The vulva is a specific topographic region of the skin with complex morphology and a variety of functions. The combination of the urinary, reproductive and gastrointestinal systems in the same area makes the vulva susceptible to a wide range of diseases that require a multidisciplinary approach. As a result of these anatomical and physiological features, vulvar diseases are difficult to diagnose and insufficiently recognized. Multimodal optical coherence tomography (MM OCT) is an optical tool that allows real-time assessment of the structure of vulvar tissues with a resolution of 10-15 μm, and also provides label-free visualization of blood and lymphatic vessels. The advantages of the MM OCT method are its non-invasiveness, the possibility of examining a large tissue area, as well as obtaining data from various parts of the organ under study. In this paper, we use cross-polarization OCT, angiography, and lymphangiography with parallel histological examination to describe the structure of normal hairless vulvar skin. Two variants of the structure of vulvar skin were identified: skin with epidermal ridges, skin without epidermal ridges. For the first time, a change in the architecture of the microvasculature in response to a change in the structure of the epidermis has been described. Knowledge and understanding of the normal processes of change in the skin of the vulva are the main study of any pathology that occurs in this organ.
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.
To develop new methods for the diagnosis and treatment of such a widespread disease as steatosis, there is still a lack of fundamental biological knowledge about various aspects of the functioning of the liver tissue at the cellular level. In our work, we assessed the metabolic state of hepatocytes, as well as the collagen content in the liver tissue with induced steatosis using the modern label-free minimally invasive methods of multiphoton laser scanning microscopy with TPEF mode (Two-Photon Excited Fluorescence) and SHG mode (Second Harmonic Generation), equipped with FLIM (Fluorescence-Lifetime Imaging Microscopy). Using multiphoton microscopy, it was shown that during the development of steatosis, it is possible to identify areas with a reduced NAD(P)H autofluorescence signal in damaged hepatocytes. Using SHG we showed a gradual accumulation of collagen in the liver tissue with induced steatosis, however, extensive areas of fibrosis were not detected even at the advanced stages of the pathology. Using FLIM, we studied the specific features of the energy metabolism of hepatocytes based on data on the lifetimes of various forms of NAD(P)H and their relative contributions. It has been revealed that there is a gradual decrease in the intensity of oxidative phosphorylation, accompanied by the rise in the intensity of lipogenesis in the liver tissue with induced steatosis. Such results are consistent with the data of histological analysis. The results obtained in this work can be useful for developing new criteria for express intraoperative assessment of liver pathology at the cellular level in the clinic.
Lichen sclerosus is recurrence chronic autoimmune disease of skin and mucosa. Multimodal OCT is a promising tool for non-invasive, label-free and real-time investigation of vulvar tissue structure and vascularization for diagnosis and therapy control.
One of the promising approaches to the treatment of injured tissue is the application of bioengineering techniques based on the introduction of cells into the damage area. An application of cells alone does not provide a complete replacement of the tissue defect. Therefore, scaffolds are used allowing organization of the cells into a structure, which is capable of the total reproduction of the damaged tissue integrity. Great number of factors, which influence the cell behavior and tissue formation at the injury site when using scaffolds are known. Here, we analyzed the effect of structural heterogeneity of scaffolds on cellular behavior and metabolism. All scaffolds were obtained by two-photon polymerization technic. It was found that colonization of heterogeneous scaffolds was insignificant less than homogeneous ones. However, there were not dead cells on heterogeneous matrix. We found that the level of free and bound NAD(P)H for the cells on the heterogeneous and homogeneous scaffolds was differ. This can indicate a different contribution of glycolysis and oxidative phosphorylation intensity in stem cells seeded on two types of scaffolds.
KEYWORDS: Optical coherence tomography, Lymphatic system, Blood, Blood vessels, In vivo imaging, Angiography, Connective tissue, Visualization, Biopsy, Medical research
The purpose of the study was in vivo assessment of the vulvar blood and lymphatic vessels characteristics in norm and lichen sclerosus by multimodal optical coherence tomography (OCT). The study was performed using a multimodal OCT setup developed at the Institute of Applied Physics RAS (Russia). OCT angiography and OCT lymphangiography are based on the analysis of speckle structure. Visualization of blood and lymph vessels does not require the use of exogenous contrast agents. A histological study of vulvar biopsy samples from two points was carried out for 10 patients without vulvar pathology and 12 patients with lichen sclerosus. 3D OCT data was obtained from six vulvar points in each patient. OCT images were verified by histological examination. It was shown that normally the vulvar mucosa has good blood supply and a well-developed network of lymphatic vessels. In the case of lichen sclerosus, the density of blood vessels in the area of hyalinosis significantly reduced and amounted to 2.5 ± 0.79% versus 3.9 ± 0.23% in norm (p = 0.0003). OCT lymphangiographic images also show a significant decrease in the number of lymphatic vessels, their density was 1.7 ± 0.75% versus 3.7 ± 0.54% in norm (p = 0.02). A direct relationship between the state of the blood and lymph vessels from the condition of the connective tissue of the vulva was shown. The number of blood and lymph vessels is sharply reduced in the area of hyalinosis and sclerosis of collagen fibers.
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.
In this work, we present the first histological in vivo and ex vivo study of effects of fractional Er fiber laser (wavelength 1550 nm, peak power 25 W) on keratinized gum and alveolar mucosa for gum regeneration. Biopsy with subsequent NBTC staining was used as primary evaluation technique. Ex vivo, porcine tissue model was used. Effects of pulse energy, beam diameter, and beam divergence were investigated in detail. It has been demonstrated that under optimal conditions columns up to 800 μm in depth could be reliably produced with 130 mJ pulses. Clinically, 2 subjects were treated and 4 punch biopsies were collected. The results were compared with ex vivo data. Both ex vivo and in vivo datasets suggest feasibility of a dental fractional system intended for gum regeneration.
In the case of infiltrative brain tumors the surgeon faces difficulties in determining their boundaries to achieve total resection. The aim of the investigation was to evaluate the performance of multimodal OCT (MM OCT) for differential diagnostics of normal brain tissue and glioma using an experimental model of glioblastoma. The spectral domain OCT device that was used for the study provides simultaneously two modes: cross-polarization and microangiographic OCT. The comparative analysis of the both OCT modalities images from tumorous and normal brain tissue areas concurrently with histologic correlation shows certain difference between when accordingly to morphological and microvascular tissue features.
Recently proposed in vivo label-free optical coherence angiography techniques based on phase and amplitude speckle variability often require additional signal pre- and post processing operations to enhance vessel-contrast. We observe here 1) contrast enhancement by optimizing the signal normalization/weighing before processing; 2) algorithm based on Kasai estimator for phase compensation between processed A-scans to reduce masking role of motion artifacts; and 3) image projection through the imaging depth for en face plotting. We demonstrate the efficiency of proposed additional algorithms as for the microcirculation imaging of hamsters cheek in vivo as for the preliminary microcirculation imaging of patients after radiotherapy. This technical framework complete in details our recent publications on M-Mode like OCT algorithms and its implementation.
We consider the capabilities of cross-polarization OCT (CP OCT) focused on comparison of images resulting
from cross-polarization and co-polarization scattering simultaneously for diagnosis of oral soft tissues. CP OCT was
done for 35 patients with dental implants and 30 patients with inflammatory intestine diseases. Our study showed good
diagnostic capabilities of CP OCT for detecting soft tissue pathology in the oral cavity. The cross-polarized images
demonstrate the ability of tissue to depolarize. CP OCT demonstrates clinical capabilities for early diagnosis of
inflammatory intestine diseases by the state of oral cavity mucosa and for early detection of gingivitis in patients above
implant.
We consider the cross-polarization OCT (CP OCT) that is focused on comparison of images resulting from
cross-polarization and co-polarization scattering simultaneously. This technique provides information about
microstructural and biochemical alterations in depolarizing tissue components (collagen). We found that mature type I
collagen gives a strong signal in orthogonal polarization. CP OCT images of benign inflammatory processes always
feature signal in orthogonal polarization, with layers and borders persisting to be well defined. In the presence of
precancerous alterations, signal in orthogonal polarization is available in the image but it is irregular, disappearing in
some areas. A CP OCT image of bladder cancer in orthogonal polarization either shows no signal at all or a weak
signal.
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