Most deaths from head and neck squamous cell cancer (HNSCC) result from tumor recurrence following radiotherapy (RT). More than 75% of HNSCC patients receive RT as part of their care and over 50% of them are at risk for developing recurrence post RT. Radio-resistance (RR) leads to poor prognosis in head and neck squamous cell cancer (HNSCC) patients. The failure of RT has been attributed to hypoxia. However, new studies found that RT-induced re-oxygenation rates alone cannot distinguish primary from recurring HNSCC tumors, as some recurrent tumors also showed re-oxygenation after RT. Overexpression of RTinduced Hypoxia-Inducible Factor-1-alpha (HIF-1α) has been shown to be associated with an increased risk of failure of RT. HIF-1α is known to regulate many growth factors to promote aerobic glycolysis and angiogenesis. We hypothesize that RT-induced HIF-1α expression and subsequent alterations in metabolism/vasculature underlie HNSCC RR. Unraveling metabolic traits of cells that evade RT and recur, and the role of the supporting vasculature, is critical to developing strategies to prevent HNSCC recurrence and improve patient survival. However, there are surprisingly few techniques available to provide a systems level view of these hallmarks together in vivo. To fill these gaps, I will build a portable multi-parametric microscope to measure the major axes of metabolism and vasculature in small animal models in vivo. I will then use these platforms to study the effect of radiation on HNSCC tumors and test our hypothesis on HNSCC RR development. This technology fills an important gap between in vitro studies on cells and whole body imaging, and is complementary to metabolomics and immunohistochemistry (IHC). I envision that this system will be well suited to study tumor RR and recurrence in patient-derived xenograft (PDX) and organoid models, which can faithfully recapitulate many micro-environmental features of patient tumors. Successful completion of the proposed studies will set the foundation for translating the optical technology to image patient derived tumor lines in PDX models, allowing us identify predictors of recurrence and develop improved radiotherapeutics (HIF-1 inhibitors) for HNSCC.