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Currently, optical biopsy technologies are being developed for rapid and label-free visualization of biological tissue with micrometer-level resolution. They can play an important role in breast-conserving surgery guidance, detection of residual cancer cells, and targeted histological analysis. For solving these problems, compression optical coherence elastography (C-OCE) demonstrated impressive results based on differences in the elasticity of different tissue constituents. However, sometimes straightforward C-OCE-based differentiation is insufficient because of the similar stiffness of certain tissue components. We present a new automated approach to the rapid morphological assessment of human breast cancer based on the combined usage of C-OCE and speckle-contrast (SC) analysis. Using the SC analysis of structural OCT images, the threshold value of the SC coefficient was established to enable the separation of areas of adipose cells from necrotic cancer cells, even if they are highly similar in elastic properties. Consequently, the boundaries of the tumor bed can be reliably identified. The joint analysis of structural and elastographic images enables automated morphological segmentation based on the characteristic ranges of stiffness (Young's modulus) and SC coefficient established for four morphological structures of breast-cancer samples from patients post neoadjuvant chemotherapy (residual cancer cells, cancer stroma, necrotic cancer cells, and mammary adipose cells). This enabled precise automated detection of residual cancer-cell zones within the tumor bed for grading cancer response to chemotherapy. The results of C-OCE/SC morphometry highly correlated with the histology-based results (r =0.96-0.98). The combined C-OCE/SC approach has the potential to be used intraoperatively for achieving clean resection margins in breast cancer surgery and for performing targeted histological analysis of samples, including the evaluation of the efficacy of cancer chemotherapy.

We report the first application of compression optical coherence elastography (C-OCE) to evaluate both linear and nonlinear elastic properties of lymph nodes (LNs) for assessing their status, which is an important factor for detecting metastasis and staging breast cancer. The reported study involved 27 excised sentinel and axillary LNs from 24 patients with breast cancer, including normal LNs (n = 6), reactive LNs with follicular hyperplasia (n = 7) and sinus histiocytosis (n = 8), and metastatic LNs (n = 6). C-OCE data were compared with co-registered gold-standard histopathology and demonstrated a significantly higher contrast in differentiation of LNs as compared to structural optical coherence tomography imaging. Normal LNs are characterized by low stiffness values in the areas of LN cortex (600 kPa). Since in terms of the tangent Young’s modulus there remained an appreciable overlap among these types of LNs, we also evaluated their elastic nonlinearity parameters. Complementary usage of both linear and nonlinear elastic parameters enabled very clear differentiation of all four main states of LNs. Thus, C-OCE imaging demonstrates high potential for future intraoperative usage to rapidly determine the LN status during breast-conserving surgery and to assess the extent of the cancer cells propagation in regional lymphatic collectors for preserving benign LNs. This is important for reducing the associated risks and complications (such as lymphedema) from excessive surgical removal of lymphoid structures.

There is a wide range of equivocal melanocytic lesions that can be clinically and dermoscopically indistinguishable from early melanoma. In the present work, we assessed the possibilities of combined using of multiphoton microscopy (MPM) and optical coherence angiography (OCA) for differential diagnosis of the equivocal melanocytic lesions. Clinical and dermoscopic examinations of 60 melanocytic lesions revealed 10 benign lesions and 32 melanomas, while 18 lesions remained difficult to diagnose. Histopathological analysis of these lesions revealed 4 intradermal, 3 compound and 3 junctional nevi in the “benign” group, 7 superficial spreading, 14 lentigo maligna and 11 nodular melanomas in the “melanoma” group and 2 lentigo simplex, 4 dysplastic nevi, 6 melanomas in situ, 4 invasive lentigo melanomas and 2 invasive superficial spreading melanomas in the “equivocal” group. On the basis of MPM, a multiphoton microscopy score (MPMS) has been developed for quantitative assessment of melanoma features at the cellular level, that showed lower score for benign lesions compare with malignant ones. OCA revealed that the invasive melanoma has a higher vessel density and thicker blood vessels than melanoma in situ and benign lesions. Discriminant functions analysis of MPM and OCA data allowed to differentiate correctly between all equivocal melanocytic lesions. Therefore, we demonstrate, for the first time, that a combined use of MPM and OCA has the potential to improve early diagnosis of melanoma.

We present a non-invasive (albeit contact) method based on Optical Coherence Elastography (OCE) enabling the in vivo segmentation of morphological tissue constituents, in particular, monitoring of morphological alterations during both tumor development and its response to therapies. The method uses compressional OCE to reconstruct tissue stiffness map as the first step. Then the OCE-image is divided into regions, for which the Young’s modulus (stiffness) falls in specific ranges corresponding to the morphological constituents to be discriminated. These stiffness ranges (characteristic "stiffness spectra") are initially determined by careful comparison of the "gold-standard" histological data and the OCE-based stiffness map for the corresponding tissue regions. After such pre-calibration, the results of morphological segmentation of OCE-images demonstrate a striking similarity with the histological results in terms of percentage of the segmented zones. To validate the sensitivity of the OCE-method and demonstrate its high correlation with conventional histological segmentation we present results obtained in vivo on a murine model of breast cancer in comparative experimental study of the efficacy of two antitumor chemotherapeutic drugs with different mechanisms of action. The new technique allowed in vivo monitoring and quantitative segmentation of (1) viable, (2) dystrophic, (3) necrotic tumor cells and (4) edema zones very similar to morphological segmentation of histological images. Numerous applications in other experimental/clinical areas requiring rapid, nearly real-time, quantitative assessment of tissue structure can be foreseen.

The aims of this study are (i) to compare ultrasound strain elastography (US-SE) and compression optical coherence elastography (C-OCE) in characterization of elastically linear phantoms, (ii) to evaluate factors that can cause discrepancy between the results of the two elastographic techniques in application to real tissues, and (iii) to compare the results of US-SE and C-OCE in the differentiation of benign and malignant breast lesions. On 22 patients, we first used standard US-SE for in vivo assessment of breast cancer before and then after the lesion excision C-OCE was applied for intraoperative visualization of margins of the tumors and assessment of their type/grade using fresh lumpectomy specimens. For verification, the tumor grades and subtypes were determined histologically. We show that in comparison to US-SE, quantitative C-OCE has novel capabilities due to its ability to locally control stress applied to the tissue and obtain local stress-strain curves. For US-SE, we demonstrate examples of malignant tumors that were erroneously classified as benign and vice versa. For C-OCE, all lesions are correctly classified in agreement with the histology. The revealed discrepancies between the strain ratio given by US-SE and ratio of tangent Young’s moduli obtained for the same samples by C-OCE are explained. Overall, C-OCE enables significantly improved specificity in breast lesion differentiation and ability to precisely visualize margins of malignant tumors compared. Such results confirm high potential of C-OCE as a high-speed and accurate method for intraoperative assessment of breast tumors and detection of their margins.

Soft biological tissues, breast cancer tissues in particular, often manifest pronounced nonlinear elasticity, i.e., strong dependence of their Young’s modulus on the applied stress. We showed that compression optical coherence elastography (C-OCE) is a promising tool enabling the evaluation of nonlinear properties in addition to the conventionally discussed Young’s modulus in order to improve diagnostic accuracy of elastographic examination of tumorous tissues. The aim of this study was to reveal and quantify variations in stiffness for various breast tissue components depending on the applied pressure. We discussed nonlinear elastic properties of different breast cancer samples excised from 50 patients during breast-conserving surgery. Significant differences were found among various subtypes of tumorous and nontumorous breast tissues in terms of the initial Young’s modulus (estimated for stress < 1 kPa) and the nonlinearity parameter determining the rate of stiffness increase with increasing stress. However, Young’s modulus alone or the nonlinearity parameter alone may be insufficient to differentiate some malignant breast tissue subtypes from benign. For instance, benign fibrous stroma and fibrous stroma with isolated individual cancer cells or small agglomerates of cancer cells do not yet exhibit significant difference in the Young’s modulus. Nevertheless, they can be clearly singled out by their nonlinearity parameter, which is the main novelty of the proposed OCE-based discrimination of various breast tissue subtypes. This ability of OCE is very important for finding a clean resection boundary. Overall, morphological segmentation of OCE images accounting for both linear and nonlinear elastic parameters strongly enhances the correspondence with the histological slices and radically improves the diagnostic possibilities of C-OCE for a reliable clinical outcome.

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.

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.

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.

In this study multiphoton tomography, based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) was used to visualize both the extracellular matrix and tumor cells in different morphological and molecular subtypes of human breast cancer. It was shown, that quantified assessment of the SHG based imaging data has great potential to reveal differences of collagen quantity, organization and uniformity in both low- and highly- aggressive invasive breast cancers. The values of quantity and uniformity of the collagen fibers distribution were significantly higher in low-aggressive breast cancer compared to the highly-aggressive subtypes, while the value representing collagen organization was lower in the former type. Additionally, it was shown, that TPEF detection of elastin fibers and amyloid protein may be used as a biomarker of detection the low-aggressive breast cancer subtype. Thus, TPEF/SHG imaging offers the possibility of becoming a useful tool for the rapid diagnosis of various subtypes of breast cancer during biopsy as well as for the intraoperative determinination of tumor-positive resection margins.

We present a novel method, which by analogy with the term "mass-spectroscopy" can be called "quasistatic elasto-spectoscopy" based on quantitative compressional Optical Coherence Elastogrphy (C-OCE). The method relies on initial determining of characteristic stiffness ranges for various morphological components of the tissue by comparing the OCE-based stiffness maps and histology. Such pre-calibration allows one to perform morphological segmentation of OCE-images demonstrating excellent correlation with segmentation of conventional histological images, but can be performed using freshly-extracted samples and even in vivo.

The possibility to assess molecular-biological and morphological features of particular breast cancer types can improve the precision of resection margin detection and enable accurate determining of the tumor aggressiveness, which is important for treatment selection. To enable reliable differentiation of breast-cancer subtypes and evaluation of resection margin, without performing conventional histological procedures, here we apply cross-polarization optical coherence tomography (CP-OCT) and compare it with a novel variant of compressional optical coherence elastography (C-OCE) in terms of the diagnostic accuracy (Ac) with histological verification. The study used 70 excised breast cancer specimens with different morphological structure and molecular status (Luminal A, Luminal B, Her2/Neo+, non-luminal and triple-negative cancer). Our first aim was to formulate convenient criteria of visual assessment of CP-OCT and C-OCE images intended (i) to differentiate tumorous and non-tumorous tissues and (ii) to enable more precise differentiation among different malignant states. We identified such criteria based on the presence of heterogeneities and characteristics of signal attenuation in CP-OCT images, as well as the presence of inclusions/mosaic structures combined with visually feasible assessment of several stiffness grades in C-OCE images. Secondly, we performed a blinded reader study of the Ac of C-OCE versus CP-OCT, for delineation of tumorous versus non-tumorous tissues followed by identification of breast cancer subtypes. For tumor detection, C-OCE showed higher specificity than CP-OCT (97.5% versus 93.3%) and higher Ac (96.0 versus 92.4%). For the first time, the Ac of C-OCE and CP-OCT were evaluated for differentiation between non-invasive and invasive breast cancer (90.4% and 82.5%, respectively). Furthermore, for invasive cancers, the difference between invasive but low-aggressive and highly-aggressive subtypes can be detected. For differentiation between non-tumorous tissue and low-aggressive breast-cancer subtypes, Ac was 95.7% for C-OCE and 88.1% for CP-OCT. For differentiation between non-tumorous tissue and highly-aggressive breast cancers, Ac was found to be 98.3% for C-OCE and 97.2% for CP-OCT. In all cases C-OCE showed better diagnostic parameters independently of the tumor type. These findings confirm the high potential of OCT-based examinations for rapid and accurate diagnostics during breast conservation surgery.

A method, based on optical coherence angiography (OCA), for the intraoperative diagnosis of blood microcirculation in the ischemic intestine has been tested. The pilot clinical study involved 18 patients who had undergone urgent operations for thrombosis of segments II or III of the superior mesenteric artery, as well as for infringement of the bowel and the mesenteric vessels. In the OCA images regions of preserved blood microcirculation in the intestinal wall were characterized by the presence of branched networks of intramural microvessels and a bright background in the OCA images. Regions of the intestine in which the intramural vessels were interrupted or could not be visualized were considered as potentially non-viable. These data were used to determine the boundaries for intestinal resection. In comparison with the traditional method of intraoperative diagnosis of intestinal blood flow, the use of such OCA criteria meant the volume of bowel resection could be decreased by 21.5 [14.1; 36.0]% (p = 0.037). This observation demonstrates the great potential of OСA in the intraoperative detection of the boundaries of damaged and healthy intestinal wall in ischemic bowel disease.

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.

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.

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.

Compressional optical coherence elastography (OCE) is the emerging tool to evaluate Young modulus of the tissue with resolution close to optical coherence tomography. Recent versions of compressional OCE based on the strain comparison within the tissue and the calibrated reference silicone layer (sensor) located between the OCT probe and tissue. For standard realizations of compressional OCE the evaluation of the strain within the tissue and the sensor performed only at one point of the stress-strain curve. In the same way the applied stress can be easily estimated in a wide range on the base on the stress-strain relation for calibrated sensor strain that allow to evaluate the stress-strain relation for the underlying tissue not in one point but in the wider range. In this report we demonstrate this approach to nonlinear parameter evaluation for wide range of the tissues. We demonstrate the application of this new tool to breast tissue, vaginal wall, bladder, brain, cartilages, coronary arteries walls in various states. The preliminary results shows that several states of the tissues such as vaginal wall state in case of pelvic organ prolapse, benign breast tumor and coronary arteries plague can be determined on the base of elastic nonlinear parameters.

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.

In this chapter we consider applications of multimodal OCT for characterizing tumorous tissues. Namely, the following extensions of OCT are discussed: (1) polarization-sensitive OCT providing complementary images in the initial and cross-polarization channels (CP OCT), (2) OCT-based label-free angiography (OCA), and (3) compressional variant of OCT-based elastography (OCE). For each modality we briefly consider the principles of realization and present examples of various biomedical applications from utilization in laboratory experiments with model tumors on animals to demonstrations of possibilities of multimodal OCT in pre-clinical studies and usage in clinical conditions. Among such applications we present the results of using CP OCT for detection of tumorous and nontumorous regions in brain and breast tissues; application of OCA for monitoring the results of photodynamic therapy of murine model tumor CT26 and patients’ basal cell carcinoma; utilization of OCA for studying mucositis on patients during radiation therapy, as well as chemically induced cancer in hamster cheek pouch. In conclusion, we consider several examples of successful realization of compressional OCE for such applications as monitoring of tumor response to chemotherapy, visualization of morphological features of breast cancer and delineation of breast cancer from non-tumor tissue.

Microvascular networks of human basal cell carcinomas (BCC) and surrounding skin were assessed with optical coherence angiography (OCA) in conjunction with photodynamic therapy (PDT). OCA images were collected and analyzed in 31 lesions pre-treatment, and immediately/24 hours/3–12 months post-treatment. Pre-treatment OCA enabled differentiation between prevalent subtypes of BCC (nodular and superficial) and nodular-with-necrotic-core BCC subtypes with a diagnostic accuracy of 78%; this can facilitate more accurate biopsy reducing sampling error and better therapy regimen selection. Post-treatment OCA images at 24 hours were 98% predictive of eventual outcome. Additional findings highlight the importance of pre-treatment necrotic core, vascular metrics associated with hypertrophic scar formation, and early microvascular changes necessary in both tumorous and peri-tumorous regions to ensure treatment success.

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.

Development of noninvasive diagnostic techniques in clinical practice, especially sufficiently fast methods suitable for intraoperative use, remains a topical and challenging problem in modern medicine. Compressional Optical Coherence Elastography (OCE) that has a high potential to be intraoperatively applied. This study reports preliminary results on application of compressional OCE for identification of tumor margins and even differentiation of breast cancer types with low- and high- histological grades, i.e., a step towards creation of a kind of OCE-based optical biopsy. The reported variant of OCE utilized B-scans with scales ~2-4 mm comparable with typical scales of histological samples. The approach is based on the ability of OCE to quantitatively visualize distribution of the Young modulus (stiffness) in studied tissue samples, which then is used to perform "elasto-spectroscopy" generically resembling the mass-spectroscopy procedures. The procedures of the OCE examination do not require any special preparation of tissue samples and thus can be applied intraoperatively on time intervals ~ a few minutes with immediately available results. In the reported study, the samples after OCE examinations were subjected to histological studies and classification made by an experienced histopathologist. Comparison between the OCE results and histological data made it possible not only to distinguish between normal tissue and tumor (for determining resection boundary), but also to formulate OCE-based criteria allowing for differentiation between carcinomas of low grades and fairly good treatment prognosis and high-grade invasive tumors with poor prognosis. Similarly, identification of not only invasive breast tumors, but also benign breast lesions, intermediate between norm and malignant tumors can be made.

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.

Cardiovascular disease remains the leading cause of mortality worldwide. Here we suggest a novel approach for tracking atherosclerosis progression based on the use of atomic force microscopy (AFM). Using AFM, we studied cross-sections of coronary arteries with the following types of lesions: Type II—thickened intima; Type III—thickened intima with a lipid streak; Type IV—fibrotic layer over a lipid core; Type Va—unstable fibrotic layer over a lipid core; Type Vc—very thick fibrotic layer. AFM imaging revealed that the fibrotic layer of an atherosclerotic plaque is represented by a basket-weave network of collagen fibers and a subscale network of fibrils that become looser with atherosclerosis progression. In an unstable plaque (Type Va), packing of the collagen fibers and fibrils becomes even less uniform than that at the previous stages, while a stable fibrotic plaque (Vc) has significantly tighter packing. Such alterations of the collagen network morphology apparently, led to deterioration of the Type Va plaque mechanical properties, that, in turn, resulted in its instability and propensity to rupture. Thus, AFM may serve as a useful tool for tracking atherosclerosis progression in the arterial wall tissue.

Emerging methods of anti-tumor therapies require new approaches to tumor response evaluation, especially enabling label-free diagnostics and in vivo utilization. Here, to assess the tumor early reaction and predict its long-term response, for the first time we apply in combination the recently developed OCT extensions - optical coherence angiography (OCA) and compressional optical coherence elastography (OCE), thus enabling complementary functional/microstructural tumor characterization. We study two vascular-targeted therapies of different types, (1) anti-angiogenic chemotherapy (ChT) and (2) photodynamic therapy (PDT), aimed to indirectly kill tumor cells through blood supply injury. Despite different mechanisms of anti-angiogenic action for ChT and PDT, in both cases OCA demonstrated high sensitivity to blood perfusion cessation. The new method of OCE-based morphological segmentation revealed very similar histological structure alterations. The OCE results showed high correlation with conventional histology in evaluating percentages of necrotic and viable tumor zones. Such possibilities make OCE an attractive tool enabling previously inaccessible in vivo monitoring of individual tumor response to therapies without taking multiple biopsies.

This paper considers valuable visual assessment criteria for distinguishing between tumorous and non-tumorous tissues, intraoperatively, using cross-polarization OCT (CP OCT) – OCT with a functional extension, that enables detection of the polarization properties of the tissues in addition to their conventional light scattering. Materials and methods. The study was performed on 176 ex vivo human specimens obtained from 30 glioma patients. To measure the degree to which the typical parameters of CP OCT images can be matched to the actual histology, 100 images of tumors and white matter were selected for visual analysis to be undertaken by three “single-blinded” investigators. An evaluation of the inter-rater reliability between the investigators was performed. Application of the identified visual CP OCT criteria for intraoperative use was performed during brain tumor resection in 17 patients. Results. The CP OCT image parameters that can typically be used for visual assessment were separated: (1) signal intensity; (2) homogeneity of intensity; (3) attenuation rate; (4) uniformity of attenuation. The degree of match between the CP OCT images and the histology of the specimens was significant for the parameters “signal intensity” in both polarizations, and “homogeneity of intensity” as well as the “uniformity of attenuation” in co-polarization. A test based on the identified criteria showed a diagnostic accuracy of 87-88%. Intraoperative in vivo CP OCT images of white matter and tumors have similar signals to ex vivo ones, whereas the cortex in vivo is characterized by indicative vertical striations arising from the “shadows” of the blood vessels; these are not seen in ex vivo images or in the case of tumor invasion. Conclusion. Visual assessment of CP OCT images enables tumorous and non-tumorous tissues to be distinguished. The most powerful aspect of CP OCT images that can be used as a criterion for differentiation between tumorous tissue and white matter is the signal intensity. In distinguishing white matter from tumors the diagnostic accuracy using the identified visual CP OCT criteria was 87-88%. As the CP OCT data is easily associated with intraoperative neurophysiological and neuronavigation findings this can provide valuable supplementary information for the neurosurgeon during tumor resection.

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.

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.

Optical coherence tomography (OCT) is a promising method of glial tumors borders diagnostics. Nowadays it is possible to use hand-held and microscope mounted OCT devices during tumor removal. But still there are no clearly visual assessment criteria of OCT images, on the basis of which good differentiation between glioma tissue and white matter can be performed. This paper presents such criteria for crosspolarization OCT (CP OCT), which can detect both the scattering and polarization properties of tissues.

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.