Summary The study of mammalian immune cells and their interactions with tissue in situ is critical for understanding how they regulate processes ranging from wound healing to autoimmune disease initiation to cancer and for designing better therapeutic strategies to treat these prevalent conditions. Intravital multiphoton microscopy (MPM) combined with a rich repertoire of fluorescent reporter mouse models and in vivo cell and tissue labeling techniques have made it possible to visualize immune cell-tissue interactions at a subcellular level in skin and other organs. However, there are significant differences in the structure and immune milieu of human skin that limits the translatability of these findings to the human cutaneous immune response. Our group has recently developed a fast large area multiphoton exoscope (FLAME), a unique imaging platform optimized for efficient clinical skin imaging to rapidly generate macroscopic images (mm to cm-scale) with microscopic resolution (0.5- 1µm) based on label-free molecular contrast (fluorescence intensity and lifetime). In this application, we leverage our extensive experience in MPM technology development and clinical imaging of more than 400 patients over the past several years to develop the first MPM-based clinical device (iFLAME) as a research imaging tool optimized for, and dedicated to, in vivo label-free imaging of immune cell populations and their dynamics in human skin. In Aim 1, we develop iFLAME as a clinical research tool for efficient in vivo label-free imaging of dermal cell populations and their dynamics in human skin. This work involves development of detection and analytic approaches as well as optical and computational methods to enable rapid fluorescence lifetime detection and analysis necessary to automate measurements of the cellular morphological and metabolic signatures. In Aim 2, we validate iFLAME performance by demonstrating in vivo characterization of immune cells in normal and inflamed human skin. In Aim 3, we develop quantitative morphological and metabolic MPM imaging endpoints to assess immune infiltrates and their dynamics in human skin in the context of monitoring wound healing. This work represents the first attempt to use intrinsic sources of MPM contrast to image, identify, and quantify key immune cells in human skin in vivo based on their optical signatures and migratory behavior. Our long-term goal is to develop iFLAME as a clinical research tool for rapid, label-free imaging of immune cells in skin based on cellular morphologic and metabolic imaging endpoints. These can be used to better understand, evaluate and optimize wound healing, autoimmune skin diseases and therapeutic responses.

Narrative In this application we develop iFLAME, a fast, large area multiphoton exoscope optimized for in vivo label-free imaging of immune cell populations and their dynamics in human skin. We validate iFLAME performance by demonstrating its ability to discriminate immune cells in vivo and develop quantitative morphological, metabolic and dynamic imaging endpoints to assess immune infiltrates in human skin during the wound healing process. Completion of these studies will establish the practical utility and value of iFLAME as a non-invasive clinical research tool for monitoring immune responses at the cellular level in human skin.