PROJECT SUMMARY In this competitive renewal for R01 CA187427, we will continue to advance label-free Fluorescence Lifetime Imaging (FLIm) to enhance the functionality of the widely-used da Vinci Surgical platform with an emphasis on Trans-Oral-Robotic-Surgery (TORS). UC Davis has pioneered the intra-operative use of FLIm and our partner, Intuitive Surgical, is the global leader in robotic-assisted surgery. To date, we have demonstrated a) the synergetic integration of a point-scanning FLIm device with the da Vinci Surgical System (first-of-its-kind); b) the potential of this approach to improve surgical decision-making during TORS without modifying conventional clinical protocols; and c) the utility of FLIM-derived parameters detecting biochemical and metabolic characteristics to distinguish oral and oropharyngeal cancer in real-time from surrounding normal tissue in patients in-situ.. We also developed innovative methods for real-time dynamic augmentation of imaging parameters on the surgical field of view. Capitalizing on the major accomplishments and knowledge gained under the current R01, the overarching objective of this renewal application is to 1) demonstrate FLIm’s diagnostic value in prospective studies by accounting for biological/experimental variables identified as critical under the current R01 and leveraging pre-operative imaging and clinical information and 2) enhance the visualization of FLIm-based classifiers on the surgeon console for real-time intraoperative feedback. To accomplish our overall objective, we propose three specific aims: Aim 1. Expand the FLIm database and develop tissue-type classifiers (retrospective analysis) accounting for biological variables (e.g., patient age/sex, tumor origin/location) and experimental situations (e.g., epithelial surface vs deep margins, tumors of an unknown primary) and the pre- operative imaging (CT, PET) features used in surgical planning. Aim 2. Develop, validate and perform regulatory activities for enhanced FLIm data visualization in support of prospective clinical studies. Aim 3. Validate FLIm as a means of real-time intraoperative TORS guidance in a prospective study analysis. In summary, this study will demonstrate the clinical feasibility and utility of FLIm for intraoperative real-time assessment of oral and oropharyngeal cancer surgical margins. The acquired FLIm parameter database will enable subsequent large clinical trials for automated tissue classification and diagnostic prediction. While the focus of this application is on TORS surgeries, the label-free FLIm-based tissue assessment, characterized by simple, fast and flexible data acquisition and display, can be broadly applied to other procedures as the da Vinci Surgical System is used in a wide range of tumor surgeries including urologic, colorectal, gynecologic, and thoracic cancers.