techniques are insufficient to precisely describe the internal structure, heterogeneous cell populations and the dynamics of biological processes of the diseased liver. Currently, multiphoton microscopy with fluorescence lifetime imaging is actively introduced to biomedical research. This technic is extremely informative and non-destructive that allows studying of a large number of processes occurring inside cells and tissues, analyzing molecular cellular composition, as well as evaluating the state of connective tissue fibers due to their ability to generate a second optical harmonic. In this study we investigated metabolic changes and collagen fibers formation in the rat liver with induced cirrhosis based on the fluorescence of the metabolic co-factors (NAD(P)H, FAD) and a second harmonic signal by multiphoton microscopy with FLIM and SHG mode. Moreover we studied ex vivo liver samples of patients with cirrhosis. We presented a separate analysis of NADH and NADPH to estimate the contribution of energy metabolism and lipogenesis in the metabolic changes. The data can be used to develop new criteria for the identification of hepatic pathology at the level of hepatocyte changes directed to personalized medicine in the future.
The clinical diagnosis of melanocytic lesions is an ongoing medical challenge. Non-invasive tools and technologies can help to distinguish equivocal lesions. The aim of the study was to improve the in vivo diagnostic possibilities for the differentiation of benign and malignant melanocytic lesions based on combination of three imaging label-free modalities (multiphoton tomography, fluorescence lifetime imaging and optical coherence angiography). Thirty-two melanocytic lesions were studied, using multiphoton tomography, fluorescence lifetime imaging and optical coherence angiography. Multiphoton tomography features of benign melanocytic nevi were characterized by the normal morphology of both the keratinocytes and the nevus cell nests surrounded by collagen. Dysplastic nevi were characterized by their increased intercellular distances and enlarged cell nuclei. Melanomas showed the presence of melanocytes and dendritic structures in all layers of the epidermis. Analysis of the metabolic state revealed that melanomas and dysplastic nevi were characterized by enhanced glycolysis. Optical coherence angiography shows that benign nevi had regular vascular networks and equal numbers of thin and thick vessels. Vascular networks of dysplastic nevi were characterized thin curved vessels. Thick irregular spiral vessels formed a dense microvascular network of melanomas.
This paper presents ex-vivo investigation of the brain tumors, namely glioblastoma and anaplastic astrocytoma, by macroscopic fluorescence lifetime imaging (FLIM) of endogenous metabolic cofactor nicotine amide dinucleotide (phosphate), NAD(P)H. The results of the study on the rat models indicate that the metabolism in brain tumors differs between tumor types and differs from normal brain tissue. It was also found that the brain tumors have specific optical metabolic signatures that differ them from most solid tumors, underling the complexity of glioma metabolism. The approach used in the experiments shows prospects to determine the surgical margins of gliomas and to investigate metabolic heterogeneity of the tumors on a macroscale. However, one has to be careful in the interpretation of the data obtained by FLIM.
The objective of the study is the quantitative analysis of the dose-time dependences of changes occurring in collagen of bladder and rectum after gamma-irradiation using optical methods [nonlinear microscopy in a second harmonic generation (SHG) detection regime and cross-polarization optical coherence tomography (CP OCT)]. For quantitative assessment of the collagen structure, regions of interest on the SHG-images of two-dimensional (2-D) distribution of SHG signal intensity of collagen were chosen in the submucosa. The mean SHG signal intensity and its standard deviation were calculated by ImageJ 1.39p (NIH). For quantitative analysis of CP OCT data, an integral depolarization factor (IDF) was calculated. Quantitative calculation of the SHG signal intensity and the IDF can provide additional information about the processes of the collagen radiation-induced degradation and subsequent remodeling. High positive correlation between the mean SHG signal intensity and the mean IDF of bladder and rectum demonstrates that CP OCT can be used as an “optical biopsy” in the grading of collagen radiation damage.
Cancer-associated fibroblasts (CAFs) are one of the key determinants in the malignant progression of cancer. The subject of this research was metabolic reorganization of CAFs and their participation in collagen cross-linking process. The metabolic differences between normal fibroblasts and CAFs were elucidated using two-photon fluorescence lifetime imaging microscopy (FLIM). Collagen structure in 3D model was assessed using second harmonic generation (SHG) microscopy. We show increased metabolic activity of fibroblasts derived from patient’s colon tumor with a shift to more oxidative metabolism compare to dermal fibroblasts. The results of the study of collagen suggest that CAFs may contribute to the tumor progression through the facilitation of collagen alignment. In general, our findings support the idea of the strong association between cancer cells and fibroblasts and extensive involvement of CAFs in modulation of tumor microenvironment.
The main purpose of this work is to evaluate the possibility to distinguish in vivo benign papilloma, severe dysplasia and squamous cell carcinoma by establishing quantitative image characteristics of multiphoton tomography (MPT) and multimodal optical coherence tomography images (MM OCT). Specific features of papillomatous outgrowths at different stages were revealed using 7,12-dimethylbenz[a]anthracen (DMBA)-induced hamster oral carcinoma. Analysis of MPT images included assessment of nuclear-cytoplasmic (NC) ratio, nuclear density and heterogeneity parameter F. Crosspolarization OCT images were quantified via the integral depolarization factor (IDF). Analysis of OCT microvascular maps enabled differential analysis based on the number of smallest-diameter blood vessels present in a particular pathology. Both MPT and MM OCT metrics showed some difference between benign papilloma, dysplastic papilloma, and squamous cell carcinoma tissue states. The results suggested that combined use of MPT and MM OCT have great potential for in vivo differentiation between benign and malignant papillomas.
This presentation describes the development of the optical macroscanner and its application for metabolic imaging of large areas of tumors in mice. The scanner allows to interrogate areas as large as 15x15mm with the lateral resolution on the order of 15 micrometers. Acquisition times range from a few seconds for low pixel numbers to
several minutes for high-resolution images. We present data for NAD(P)H imaging of tumor with genetically encoded mKate2. In addition, using macroscanner we demonstrated the possibility of visualizing caspase-3 activity using the FRET-biosensor TR23K, which is based on a pair of proteins - a red fluorescent protein as a donor and a chromoprotein as an acceptor. The in vivo assay was noninvasive and could be applied in strongly and weakly fluorescent subcutaneous xenografts in mice using the FLIM-FRET method.
Cisplatin is an effective anticancer drug commonly used in the treatment of solid tumors. Although DNA is considered as the primary target, the cisplatin action at the cellular level remains unknown. Advanced fluorescence microscopy techniques allow probing various physiological and physicochemical parameters in living cells and tissues with unsurpassed sensitivity in real time. This study was focused on the investigation of cellular bioenergetics and cytosolic pH in colorectal cancer cells during chemotherapy with cisplatin. Special attention was given to the changes in cisplatininduced apoptosis that was identified using genetically encoded FLIM/FRET sensor of caspase-3 activity. Metabolic measurements using FLIM of the metabolic cofactor NAD(P)H showed decreased contribution from free NAD(P)H (a1, %) in all treated cells with more pronounced alterations in the cells undergoing apoptosis. Analysis of cytosolic pH using genetically encoded fluorescent sensor SypHer1 revealed a rapid increase of the pH value upon cisplatin exposure irrespective of the induction of apoptosis. To the best of our knowledge, a simultaneous assessment of metabolic state, cytosolic pH and caspase-3 activity after treatment with cisplatin was performed for the first time. These findings improve our understanding of the cell response to chemotherapy and mechanisms of cisplatin action.
A cholestatic liver disease presents one of the most common liver diseases and can potentially progress to cirrhosis or even cholangiocarcinoma. Conventional techniques are insufficient to precisely describe the complex internal structure, heterogeneous cell populations and the dynamics of biological processes of the liver. Currently, the methods of multiphoton and fluorescence lifetime imaging microscopy are actively introducing to biomedical research. Those methods are extremely informative and non-destructive that allows studying of a large number of processes occurring inside cells and tissues, analyzing molecular cellular composition, as well as evaluating the state of connective tissue fibers due to their ability to generate a second optical harmonic. Multiphoton and FLIM microscopy do not need additional staining of samples or the incorporation of any markers to study metabolism, lipid composition, microstructure analysis, evaluation of fibrous structures. These parameters have pronounced changes in hepatocytes of liver with common pathological diseases. Thereby in this study we investigated metabolic changes in the healthy and cholestatic liver based on the fluorescence of the metabolic co-factors NAD(P)H and FAD by multiphoton microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH was presented. The data can be used to develop new criteria for the identification of hepatic pathology at the level of hepatocyte changes directed to personalized medicine in the future.
Nonlinear optical microscopy combined with fluorescence lifetime imaging is a non-invasive imaging technique, based on the study of fluorescence decay times of naturally occurring fluorescent molecules, enabling a noninvasive investigation of the biological tissue with subcellular resolution. Cancer exhibits altered cellular metabolism, which affects the autofluorescence of metabolic cofactors NAD(P)H and FAD. In this study features of tumor metabolism in different systems of organization (from cell culture to patient lesion) was showed. The observed differences in the relative contributions of free NAD(P)H and FAD testify to an increased a glycolytic metabolism in cancer cells compare to fibroblasts. In 3D spheroids, the cells of the proliferating zone had greater a1 and lower tm values than the cells of the quiescent zone, which likely is a consequence of their higher glycolytic rate. During the growth of colorectal cancer in the experimental mouse model, the contribution of the free component of NAD(P)H was increased. Dysplastic nevus and melanoma is characterized by raised contribution of free NADH compare to healthy skin. Therefore, melanoma cells had very short value of τ1.
Fluorescence lifetime imaging microscopy (FLIM) is a promising non-invasive highly sensitive technique for probing multiple physiological and physicochemical parameters in living cells and tissues. The present study is focused on the investigation of bioenergetics and microscopic viscosity of cultured cancer cells and animal tumors using FLIM during natural growth and chemotherapy. Fluorescence lifetime measurements of the metabolic cofactor NAD(P)H revealed a decrease of the relative amplitude of free NAD(P)H after cisplatin treatment, indicating a change towards a more oxidative metabolic state. Microviscosity mapping performed with the use of fluorescent molecular rotor BODIPY-2 showed a pronounced increase in the plasma membrane viscosity in cancer cells exposed to cisplatin. Although biochemical mechanisms underlying the metabolic and viscosity alterations during chemotherapy have yet to be clarified, our data suggest that the cisplatin-induced changes in cellular metabolism and membrane viscosity play a role in the cytotoxicity of the drug. The results of the study contribute to an understanding of mechanisms of cisplatin action and will be useful for development new approach for assessing response to a therapy.
The aim of the study was to estimate energy metabolism in human cervical cancer cells HeLa Kyoto after chemotherapy in vitro and in vivo using two-photon fluorescence lifetime microscopy (FLIM). Cellular metabolism was examined by monitoring of the fluorescence intensities and lifetimes of metabolic cofactors NAD(P)H and FAD. Cancer metabolism was analyzed in dynamics after treatment with cisplatin. Two-photon fluorescence and second harmonic generation microscopies as well as standard histopathology with hematoxylin and eosin were used to characterize cancer tissue structure. We showed an increase of the optical redox ratio FAD/NAD(P)H in cancer cells in vitro and decrease of the relative contribution of free NAD(P)H (ɑ1) in vitro and in vivo, which presumably indicate a shift to more oxidative metabolism after chemotherapy. These data demonstrate the possibility to detect response of cancer cells to chemotherapy using optical metabolic imaging.
Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities
A combination of approaches to the image analysis in cross-polarization optical coherence tomography (CP OCT) and high-resolution imaging by nonlinear microscopy and atomic force microscopy (AFM) at the different stages of atherosclerotic plaque development is studied. This combination allowed us to qualitatively and quantitatively assess the disorganization of collagen in the atherosclerotic arterial tissue (reduction and increase of CP backscatter), at the fiber (change of the geometric distribution of fibers in the second-harmonic generation microscopy images) and fibrillar (violation of packing and different nature of a basket-weave network of fibrils in the AFM images) organization levels. The calculated CP channel-related parameters are shown to have a statistically significant difference between stable and unstable (also called vulnerable) plaques, and hence, CP OCT could be a potentially powerful, minimally invasive method for vulnerable plaques detection.
In this study we demonstrate the capability of cross-polarization optical coherence tomography (CP OCT) to assess collagen and elastin fibers condition in atherosclerotic plaques basing on ratio of the OCT signal levels in cross- and со- polarizations. We consider the depolarization factor (DF) and the effective birefringence (Δn) as quantitative characteristics of CP OCT images. We revealed that calculation of both DF and Δn in the region of interest (fibrous cap) yields a statistically significant difference between stable and unstable plaques (0.46±0.21 vs 0.09±0.04 for IDF; (4.7±1.0)•10–4 vs (2.5±0.7)•10–4 for Δn; p<0.05). In parallel with CP OCT we used the nonlinear microscopy for analysis of thin cross-section of atherosclerotic plaque, revealing the different average isotropy index of collagen and elastin fibers for stable and unstable plaques (0.30 ± 0.10 vs 0.70 ± 0.08; р<0.001). The proposed approach for quantitative assessment of CP OCT images allows cross-scattering and birefringence characterization of stable and unstable atherosclerotic plaques.
The aim of the present work was to study energy metabolism in human cervical carcinoma (HeLa) cells in vitro and in vivo using two-photon FLIM. Cellular metabolism was examined by monitoring of the fluorescence lifetimes of free and protein-bound forms of NAD(P)H and FAD and their relative contributions. Two-photon fluorescence and second harmonic generation microscopy as well as standard histopathology with hematoxylin and eosin were used to characterize tissue structure. Cellular metabolism was analyzed in cancer cells co-cultured with human fibroblasts and in tumor xenografts transplanted to nude mice. In the HeLa-huFB co-culture we observed a metabolic shift from OXPHOS toward glycolysis in cancer cells, and from glycolysis to OXPHOS in fibroblasts, starting from Day 2 of co-culturing. In the tumor tissue we detected metabolic heterogeneity with more glycolytic metabolism of cancer cells in the stroma-rich zones. The results of the study are of a great importance for understanding metabolic behavior of tumors and for development of anticancer drugs targeted to metabolic pathways.
Non-invasive imaging of cell metabolism is a valuable approach to assess the efficacy of stem cell therapy and understand the tissue development. In this study we analyzed metabolic trajectory of the mesenchymal stem cells (MCSs) during differentiation into adipocytes by measuring fluorescence lifetimes of free and bound forms of the reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD). Undifferentiated MSCs and MSCs on the 5, 12, 19, 26 days of differentiation were imaged on a Zeiss 710 microscope with fluorescence lifetime imaging (FLIM) system B&H (Germany). Fluorescence of NAD(P)H and FAD was excited at 750 nm and 900 nm, respectively, by a femtosecond Ti:sapphire laser and detected in a range 455-500 nm and 500-550 nm, correspondingly. We observed the changes in the NAD(P)H and FAD fluorescence lifetimes and their relative contributions in the differentiated adipocytes compare to undifferentiated MSCs. Increase of fluorescence lifetimes of the free and bound forms of NAD(P)H and the contribution of protein-bound NAD(P)H was registered, that can be associated with a metabolic switch from glycolysis to oxidative phosphorylation and/or synthesis of lipids in adipogenically differentiated MSCs. We also found that the contribution of protein-bound FAD decreased during differentiation. After carrying out appropriate biochemical measurements, the observed changes in cellular metabolism can potentially serve to monitor stem cell differentiation by FLIM.
We combined cross-polarization optical coherence tomography (CP OCT) and non-linear microscopy based on second
harmonic generation (SHG) and two-photon-excited fluorescence (2PEF) to assess collagen and elastin fibers in the
development of the atherosclerotic plaque (AP). The study shows potential of CP OCT for the assessment of collagen
and elastin fibers condition in atherosclerotic arteries. Specifically, the additional information afforded by CP OCT,
related to birefringence and cross-scattering properties of arterial tissues, may improve the robustness and accuracy of
assessment about the microstructure and composition of the plaque for different stages of atherosclerosis.
Holographic scanning microscopy - novel technique both in laser scanning microscopy and digital holographic microscopy allow multimodal approach to cell and tissue investigation in biomedical applications promising new advantages (quantitative phase imaging, superresolution, computerized tomography), but regular reconstruction leads to incorrectness. Analysis of light propagation through the schematics allows to offer reconstruction procedures depending on recording conditions.
In this paper we report on our recent achievement in application of conventional and cross-polarization OCT (CP OCT) modalities for in vivo clinical diagnostics in different medical areas including gynecology, dermatology, and stomatology. In gynecology, CP OCT was employed for diagnosing fallopian tubes and cervix; in dermatology OCT for monitoring of treatment of psoriasis, scleroderma and atopic dermatitis; and in stomatology for diagnosis of oral diseases. For all considered application, we propose and develop different image processing methods which enhance the diagnostic value of the technique. In particular, we use histogram analysis, Fourier analysis and neural networks, thus calculating different tissue characteristics as revealed by OCT’s polarization evolution. These approaches enable improved OCT image quantification and increase its resultant diagnostic accuracy.
This work is aimed at the development of new approach to register intracellular pH with genetically encoded ratiometric sensor. Intracellular pH of cancer cells was studied in vitro and in vivo. Changes of intracellular pH under conditions of co-culturing with fibroblast were investigated.
Optical methods for study biological tissue and cell at micro- and nanoscale level step now over diffraction limit. Really it is single molecule localization techniques that achieve the highest spatial resolution. One of those techniques, called bleaching/blinking assisted localization microscopy (BaLM) relies on the intrinsic bleaching and blinking behavior characteristic of commonly used fluorescent probes. This feature is the base of BaLM image series acquisition and data analysis. In our work blinking of single fluorescent spot against a background of others comes to light by subtraction of time series successive frames. Then digital estimation gives the center of the spot as a point of fluorescent molecule presence, which transfers to other image with higher resolution according to accuracy of the center localization. It is a part of image with improved resolution. This approach allows overlapping fluorophores and not requires single photon sensitivity, so we use 8,8 megapixel CMOS camera with smallest (1.55 um) pixel size. This instrumentation on the base of Zeiss Axioscope 2 FS MOT allows image transmission from object plane to matrix on a scale less than 100 nm/pixel using 20x-objective, thereafter the same resolution and 5 times more field of view as compared to EMCCD camera with 6 um pixel size. To optimize excitation light power, frame rate and gain of camera we have made appropriate estimations taking into account fluorophores behaviors features and equipment characteristics. Finely we have clearly distinguishable details of the sample in the processed field of view.
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