This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objectives: Early detection of cancer and its curable precursors remains the best way to ensure patient survival and quality of life. Despite significant advances in treatment, oral cancer still results in 10,000 U.S. deaths annually, mainly due to the late detection of most oral lesions. Specific aim: Using a combination of non-invasive optical in vivo technologies to test a multi-modality approach to non-invasive diagnostics of oral premalignancy and malignancy in human subjects. Methods: In 30 patients with healthy, dysplastic, and/or malignant oral tissues, in vivo optical coherence tomography (OCT) and optical Doppler tomography (ODT) mapped epithelial, subepithelial and vascular change in specific, marked sites prior to routine biopsy. Ex vivo multi-wavelength multi-photon (MPM) and second harmonic generated (SHG) fluorescence techniques provided parallel data on surface and subsurface tissue structure, specifically collagen presence and structure, cellular presence, and vasculature. Images were diagnosed by 2 blinded, pre-standardized investigators using a standardized scale from 0-6 for all modalities. Histopathological sections were prepared and pathology evaluated on a scale of 0-6. ANOVA techniques compared imaging diagnostics with histopathology. 95% confidence limits of the sensitivity and specificity were established for the diagnostic capability of OCT/ODT+ MPM/SHG using ROC curves and kappa statistics. Results: Imaging data were reproducibly obtained with good accuracy. Dysplasia- and malignancy-related structural and vascular changes were clearly visible to tissue depths of 2mm. Sensitivity (OCT/ODT alone: 72-89%;OCT+MPM/SHG: 76-90%) and specificity (OCT alone: 68-84%;OCT+MPM/SHG: 74-88%) compared well with conventional techniques. Conclusions: OCT/ODT and MPM/SHG are promising non-invasive in vivo diagnostic modalities for oral dysplasia and malignancy.