In vivo label free optical imaging of immune cells in human skin

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Summary. The study of mammalian immune cells and their interactions under native conditions is critical for understanding the mechanisms initiating many diseases, including cancer, and for improving their treatments. Intravital multiphoton microscopy (MPM), fluorescent reporter mouse models and in vivo cell and tissue labeling techniques have made possible the investigation of immune cells interactions at a cellular/subcellular level in their native environment. However, the significant differences in the skin and the immune system of mice and humans preclude studies of uniquely human immune responses. The goal of this proposal is to evaluate the ability of intravital MPM imaging to identify and distinguish immune cell populations in human skin, based on label-free molecular contrast. We will utilize a fast, large area multiphoton exoscope (FLAME), an imaging platform recently designed and developed by our group to have unique performance features optimized for clinical skin imaging. FLAME has the ability to generate in vivo 3D images of human skin over macroscopic areas (up to 8x8 mm2) with microscopic resolution (0.5-1µm) at fast acquisition rates (tens of seconds). FLAME features label-free specificity for melanin and NADH based on their spectral and rapid time-resolved endogenous fluorescence detection. Selective detection of melanin allows for imaging of pigment-rich cells such as melanophages with high specificity, while time-resolved NADH fluorescence detection reports on the protein binding activity of these molecules within the immune cells and on their metabolic heterogeneity. We have demonstrated the ability of FLAME to detect resident immune cells in normal human skin as well as an activation of a cellular immune response, based on endogenous molecular contrast. In this proposal, we will develop methods for the in vivo characterization of the spatial and metabolic heterogeneity of immune cells in human skin by using atopic dermatitis as an inflammatory skin condition model and Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA) for validation. We will also evaluate the feasibility of FLAME to quantify changes in immune cell metabolism and spatiotemporal heterogeneity in response to immune activation. If successfully validated in a larger clinical study, this approach would be a critical first step in making it possible to characterize cellular-level immune responses in human skin at the bedside, with broad applications ranging from detecting early immune reactions to developing improved cancer treatments.