Multi-photon excited intensity and lifetime fluorescence images relying on endogenous contrast can be analyzed to quantify contributions from key metabolic co-enzymes and associated metabolic function and mitochondrial organization metrics. The high spatio-temporal resolution and context of these non-destructive measurements can be used to provide important insights related to a wide range of samples, conditions and disease models. Corresponding images are acquired from mitochondria, engineered tissues, excised and in vivo human tissues. Recent studies highlight the value of multi-parametric, label-free, metabolic assessments to improve our understanding of traumatic brain injury, (pre)cancer development, and vitiligo lesions.
Wound re-epithelialization is complex and imperfect. Understanding which cells are involved in this process, how they are spatially arranged, and when they contribute to wound healing is a longstanding scientific and clinical challenge. We used a recently developed fast large area multiphoton exoscope for in vivo imaging of human skin to study the process of wound healing in vivo in human skin. We monitored the re-epithelization of wounds generated by removal of the epidermis following a suction blistering procedure and identified the morphological and metabolic signatures of epidermal and dermal cells involved in the healing process.
Our group and others have demonstrated the strong potential of the multiphoton microscopy for a broad range of applications from advancing the understanding of skin biology to non-invasive diagnosis of skin diseases and monitoring therapy effects. We have recently reported on a fast large area multiphoton exoscope for rapidly mapping out macroscopic tissue areas with microscopic resolution and enhanced contrast for selective melanin detection. We will describe the technical abilities of this instrument and demonstrate its feasibility for early melanoma diagnosis based on a pilot study on ex-vivo and in-vivo imaging of pigmented lesions suspicious of melanoma in human skin.
Vitiligo is an immune skin disorder consisting of depigmented skin patches caused by the destruction of epidermal melanocytes. Vitiligo treatment represents a clinical challenge since the re-pigmentation mechanism is not fully understood. In this pilot study, we employ in-vivo multiphoton microscopy to evaluate epidermal keratinocyte metabolic state before and during treatment, in-vivo reflectance confocal microscopy to track melanocyte migration after treatment initiation, and single cell transcriptomics to identify unique cell populations more abundant in stable vitiligo lesions compared to normal skin. The findings provide insights into the role of certain cell populations in the viability of micro-grafting treatments.
Vitiligo is a disorder characterized by the loss of skin pigment due to the autoimmune destruction of epidermal melanocytes. Treatments that aim to repigment vitiligo regions are long, incomplete, and suffer from a highly variable success rate. This study aims to use non-invasive, depth-resolved, label-free, two-photon detection of reduced nicotinamide adenine dinucleotide (NADH) to quantitatively evaluate mitochondrial dynamics in vitiligo micrografting patients during the repigmentation process. This study indicates differences in the depth-dependent mitochondrial profiles between healthy and vitiligo tissues. This study also indicates that vitiligo regions responsive to treatment exhibit depth-dependent shifts in mitochondrial dynamics prior to repigmentation.
Vitiligo is characterized by white patches on skin due to the loss of melanocytes. Treatments are not uniformly successful and re-pigmentation is rarely complete. Optical laser scanning microscopy techniques have great potential to advance our understanding of the repigmentation process of vitiligo.
In this pilot study, we employ in-vivo multiphoton microscopy (MPM) to assess potential changes in the metabolic state of epidermal keratinocytes involved in vitiligo before and throughout treatment, and in-vivo reflectance confocal microscopy (RCM) to assess the initiation of the re-pigmentation process and monitor wound healing after micro-grafting treatment.
Multiphoton microscopy (MPM) can provide sub-micron resolution images of living tissues in their native environment with contrast from multiple modalities, including second harmonic generation (SHG) and two-photon excited fluorescence (TPEF). Recent advances of MPM in clinical skin imaging demonstrated the unique potential of this technology as a label-free research and clinical tool for a broad area of applications such as melanoma and non-melanoma skin cancer detection, monitoring pigmentary skin disorders, characterizing keratinocyte metabolism, etc. In this contribution we demonstrate the ability of this microscope to provide sub-micrometer resolution ex-vivo images of large areas of skin tissue (up to 5x5 mm2) in <1 minute. We demonstrate the importance of high-speed, high-resolution mesoscopic imaging on cancerous skin tissues that present heterogeneous morphology to show the ability of the instrument to capture both benign and malignant areas of the lesion.
Vitiligo is a skin condition in which pigment-producing cells are removed by the immune system, leading to patches of white skin on different parts of the body. Treatments, including UVB light therapy and skin micro-grafting, may lead to repigmentation of the skin; however, treatments are not uniformly successful, and it is currently unclear why some vitiligo areas repigment more rapidly than others. An optical imaging technique that allows non-invasive visualization of melanocytic activity in skin may advance the knowledge about this skin condition and help understand treatment impact. In this pilot study, we employ in-vivo multiphoton microscopy (MPM) to evaluate architectural and structural features of the melanocytes that repigment vitiligo skin. MPM is a nonlinear laser scanning microscopy technique that features sub-cellular resolution and label-free molecular contrast. MPM contrast in skin is derived from two-photon excited fluorescence of NADH/FAD+, keratin, melanin, and elastin, and second-harmonic generation of collagen. We employed a clinical MPM tomograph (MPTflex, JenLab, Germany) to image vitiligo and adjacent normal areas in 10 patients undergoing treatment. The treatment consisted of either UVB light therapy or skin micro-grafting treatment followed by UVB light therapy. We visualized pigment producing melanocytes near hair follicles, migrating melanocytes within the human epidermis, newly pigmented keratinocytes in the basal layer, and epidermal melanin granules. The overarching goal is to use this technology to better define the phenotypic characteristics of migrating melanocytes in the hope of improving transplantation therapies for vitiligo.
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