Non-healing wounds affect millions of Americans and require care with annual costs in the billions of dollars. These chronic wounds are often attributed to pathologies, such as cardiovascular disease and diabetes, which can alter cell metabolism within the wound and produce a host of other pathological sequelae. The long-term goal of this research plan is to establish non-invasive, real-time, quantitative optical biomarkers to identify and characterize impaired healing in vivo. The objective of this proposal is to develop biomarkers of altered metabolism and tissue microstructure using in vitro and in vivo models of impaired wound healing. Specifically, we plan to use non-linear optical microscopes capable of two photon excited fluorescence, second harmonic generation, and confocal reflectance to define dermal and epidermal cell redox states, collagen organization, and angiogenesis using only endogenous sources of contrast. The central hypothesis is that altered cell metabolism can provide an early marker of impaired wound healing, and quantitative biomarkers for collagen remodeling, angiogenesis, and re-epithelialization can provide context for developing appropriate treatment regimens. To initially test this hypothesis, and develop biomarkers of impaired healing, three- dimensional human skin equivalent models will be utilized in Aim 1 to evaluate the sensitivity of an optical redox ratio to changes in oxygen and glucose levels. In Aims 2 and 3, impaired healing will be evaluated in vivo using a diabetic mouse model of wound healing. In addition to the metabolic biomarkers established in vitro in Aim 1, techniques will be developed to quantify altered collagen matrix organization and reduced angiogenesis in diabetic wounds. Collectively, these aims will provide a suite of optical biomarkers to enable the early detection of altered cell metabolism in the wound, and a variety of other microstructural and biochemical outcomes that can be used to guide wound care. These non-invasive 3D imaging techniques will enable spatiotemporal assessments of wound metabolism and organization not currently available in the laboratory or clinic. With non-linear microscopy technology already available for the clinic, the imaging methods developed in this proposal will enable immediate clinical application to chronic wound assessments. Through the completion of the research and training plans, the applicant will gain extensive experience in different wound healing research methods from collaborators at Tufts, MIT, and Beth Israel Deaconess Medical Center. This intensive training and career development plan will supplement the applicant's background in non-linear optics and enable the successful transition to independent investigator.