Early diagnosis of melanoma is an ongoing challenge in dermatology and oncology. There is a special subgroup of melanocytic lesions that can be clinically and dermoscopically indistinguishable from early melanoma. The aim of the study was to improve the in vivo diagnostic possibilities for the differentiation of dermoscopic equivocal melanocytic lesions based on combination of multiphoton tomography (MPT) and optical coherence angiography (OCA). A multiphoton optical score (MPOS) for quantitative assessment of the melanoma features revealed by MPM was developed. OCA images were processed to calculate the vessel densities and the total lengths thin and thick vessels. Histopathological analysis separated the equivocal lesions into benign, melanoma in situ, and invasive melanoma. The MPOS value of benign lesions was significant lower than for the malignant ones. Quantitative analysis of OCA images revealed that the invasive melanoma type has the highest vessel density. The combined use of multiphoton tomography with MPOS calculation and quantification of optical coherence angiography data demonstrated a potential to discriminate all dermoscopic equivocal melanocytic lesions in vivo.
Two pronounced absorption peaks in blue and red ranges of the chlorin-based photosensitizer (PS) absorption spectrum provide additional benefits in photodynamic therapy (PDT) performance. Differing optical properties of biological tissues in these ranges allow for both dual-wavelength diagnostics and PDT performance. We provide a comparative analysis of different PDT regimes performed with blue and red lights and their combination, with doses varying from 50 to 150 J / cm2. The study was performed on the intact skin of a rabbit ear inner surface, with the use of chlorin e6 as a PS. PDT procedure protocol included monitoring of the treated site with fluorescence imaging technique to evaluate PS accumulation and photobleaching, as well as with optical coherence tomography (OCT) to register morphological and functional responses of the tissue. Optical diagnostic observations were compared with the results of histopathology examination. We demonstrated that PDT procedures with the considered regimes induce weaker organism reaction manifested by edema in normal tissue as compared to irradiation-only exposures with the same light doses. The light doses delivered with red light induce weaker tissue reaction as compared to the same doses delivered with blue light only or with a combination of red and blue lights in equal parts. Results of in-vivo OCT monitoring of tissue reaction are in agreement with the results of histopathology study.
Dual-wavelength photodynamic therapy is a photodynamic therapy (PDT) modality combining therapeutic effects of irradiation at two wavelengths of different region of visible range. In this paper we report on comparative analysis of single- and dual-wavelength PDT regimes based on multimodal optical monitoring of tissue response with histologic verification. Morphological and functional tissue responses to PDT procedure with chlorin-based photosensitizer (PS) were studied at the normal skin of a rabbit ear inner surface. Multimodal optical monitoring was performed by OCT and dual-wavelength fluorescence technique. The studied doses vary from 50 to 150 J/cm2 delivered separately at 405 or 660 nm, or at both wavelengths together in the equal dose.
In this study we present a complex approach to photodynamic therapy (PDT) with chlorin based photosensitizers including Monte Carlo based planning and prediction of optical diagnostics results, intra-procedure dual-wavelength fluorescence monitoring allowing to evaluate PS accumulation and photobleaching, and monitoring of tissue response with optical coherence tomography (OCT). The approach was employed to compare the effects of different PDT regimens in normal and tumor tissues and the results of non-invasive optical diagnostics were matched with results of histologic examination, including hematoxylin-eosin and Mallory staining. The considered doses are in the range 50-150 J/cm2 for normal tissues and 150-275 J/cm2 for tumor tissues, single wavelength (405 and 660 nm) and dual-wavelength regimes are studied.
The clinical diagnosis of melanocytic lesions is an ongoing medical challenge. Non-invasive tools and technologies can help to distinguish equivocal lesions. The aim of the study was to improve the in vivo diagnostic possibilities for the differentiation of benign and malignant melanocytic lesions based on combination of three imaging label-free modalities (multiphoton tomography, fluorescence lifetime imaging and optical coherence angiography). Thirty-two melanocytic lesions were studied, using multiphoton tomography, fluorescence lifetime imaging and optical coherence angiography. Multiphoton tomography features of benign melanocytic nevi were characterized by the normal morphology of both the keratinocytes and the nevus cell nests surrounded by collagen. Dysplastic nevi were characterized by their increased intercellular distances and enlarged cell nuclei. Melanomas showed the presence of melanocytes and dendritic structures in all layers of the epidermis. Analysis of the metabolic state revealed that melanomas and dysplastic nevi were characterized by enhanced glycolysis. Optical coherence angiography shows that benign nevi had regular vascular networks and equal numbers of thin and thick vessels. Vascular networks of dysplastic nevi were characterized thin curved vessels. Thick irregular spiral vessels formed a dense microvascular network of melanomas.
Employment of chlorin-based photosensitizers (PSs) provides additional advantages to photodynamic therapy (PDT) due to absorption peak around 405 nm allowing for superficial impact and efficient antimicrobial therapy. We report on the morphological and clinical study of the efficiency of PDT at 405 nm employing chlorin-based PS. Numerical studies demonstrated difference in the distribution of absorbed dose at 405 nm in comparison with traditionally employed wavelength of 660 nm and difference in the in-depth absorbed dose distribution for skin and mucous tissues. Morphological study was performed at the inner surface of rabbit ear with histological examinations at different periods after PDT procedure. Animal study revealed tissue reaction to PDT consisting in edema manifested most in 3 days after the procedure and neoangiogenesis. OCT diagnostics was confirmed by histological examination. Clinical study included antimicrobial PDT of pharynx chronic inflammatory diseases. It revealed no side effects or complications of the PDT procedure. Pharyngoscopy indicated reduction of inflammatory manifestations, and, in particular cases, hypervascularization was observed. Morphological changes were also detected in the course of monitoring, which are in agreement with pharyngoscopy results. Microbiologic study after PDT revealed no pathogenic bacteria; however, in particular cases, saprophytic flora was detected.
Nonlinear optical microscopy combined with fluorescence lifetime imaging is a non-invasive imaging technique, based on the study of fluorescence decay times of naturally occurring fluorescent molecules, enabling a noninvasive investigation of the biological tissue with subcellular resolution. Cancer exhibits altered cellular metabolism, which affects the autofluorescence of metabolic cofactors NAD(P)H and FAD. In this study features of tumor metabolism in different systems of organization (from cell culture to patient lesion) was showed. The observed differences in the relative contributions of free NAD(P)H and FAD testify to an increased a glycolytic metabolism in cancer cells compare to fibroblasts. In 3D spheroids, the cells of the proliferating zone had greater a1 and lower tm values than the cells of the quiescent zone, which likely is a consequence of their higher glycolytic rate. During the growth of colorectal cancer in the experimental mouse model, the contribution of the free component of NAD(P)H was increased. Dysplastic nevus and melanoma is characterized by raised contribution of free NADH compare to healthy skin. Therefore, melanoma cells had very short value of τ1.