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.
The aim of this study is to compare the results of tissue stiffness estimates given by compression (strain) ultrasound elastography (S-USE) and compression optical coherence elastography (C-OCE) beyond the difference in resolution and penetration depth. Namely, the focus of this work is on the contribution of elastic nonlinearity and mechanical inhomogeneity of the tissues to the stiffness estimates and resultant diagnostic performance of these techniques. We demonstrate that in comparison with S-USE despite basically the same compression principle, the applied realization of quantitative C-OCE have novel capabilities due to its ability to obtain spatially-resolved local pressure control and the local stress-strain curve mapping within the tissue.
In the study the multimodal optical coherence tomography (MM OCT) including microstructural cross-polarization OCT (CP OCT) imaging with the application of attenuation coefficients combined with compression OCT-elastography (OCE) with quantitative morphological segmentation based on specific stiffness ranges for delineation of breast cancer margins was applied. The research was carried out on different morphological and molecular subtypes of human breast cancer. The findings of this study suggest that OCE and CP OCT of breast cancer images may, in the future, enable real-time feedback to the surgeon about accurate resection margin location in patients with breast cancer.
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.
We demonstrate possibilities of multimodal OCT for quick distinguishing of uninformative necrotic zones from other morphological structures in freshly-excised breast cancer samples to improve information value of subsequent histological examination.
The possibilities of optoacoustic microscopy for comparison of vascular network of different tumor models as well as for investigation of tumor vessels response to radiation therapy were demonstrated.
The aim of this study was to evaluate the application of attenuation coefficients calculated from the cross-polarization optical coherence tomography (CP OCT) data for differentiation of breast cancer from non-tumorous breast tissues. For this purpose, surgically obtained breast specimens from 45 patients were examined using CP OCT in order to construct color-coded en-face OCT maps based on calculation of three optical coefficients (the commonly used rate of attenuation in the co- polarization channel; and, additionally, the attenuation in the cross- polarization channel; and the interchannel attenuation difference). It was shown that the use of these optical coefficients significantly increased the amount of obtained information from the OCT data in comparison with unprocessed images, enabling objective quantification for differentiating non-tumorous and tumorous tissue (adipose tissue, normal stroma, tumor stroma and agglomerates of tumor cells). The attenuation in the cross- polarization channel and the interchannel attenuation difference provided greater contrast for the visualization of the different breast cancer structures compared to the attenuation coefficient in the co- polarization channel. The findings of this study suggest that assessment of CP OCT images of breast cancer based on optical coefficients calculation may in perspective enable real-time feedback about accurate resection margin in patient with breast cancer to the surgeon.
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.
An important factor in assessing tumor status for determining therapeutic and surgical tactics is the study of its morphological structure. In particular, preoperative differentiation/grading of colorectal adenocarcinomas is very important for the adjustment of neoadjuvant therapy, surgical technique choice and survival prediction. To achieve the maximum benefit and to avoid unnecessary side effects, the ability to distinguish high-grade from low-grade colorectal adenocarcinoma, as well as identification of patients with high recurrence risk before the possible initiation of treatment will increase the quality and duration of patients' life. Taking into account the peculiarities of the biology of intestinal cancer, optical coherence elastography, as a method of studying tissue structure at a shallow depth, may be promising in the study of colorectal adenocarcinoma. Possibilities of optical coherence elastography in differentiating low-grade and high-grade types of colorectal adenocarcinoma are demonstrated in this study. The reported results are based on revealed qualitative criteria for assessment of OCE images in combination with quantitative statistically significant differences in characteristic stiffness values for low-grade and high-grade colorectal adenocarcinomas.
Development of a number of diseases is accompanied by changes in the blood vessels’ structure and the investigation of tissue vascular pattern remains one of the most essential problems in experimental and clinical medicine. Hybrid methods of optoacoustic (OA) imaging enable label-free optical-contrast angiography at optical penetration depths with ultrasonic resolution. We used OA to study vascular network of experimental tumor during growth and after treatment as well as to study the dynamics of blood content of human skin during functional tests. Optoacoustic angiography was performed using raster-scan system in reflection mode with 532 nm laser source and wideband PVDF detector. The sensitivity of the system allowed to visualize 50 µm blood vessels at up to 2.1 mm depth. Minimally detected diameter of blood vessel located at the surface of a soft tissue was 15 µm. Imaging of colon tumor models CT26 and HT-29 revealed peculiarities of vascular system development. Irradiation-induced increase of small tumor vessels segments number and parallel decrease in the number of large hemoglobin-containing structures were demonstrated. The fraction of blood-filled vessels of the human skin was assessed during cuff occlusion and temporarily filling with blood became clearly visible on OA images. To study the effects of local mechanical compression on human skin vasculature we varied external pressure and revealed the gradual drop in OA signal from blood vessels. We demonstrated the possibilities of raster-scan angiography for in vivo analysis of vessels structure, for monitoring of neoangiogenesis and for dynamical investigation of blood content under external actions.
This report presents an overview of recent progress in the development of novel OCE-based technologies enabling non-invasive morphological segmentation of tumor tissues during their natural development and response to therapies. The approach is based on preliminary quantitative assessment of characteristic elasticity ranges corresponding to the segmented morphological constituents (“quasistatic elasto-spectroscopy”). It has been found that such constituents are characterized by fairly well separated ranges of the Young’s modulus, which makes it possible to automatically segment up to 4-6 such zones in OCE-based elasticity maps. High consistency of such OCE-based procedure with conventional morphological segmentation of histological slides in terms of percentages of the segmented-zone areas has been demonstrated. Unlike invasive, time-consuming and laborious histological studies the developed OCE-based segmentation is non-invasive and can be performed even in vivo. High accuracy and sensitivity of the OCE-based-segmentation was demonstrated in vivo for two different types of model tumors (4T1 and Ct26) treated by PDT and chemotherapies. The OCE-based analysis can readily be combined with such OCT-based extensions as texture analysis and OCT-based angiography. The latter opens the possibility to combine morphological assessment with functional characterization of tumor response to therapies. OCE-based characterization of mechanical heterogeneity and elasticity spectra of tumors has also proven to be efficient for express assessment of patients’ breast-cancer subtypes. Overall, the developed OCE-based assessment in many applications suggests a rapid and in vivo feasible alternative to conventional histology and can be efficiently used in a wide range of biomedical problems.
The study is devoted to in vivo assessment of the vulvar tissue affected by lichen sclerosus and determining the severity of the disease by the state of connective tissue, blood flow and lymph flow using multimodal optical coherence tomography. This research will open up the possibility of developing and applying an effective noninvasive high-resolution method for visualization the state and depth of connective tissue lesions by scattering and polarization properties of the vulvar tissue, the blood flow and lymph flow based on the method of spectral multimodal OCT to assess the severity of lichen sclerosus. The in vivo OCT monitoring both the early dynamics of the response of all components of the vulvar tissue to low-level laser therapy (LLLT), and the degree of structure reparation after the treatment was carried out. Different response to LLLT was revealed: 3 of 10 patients had complete resolution of symptoms, 4 patients noted improvement in symptoms and 3 patients had no change in symptoms within 6 months of treatment and it was visualized by OCT.
Dystrophic diseases of the vulva represent a large group of dermatoses. The clinical picture of these lesions is often nonspecific, and non-invasive diagnosis is ineffective. The in vivo diagnosis of dystrophic diseases is difficult due to the lack of informative imaging methods for examining the vulvar mucosa. The new method of multimodal optical coherence tomography (OCT) is a non-invasive approach for biological tissues visualization with high spatial resolution, which does not require contrasting agents. Here we report the application of multimodal OCT to in vivo study the vulvar mucosa structure and its lymphatic and blood microcirculation. Multimodal OCT showed that normally the mucous membrane of the vulva has a good blood supply and a well-developed lymphatic vessels network. The difference in the diameter of the vessels in depth is clearly traced. In the subepithelial zone, only blood capillary loops are visualized. Lymphatic capillaries are visualized starting from submucosa.
As was shown earlier in the animal experiments, compression optical coherence elastography (OCE) is a highly sensitive method for studying the morphological features of biological tissues. An attempt was made to study the possibilities of the OCE method for identifying and differentiating from each other various morphological structures in human tissues. Postoperative samples of human breast cancer and human colon cancer were studied. Histological examination identified areas of morphological structures - (I) adipose tissue, (II) stroma and (III) tumor tissue in breast cancer samples; and areas of (I) mucous membrane and (II) tumor tissue in colon cancer samples. For these areas, an accurate comparison was made with OCE images and stiffness ranges for morphological structures were obtained. This observation allows us to make a conclusion that OСE can be a promising method for studying the structure of human tissue in clinics.
This study presents results on application of compressional optical coherence elastography (OCE) for visualization of a transitional zone between peri-tumoral (normal) and tumor region of breast tissue. The assessment of non-tumor/tumor regions is made based on percentage of pixels with different characteristic stiffness (“stiffness spectrum”) in the OCE image, also taking into account spatial localization of different-stiffness regions. The OCE-based stiffness maps and corresponding histograms of normalized stiffness spectrum demonstrate clear the boundary between normal (peritumoral) tissue and an invasive tumor. The normal breast tissue is fairly heterogeneous on the OCE images with increased stiffness in the region of the lobular structures and lower stiffness in the surrounded soft fibrous and especially adipose tissue of the mammary gland. The invasive breast cancer includes mechanically more dense stiffer accumulations of tumor cells, as well as less stiffer hyalinized tumor stroma. The corresponding histograms of stiffness show clearly dominating and ever increasing percentage of stiff regions in the tumor tissue showing gradually diminishing share of the normal stromal component in tissue. Thus, analysis of the so-obtained normalized histograms (“stiffness spectrum”) may be used to assess the negative surgical margin of resection in the future.
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.
We report a new application of compressional optical coherence elastography (OCE) to discriminate morphological constituents of biological tissues by analyzing OCE images obtained either in vivo or for freshly excised samples. The new technique enables quantitative morphological segmentation of OCE images with delineation of several (~4-6) tissue constituents. As the first step, the method uses compressional OCE to reconstruct stiffness maps for a pre-chosen standardized pressure over the entire area of the OCE image. Then specific stiffness ranges (characteristic "stiffness spectra") are initially determined by careful comparison of the OCE-based stiffness maps with the results of segmentation of "gold-standard" histological slices. After such pre-calibration, the stiffness maps can be automatically segmented into regions, for which the Young’s modulus (stiffness) falls in specific ranges corresponding to the morphological constituents to be discriminated. The results of such automated segmentation of OCE-images demonstrate a striking correlation with the results of conventional segmentation of histological slices in terms of percentages of the segmented zones. High sensitivity of the OCE-method to histological alterations was demonstrated in vivo in comparative studies of various types of anti-tumor therapies using murine tumor models. Studies of >100 samples of freshly excised breast cancer samples revealed strong correlation between the tumor-tissue subtype and its morphological composition determined by the developed OCE method. Thus, the developed approach can be used as a basis for express OCE-based biopsy (feasible intraoperatively). Longitudinal in vivo monitoring of morphological alterations in tumors under therapy or during natural development is also possible for locations accessible to OCT.
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.
Lichen Sclerosus is a chronic recurrent inflammatory dermatosis of unknown etiology, which affects both the skin and also the mucous membrane and is usually localized at the genital area. OCT angiography is a promising tool for microcirculation mapping in 3D with ~micrometer resolution. During the study, we developed and implemented a robust real time OCT angiography realization for routine clinical practice. This allows clinicians to combine structural OCT imaging with angiograms during routine clinical practice. Normal vulvar mucosa and dystrophic vulvar mucosa were analyzed by multimodal OCT. In the structural OCT scans of connective tissue, lymphatic vessels were visualized as contrast, low-signal regions. The angiographic images of the normal mucosa show dense, uniform network, mostly consisting of relatively thin vessels. Lymphatic vessel network presented by rare, very thick vessels. Further realization of OCT lymphangiography in real-time in parallel with OCT blood-vessel angiography can improve diagnostic capabilities of the multifunctional OCT in real clinical practice.
A multimodal optical coherence tomography (MM OCT) combining microstructural cross-polarization (CP) imaging and compression OCT-elastography (OCE) was employed in this study. The research was carried out on the noninvasive and invasive subtypes of human breast cancer. This study demonstrates how CP OCT combined with OCTelastography identifies an example of DCIS cancer that progresses into invasive breast cancer. OCE images more clearly show the cross sections of the ducts filled with tumor cells for DCIS (high-contrast structures with clear boundaries and high stiffness values) surrounded by areas with lower stiffness corresponding to fibrous stroma regions. The regions of invasive breast cancer in OCE-scans look as zones with strongly increased stiffness, which well agrees with the histology. Percentage of pixels with different characteristic stiffness ("stiffness spectrum") in the OCTelastography images was assessed to quantitatively visualize stiffness of tumor. The demonstrated ability of MM OCT imaging for breast cancer subtypes differentiation and breast cancer margin assessment is important for improved managements of patients.
The objective of the research was a multifaceted study of strangulated small intestine to reveal the optical, morphofunctional and biochemical signs of small bowel ischemia. The study was carried out in vivo using an artificially induced strangulation model of the small intestine (together with its mesentery and blood vessels) in 12 Wistar rats. Over a period of 120 minutes following the bowel ligation, changes in the density of the intramural vasculature and intestinal wall microstructure were detected using multimodal optical coherence tomography (MM OCT). Fluorescence lifetime changes of endogenous fluorophores were also measured using macro-FLIM of the strangulated loop and the adjacent segments of the intestine. At the end of the experiment, a morphometric study of the thickness of the layers and the prevalence of necrosis in the intestinal wall was carried out. A comprehensive analysis of the results of the OCT, FLIM and morphometry of the ischemic wall of the small intestine made it possible to determine the correlating morphofunctional and biochemical manifestations that are specific to this model of mesenteric blood flow disturbance.
An introduction of innovative minimally invasive methods like multimodal optical coherence tomography (OCT) with polarization-sensitivity and angiography modes for intrasurgical guidance appears to be a critical challenge for the modern neurooncology. This study aimed to develop a new instrument for targeted stereotactic brain biopsy based on cross-polarization (CP) OCT realized in standard biopsy needle to increase the safety of ongoing neurosurgical procedures. Experimental studies in vivo on the healthy rat’s brain allow detecting blood vessels in the process of moving the biopsy needle along the brain surface and detect tissue type (cerebral cortex or white matter) when the needle went inside the brain. The images was assessed by visual criteria. However, CP OCT signal quantification methods will increase the sensitivity/specificity for the tissue type differentiation and blood vessels detection. The potential of CP OCT as an effective instrument for OCT-guided stereotactic biopsy of brain tumors was demonstrated.
Photodynamic therapy (PDT) is emerging as a common and efficacious method for basal cell carcinoma (BCC) treatment, and new non-invasive imaging technologies can further enhance it. Optical coherence angiography (OCA) was employed in this study. OCA is a non-invasive, label-free, real-time bioimaging method that has proven to be a helpful tool for visualizing normal and pathological vasculature, including vascular damage evaluation after using a vasculature-targeted therapy for predicting its success. In experimental study, it was shown that both the tumor and peritumorous vessels stasis (disappearance in OCA images) in 24 hours post treatment play significant roles in PDT success. On controllable mouse ear tumor model the following practical and robust OCA-based criterion of PDT success was formulated: there should be no visibly perfused vessels on OCA images inside the tumor borders, whereas in the 2 mm near-tumor proximity regions the vascular density should not exceed 1% from OCA image area in 24 hours post PDT. The criterion obtained on the experimental model was translated to clinical study. OCA monitoring of basal cell carcinoma reaction to PDT has shown that dramatic decrease in the vascular density in the tumor in 24 hours post PDT can predict tumor non-recurrence with high diagnostic accuracy for 12 months follow-up. The strong reaction of peritumorous vessels in 24 hours post PDT is associated with hypertrophic scar formation in 3-6 months, but the weak reaction of peri-tumorous vascular reaction leads to normotrophic scar formation.
Raster-scan optoacoustic angiography at 532 nm wavelength with 50 μm lateral resolution at 2 mm diagnostic depth was used for quantitative characterization of neoangiogenesis in colon cancer models. Vessels of subcutaneously growing murine colon carcinoma (СT26) was imaged from 5th to 13th day of growth. The values of vascular density were calculated from the optoacoustic data. Inhomogeneous distribution of areas with high and low vascularization was demonstrated in the tumors. During tumor development vessel growth from the periphery to the center of the tumor was shown. Increase of vascular density precedes the increase of tumor volume. The obtained results may be important for the investigation of tumor development and for improvement of cancer treatment strategies.
Normal and pathological tissues are different in their elastic properties. Thus, elastic characteristics of tissues can serve as an additional diagnostic marker to distinguish altered tissues. A number of studies have demonstrated advantages of using optical coherent tomography in clinical practice and research. Here we report the application of optical coherence elastography (OCE) for studying elastic properties of murine breast cancer 4T1. It is known that 4T1 model is convenient for in vivo tumor imaging and is highly relevant to studies of tumor process. In this study the therapy was performed with a cytostatic chemotherapeutic drug cisplatin. We developed the protocol of compressional OCT elastography for long-term in vivo monitoring of tumor response to anti-tumor therapy. The ear tumor model was chosen as optimal for this task. Histological analysis demonstrated good agreement with OCE-based stiffness-mapping results.
We present an optical coherence tomography (OCT) lymphangiography visualization approach based on analysis of speckle statistics. For in-vivo experimentation, normal and tumor tissues are examined in mouse dorsal skin window chamber model. In order to evaluate the speckle statistics, OCT datasets are acquired in 3 spatial and 1 temporal dimensions to be divided then into smaller volumes of interests. In temporal dimension, repeated same-location scanning is performed for simultaneous blood vessel detection through speckle variance processing. Speckle statistics in each volume of interest are tested for similarity to known distributions corresponding either to noise or to tissue. We show that lymphatic vessels could be detected using a specific parameter range of speckle statistics as a filter, to separate the surrounding tissues and blood vessels. The proposed approach does not require numerous post-processing steps that are often used in lymphatic detection methods that are based on low signal amplitude regions (e.g., OCT signal attenuation compensation, inversion, amplitude thresholding etc.). Instead, we use a fast 2-step filtering procedure to reveal lymphatic vessels in imaged tissues.
In this study we combined cross-polarization optical coherence tomography (CP OCT), multiphoton tomography (MPT), based on second harmonic generation, and two-photon-excited fluorescence to visualize collagen fibers and tumor cells in the various morphological subtypes of breast cancer. The ability of CP OCT to visualize tissue birefringence and cross-scattering adds new information about the microstructure of such breast cancers, while the MPT provides verification of this microstructure. Mammary glands, both normal and tumorous, were assessed by MPT and CP OCT to establish the relationships between spatial organization features of the cellular component and the intercellular matrix. It was shown, that such multimodal optical imaging has great potential for distinguishing various breast cancer morphological subtypes and could provide useful tools for identifying positive breast cancer margins for surgery.
Collageneus tissues manifest strongly pronounced viscoelastic behavior. Namely, viscosity leads to time-dependence of
the deformation processes. This concerns both compression (loading) and unloading of the tissue. Both processes can be
characterized by a relaxation time - the time during which the strain changes e-times. We demonstrate the applicability
of the OCT-based 2D relaxography to characterize local relaxation time of collageneous tissues. The developed
technique can be used for further investigation of the viscoelastic properties of healthy and pathological collageneous
tissues.
In this report we discuss some practical obstacles/pitfalls arising in realization of quantitative compressional OCE, as
well as discuss possible ways of their resolution. More specifically we consider (i) complications in quantification of the
Young modulus of tissues related to influence of partial adhesion between the OCT probe and pre-calibrated reference
layers - "compliant sensors", (ii) distorting influence of surface curvature/corrugation on strain distribution in the tissue
bulk, (iii) ways of enhancement of effective SNR in OCT-based strain mapping without periodic averaging, and (iv)
potentially significant influence of nonlinearity of the elastic response of biological tissues on quantification of their
stiffness.
Soft biological tissues manifest strongly pronounced nonlinear elastic behavior. Namely, the Young modulus for some tissues strongly depends on the applied stress. This property can be evaluated by compressional OCT elastography. We demonstrate the evaluation of nonlinear elastic properties on the samples of coronary arteries, breast cancer and cornea. The developed technique can be used for further investigation of the nonlinear properties of healthy and pathological tissues.
Introduction of innovative noninvasive diagnostic techniques in clinical practice remains an actual topic of modern medicine. In the study, we assessed feasibility of multimodal OCT (MM OCT) that combines crosspolarization imaging and elastographic stiffness mapping to assess spatial structural organization and heterogeneity of breast cancer in the tumor center in comparison with normal mammary gland tissue. The research was carried out using human breast tissue mastectomy surgical samples including different histological types of breast cancer: ductal carcinoma in situ, invasive lobular and ductal carcinoma, fibroadenoma and normal tissue. The histological subtypes of breast cancer showed different structural and stiffness features of tumor tissue. In the case of invasive ductal carcinoma, the cross-polarization OCT image shows a more heterogeneous high-level OCT signal and higher stiffness in comparison with lobular breast cancer or fibroadenoma. These results indicate that assessing microstructures and elasticity changes yields complementary information about the microstructural features of breast cancer, which is important for selection of treatment tactics.
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.
OCT-based local strain relaxation/creep evaluation is an emerging tool for tissue viscoelasticity characterization. We present a tool for 2D visualization of local strain relaxation and creep time/rate inside the tissue.
Multimodal OCT is a promising tool for monitoring of individual tumor response to antitumor therapies. The
changes of tumor cells, connective tissue, microcirculation and stiffness can be estimated simultaneously in
real time with high resolution.
We discuss the application of compressional OCT-based elastography for measuring elastic response of biological
tissues in the strain range from fractions of one per cent up to strains over ~10% and greater. Such fairly large strains are
not typical of phase-sensitive OCT-based elastographic methods and can be measurable due to application of interframe
strain-estimation method in which interframe phase gradients are estimated without the necessity of preliminary
measuring total displacements of scattering particles in combination with summation of interframe strains. For
estimating applied stresses, intervenient soft layer of translucent silicone is used a a reference fairly linear material. The
obtained results based on comparison of strains in the reference layer and tissue demonstrate highly nonlinear character
of elasticity of biological tissues even for rather moderate strains on order of several per cent.
This work is dedicated to development of the OCT system with angiography for everyday clinical use. Two major problems were solved during the development: compensation of specific tissue displacements, induced by contact scanning mode and physiological motion of patients (e.g. respiratory and cardiac motions) and on-line visualization of vessel net, to provide the feedback for system operator. The performance of the resulting OCT-based microangiography device with hand-held probe was evaluated by visualization of vessels nets of volunteers oral mucosa and skin on different locations (hands, face, abdomen etc.). Success-rate more than 90% was demonstrated during the experiments.
The main goal of the study was a vasculature targeted PDT. A new approach named M-mode-like OCT (MML OCT)
was applied to monitor early response to PDT. Due to the chosen filtering parameters (96 Hz filter threshold), this
approach visualizes only vessels with flowing blood. Without flowing blood even filled vessels are not visualized
because flow-less blood causes speckle variations with significantly lower frequencies (<50 Hz corresponding to speckle
decorrelation time for stationary blood). This feature allows us to detect thrombosis of blood vessels, the results of MML
OCT and histological examination being perfectly coinciding. The advantages of MML OCT such as a simple and fast
process of obtaining microvasculature images and label-free nature of the visualization makes this method perspective in
routine clinical monitoring of antitumor therapies.
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.
Multimodal optical coherence tomography (OCT) is an emerging tool for tissue state characterization. Optical coherence elastography (OCE) is an approach to mapping mechanical properties of tissue based on OCT. One of challenging problems in OCE is elimination of the influence of residual local tissue relaxation that complicates obtaining information on elastic properties of the tissue. Alternatively, parameters of local relaxation itself can be used as an additional informative characteristic for distinguishing the tissue in normal and pathological states over the OCT image area. Here we briefly present an OCT-based approach to evaluation of local relaxation processes in the tissue bulk after sudden unloading of its initial pre-compression. For extracting the local relaxation rate we evaluate temporal dependence of local strains that are mapped using our recently developed hybrid phase resolved/displacement-tracking (HPRDT) approach. This approach allows one to subtract the contribution of global displacements of scatterers in OCT scans and separate the temporal evolution of local strains. Using a sample excised from of a coronary arteria, we demonstrate that the observed relaxation of local strains can be reasonably fitted by an exponential law, which opens the possibility to characterize the tissue by a single relaxation time. The estimated local relaxation times are assumed to be related to local biologically-relevant processes inside the tissue, such as diffusion, leaking/draining of the fluids, local folding/unfolding of the fibers, etc. In general, studies of evolution of such features can provide new metrics for biologically-relevant changes in tissue, e.g., in the problems of treatment monitoring.
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 describe a modification of a recently proposed unconventional OCT approach to 3D microvasculature imaging based on high-pass filtering of B-scans in the lateral direction. The B-scans are acquired in M-mode-like regime with highly overlapped A-scans. The goal of the described modification is to suppress non-fluid artifacts in the resultant microcirculation images. The modification is based on the amplitude normalization procedure of complex-valued OCT signal before subsequent processing. This allows one to efficiently suppress imaging degradation due to the influence of very bright spots/lines (e.g. from hairs on the surface) and retain images of real flows inside the tissue without any artificial cut-off of the surface signal, or application of pixel-intensity thresholds, or signal classification approaches.
In this work we demonstrate an efficiency of transillumination fluorescence imaging for intravital study of
photosensitizers pharmacokinetics in tumor-bearing mice. Experiments were performed on CBA mice with
subcutaneously transplanted cervical carcinoma. Photosensitizer fotoditazin was used in the therapeutic dose of 10
mg/kg, i.v. Measuring the fluorescence in the whole tumors at different times after injection of photosensitizer, we
obtained in vivo data about the drug accumulation and elimination. It was found in vivo that the period of maximum
uptake of the photosensitizer in the mouse tumor is from 1 to 8 h post-injection, and its concentration in the tumor tissue
at this time is about 2 μg/g. An accumulation of the photosensitizer in the tumor was confirmed by standard methods ex
vivo. Noninvasive assessment of pharmacokinetics by transillumination imaging setup provides an opportunity for rapid
and cost-effective studies of newly developed photosensitizers.
The possibility of in vivo investigation of the pharmacokinetics of photosensitizers by means of fluorescence transillumination imaging is demonstrated. An animal is scanned in the transilluminative configuration by a single source and detector pair. Transillumination is chosen as an alternative approach to reflection imaging. In comparison with the traditional back-reflection technique, transillumination is preferable for photosensitizer detection due to its higher sensitivity to deep-seated fluorophores. The experiments are performed on transplantable mouse cervical carcinomas using three drugs: photosens, alasens, and fotoditazin. For quantitative evaluation of the photosensitizer concentration in tumor tissue the fluorescence signal is calibrated using tissue phantoms. We show that the kinetics of photosensitizer tumor uptake obtained by transillumination imaging in vivo agree with data of standard ex vivo methods. The described approach enables rapid and cost-effective study of newly developed photosensitizers in small animals.
In the current paper we present preliminary data demonstrating therapeutic efficiency of local laser hyperthermia of
mouse tumors with gold nanoparticles. Measuring the tumor temperatures both superficial and inner by means of
standard NIR-thermograph and original acoustic thermometer correspondingly we show that the gold nanoparticles
increase thermal sensitivity of tumor tissue. Transmission electron microscopy and histopathology of the tumor tissue
indicated that the mechanism of apoptotic death of tumor cells is triggered following the laser treatment. 5 days after the
treatment tumor growth inhibition was 104 %.
The study was performed on 16 CBA-line female mice with transplanted cervical cancer. 0.2 ml of gold
nanoparticle solution with a concentration of 109 particles/ml were injected into the animals intravenously. The particles
were 200-250 nm in size; the plasmon-resonance related extinction maximum was at the wavelength of 850-950 nm.
Accumulation of the nanoparticles into tumor node was visualized by the method of optical coherence tomography
(OCT). When the accumulation of nanoparticles in the tumor was maximal, hyperthermia was accomplished using the
LSP-AZOR laser setup generating cw radiation at 810 nm. The duration of exposition was 20 min. The therapeutical
effect was assessed by the rate of tumor growth inhibition (TGI, %).
Determining the instant when nanoparticle concentration in tumor tissue reaches its maximum enables more
efficient laser impact. The use of nanoparticles decreases laser irradiation power and ensures local action.
KEYWORDS: Nanoparticles, Skin, Optical coherence tomography, Monte Carlo methods, Gold, Particles, In vivo imaging, Optical properties, Scattering, Titanium dioxide
Contrasting of skin forming elements in optical coherence tomography (OCT) images after application of silica/gold
nanoshells or titanium dioxide nanoparticles in solution is discussed. The study is performed both by Monte Carlo
simulations and in vivo on animals. The result show that application of both types of nanoparticles produces contrast
increase in the OCT images of skin. The increase in OCT signal level originates from the higher backscattering on
nanoparticles compared to that on skin forming elements. The increase of contrast in the OCT images originates from the
difference in nanoparticles concentration within different skin constituents. These experimental results are confirmed
qualitatively by Monte Carlo simulations based on multilayer skin model.
KEYWORDS: Nanoparticles, Skin, Gold, Optical coherence tomography, Monte Carlo methods, Particles, Scattering, In vivo imaging, Coherence imaging, Titanium dioxide
The effect of silica/gold nanoshells and titanium dioxide nanoparticles on the optical properties of skin is studied. By implementing in vivo measurements and Monte Carlo simulations, we analyze the efficiency of using these nanoparticles as contrasting agents for optical coherence tomography (OCT) imaging of skin. In vivo measurements are performed on pig skin, where nanoparticle suspension drops have been applied. The identification of skin layers is performed by comparison with corresponding histology images. Experimental results exhibit an increase in contrast of the obtained OCT images after a single nanoparticles application. Multiple applications do not lead to increase in the obtained contrast. To interpret the obtained experimental OCT images of skin and understand the mechanisms of contrasting, a set of Monte Carlo calculations is performed. The results of the simulations exhibit good qualitative agreement with the experimental images, and prove that the contrasting originates from the nanoparticles added, while the contrast of inclusion originates from the absence of nanoparticles within it and their presence in the surrounding area.
Dynamics of nanoparticles penetrating and accumulating in healthy (skin) and pathologically altered (tumor)
tissue was investigated in vivo by the method of optical coherence tomography (OCT). Gold nanoshells having the size
of 130/15 nm and titanium dioxide nanoparticles 40-100 nm in size were studied. Nanoparticles accumulation in
biotissue was accompanied by the change of optical effects in OCT images. Continuous OCT monitoring of the process
of nanoparticles penetration into skin showed that optical effects appeared 30 minutes after application of nanoparticles
on the surface; maximal effect of nanoparticles accumulation in the skin was recorded in the observation period of 1.5-5
hours.
Nanoparticles accumulation in neoplastic tissue at passive delivery was studied in vivo. Accumulation maximum
was 4-6 hours after intravenous introduction.
The transmission electron microscopy technique confirmed accumulation of nanoparticles in biotissues.
KEYWORDS: Optical coherence tomography, Skin, Gold, Monte Carlo methods, In vivo imaging, Nanoparticles, Optical properties, Scattering, Tissue optics, Computer simulations
We have investigated the effect of application of gold nanoshells with a 150 nm silica core size and 25 nm thick gold
coating on optical properties of skin. We have analyzed the possibility of using these particles as a contrasting agent for
optical coherence tomography (OCT). A set of Monte Carlo calculations was performed in order to simulate the images
of skin before and after application of the nanoshells for a skin model close to that in vivo. We investigated the
mechanisms of boundary contrasting between tissue layers with different optical properties in the presence of gold
nanoshells on two-layer agar gel phantom. Gold nanoshells were also applied on the skin surface in vivo. Gold-silica
nanoshells caused an increase in the intensity of OCT signal, brightness of the superficial part of the dermis, contrast
between dermis layers and contrast of hair follicles and glands in the OCT image. The contrasting effects of the gold
nanoshells lasted up to 24 hours of observation.
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