Imaging techniques that can enhance tumor contrast against non-specific background can significantly impact diagnostic and surgical applications in oncology. Fluorescence optical imaging is being evaluated for disease diagnosis and surgical treatment, using probes that either preferentially accumulate in tumors, or are antibody conjugated to label tumor-specific receptors. While there has been a significant progress in the development of molecularly targeted near infrared fluorescent probes, background fluorescence from non-specific probe accumulation remains a major confound that reduces sensitivity and specificity for tumor detection. Even when the probe clearance is rapid, tissue autofluorescence can be significant compared to the tumor fluorescence. Existing clinical optical imaging systems primarily employ fluorescence intensity-based imaging. Fluorescence intensity strongly depends on tissue attenuation and experimental factors such as excitation light intensity, and therefore cannot distinguish tumor bound probe from non-specific probe or tissue autofluorescence on an absolute scale. Our preclinical and clinical studies indicate that the fluorescence lifetimes of tumor and normal tissue in subjects injected with cancer targeted probes are distinct and independent of experimental parameters under typical conditions. We have shown that this FLT contrast dramatically improves the accuracy for tumor vs. normal classification compared to intensity-based imaging using the FDA-approved near infrared fluorescent dye, Indocyanine green (ICG). Building on this exciting finding, the goal of this proposal is to robustly validate fluorescence lifetime as a contrast mechanism for tumor identification in oral cancers using ICG. We will develop a portable time domain imaging system for concurrent intraoperative imaging and surgical specimen mapping and optimize the system using preclinical models. Subsequently, we will validate the system for intraoperative imaging and specimen margin assessment using clinical studies in head and neck cancer surgery patients systemically injected with ICG, and determine the dosage and injection time points that provide optimal accuracy for tumor vs normal classification. This proposal will also lead to future applications of lifetime contrast to enhance accuracy of tumor detection in other cancers, using ICG and novel cancer targeted probes currently under development.