[Possibilities of multiphoton microscopy for the diagnosis of the vulvar lichen sclerosus]. / Vozmozhnosti mul'tifotonnoi mikroskopii dlya diagnostiki skleroticheskogo likhena vul'vy.

BACKGROUND: Vulvar lichen sclerosus (VLS) is a chronic and recurrent dermatosis of an inflammatory nature with severe focal atrophy of the skin. Connective tissue changes are polymorphic and are still not taken into account in histological diagnostics due to the difficulty of interpreting routine histological methods. In this work, we use multiphoton microscopy (MPM) as a new imaging technique that provides detailed information about the organization of collagen fibers in the dermis based on a non-linear second harmonic generation (SHG) process. OBJECTIVE: To determine the degree of connective tissue damage in lichen sclerosus using standard histological techniques and to reveal the diagnostic capabilities of multiphoton microscopy. MATERIAL AND METHODS: We studied 42 biopsies with a histopathological diagnosis of VLS and 10 biopsies of normal vulvar skin. Histological, histochemical and immunohistochemical evaluation was used in comparison with MPM data. Quantitative analysis included the determination of the thickness, length of collagen fibers and the average intensity of the SHG signal. RESULTS: A comprehensive study of the skin showed 4 groups of changes that can be regarded as the degree of the dermis damage: initial, mild, moderate, severe. The affected area at the initial and mild degree has subtle changes, however, it is reliably identified by quantitative analysis of the SHG signal. So, the initial degree is characterized by thin (1.3-1.8 µm) long (56-69 µm) collagen fibers, with a moderate degree, the fibers are thickened (3.4-4.3 µm) and fragmented (22-37 µm). The affected area in moderate and severe cases undergoes homogenization, which is associated with the deposition of extremely thin (0.6-0.9 µm) short (16-28 µm) collagen fibers and the expression of type V collagen. CONCLUSION: Multiphoton microscopy in the second harmonic generation mode is a reliable method for identifying collagen fibers in tissues. The study made it possible to identify 4 degrees of the dermis damage in vulvar lichen sclerosus.

Experimental Models for Studying Structural and Functional State of the Pathological Liver (Review).

Liver pathologies remain one of the leading causes of mortality worldwide. Despite a high prevalence of liver diseases, the possibilities of diagnosing, prognosing, and treating non-alcoholic and alcoholic liver diseases still have a number of limitations and require the development of new methods and approaches. In laboratory studies, various models are used to reconstitute the pathological conditions of the liver, including cell cultures, spheroids, organoids, microfluidic systems, tissue slices. We reviewed the most commonly used in vivo, in vitro, and ex vivo models for studying non-alcoholic fatty liver disease and alcoholic liver disease, toxic liver injury, and fibrosis, described their advantages, limitations, and prospects for use. Great emphasis was placed on the mechanisms of development of pathological conditions in each model, as well as the assessment of the possibility of reconstructing various key aspects of pathogenesis for all these pathologies. There is currently no consensus on the choice of the most adequate model for studying liver pathology. The choice of a certain effective research model is determined by the specific purpose and objectives of the experiment.

Multiphoton Microscopy and Mass Spectrometry for Revealing Metabolic Heterogeneity of Hepatocytes in vivo.

The aim of the investigation was to study the possibility of revealing the heterogeneity of normal liver hepatocytes in terms of metabolic status using the modern methods of multiphoton microscopy and mass spectrometry. Materials and Methods: Heterogeneity of hepatocytes in terms of total metabolic activity was assessed using multiphoton microscopy based on the autofluorescence intensity of intracellular cofactors NAD(P)H and FAD. Hepatocyte heterogeneity in terms of intensity of intracellular metabolic processes was determined using the fluorescence lifetime imaging (FLIM) method based on the data about fluorescence lifetime contributions of various forms of NAD(P)H. The method of time-of-flight secondary ion mass spectrometry (TоF-SIMS) was used to study the lipid and amino acid composition of hepatocytes. Results: It has been revealed using multiphoton microscopy that hepatocytes are heterogeneous in terms of general metabolic activity. Using FLIM, it was established that the heterogeneity degree was high in terms of intensity of oxidative phosphorylation, glycolysis, and synthetic processes (lipogenesis, nucleic acid synthesis, and the pentose phosphate pathway). The TоF-SIMS method revealed the presence of hepatocyte heterogeneity in terms of amino acid and lipid composition, which points to various intensities of synthetic processes in individual hepatocytes. Moreover, differences in the content of PO3 ions were revealed. The results of ToF-SIMS study correlate with the data obtained by multiphoton microscopy and FLIM, confirming the revealed heterogeneity of hepatocytes in terms of general metabolic activity and intensity of intercellular metabolic processes. Conclusion: The latest methods of fluorescence bioimaging and mass spectrometry proved to be effective in revealing hepatocyte heterogeneity in terms of metabolic status. The presence of heterogeneity should be taken into account in studying the liver tissue under various conditions with the application of fluorescence bioimaging methods.

Trans-Serosal Multimodal Optical Coherence Tomography for Visualization of Microstructure and Blood Circulation of the Small Intestine Wall.

The aim of the study was to evaluate the performance of trans-serosal multimodal OCT (MM OCT) in in vivo detecting of changes in microstructure and blood circulation of the small intestine wall caused by arteriovenous ischemia resulted from intestine strangulation. MATERIALS AND METHODS: In experiments on Wistar rats (n=22), we examined the small intestine wall in vivo using MM OCT; the access to the intestine was reached through laparotomy. The microvasculature and microstructure of the wall were studied before and after acute arteriovenous ischemia created by ligation of a small bowel segment. The results were then added with data obtained from histological and intravital microscopic examination. RESULTS: Trans-serous MM OCT allowed us to visualize the bowel wall to its entire thickness, distinguish between the serous-muscular and mucous-submucosal layers, and detect the villi and functioning blood vessels. The structures were best seen after a fat emulsion had been administered into the bowel lumen. In OCT images made in the optical coherent angiography (OCA) mode, large paired vessels (arteries and veins) and micro-vessels with a diameter of >15 µm could be seen. Most of the blood vessels were imaged in the depth range of 80-300 µm from the surface. Capillaries with a diameter of 7-10 µm were not seen, but they produced an overall bright background. In the OCA images reconstructed from a volume of 2.4×2.4×1.8 mm, the total length of the vascular bed before ischemia was 18.3 [16.6; 19.8] mm.Strangulation of the intestinal loop was associated with changes in the CP OCT picture: the villi-associated vertical pattern and shadows of blood vessels disappeared and the depth of tissue visualization in the cross-channel decreased. The optical equivalents of the serous-muscular layer were preserved; after 180±12 min of ischemia, their proportion in the intestinal wall thickness increased from 25 [18; 32] to 42 [31; 55]% (p=0.031). At that time-point, OCA images of the strangulated bowel loop looked all similar: a uniform dark background with isolated fragmentary large vessels and no signs of blood flow in the microvascular network. CONCLUSION: Trans-serous MM OCT provides for in vivo visualization of microstructures critical for surgical gastroenterology: the intestinal wall layers including villi and blood vessels of each layer, as confirmed by histological analysis. Destructive processes in the intestinal wall resulting from bowel ligation bring about optical changes, which can be detected using real-time MM OCT.

[Use of optical coherence tomography for prognosis of the severity of oral mucositis].

The results of oral mucosa monitoring by optical coherence tomography (OCT) in the course of radiochemotherapy of 18 cases of oropharyngeal cancer are discussed. Damage to the mucosa was mainly assessed using contrast characteristics deterioration and reduction of epithelial layer thickness. Significant variation in OCT image characteristics was identified vs. mucositis grade and prognostic criteria for individual mucosal radiosensitivity worked out intact contrast on day 1 of mucositis should be interpreted as a sign of relatively mild complication. Blurred contrast would indicate mucositis stage III-IV development. Numerical analysis of OCT image patterns confirmed the contribution of the endothelial blood vessels and connecting tissue to radiation-induced damage to the mucosa.