Project Summary: Age-related delays in wound healing have been attributed to altered dermal microstructure, mitochondrial dysfunction, and reduced cellular proliferation. However, an inability to non-destructively measure these characteristics has limited their use in guiding wound care and product development. The long-term goal of this project is to establish non-invasive, real-time, quantitative optical biomarkers to predict age-related delays in skin wound healing. The specific objective of this proposal is to utilize label-free multiphoton microscopy techniques, including two-photon excited fluorescence, fluorescence lifetime imaging, and second harmonic generation to identify and develop metabolic and microstructural biomarkers of aged skin that are associated with delayed closure and susceptibility to mechanical re-injury. Our central hypothesis is that the natural fluorescence of cells and their surrounding matrix can be quantified to provide sensitive biomarkers of age- related wound healing impairment. To test this hypothesis and evaluate the effect of aging, controlled pre- clinical studies will be performed using mice with ages ranging from 12.5-75% of their life expectancy, independent of common chronic wound comorbidities such as diabetes mellitus. In Aim 1, we will non- invasively monitor wound healing dynamics in individual live mice and identify differences in wound metabolism during the healing process using the natural fluorescence of NADH and FAD. In Aim 2, we quantify extracellular matrix organization and composition using multiphoton microscopy to predict wound strength through image-based multiscale modeling. In Aim 3, we will evaluate the effect of diabetes mellitus on the age-related optical biomarkers of wound healing developed in Aims 1 and 2. Quantitative comparisons of cellular and extracellular biomarkers will be made across different ages and validated against histology and immunohistochemistry. The expected outcome of this project is a set of candidate biomarkers for use in guiding therapy that are capable of discriminating age-related alterations and common comorbidities known to impair healing. With multiphoton microscopy technology already making its way into the clinic, the imaging methods developed here have the potential for immediate impacts in the clinical assessment and management of wounds.