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. Despite significant advances in treatment, oral cancer still results in 10,000 U.S. deaths annually. Early detection of cancer and its curable precursors remains the best way to ensure patient survival and quality of life. A highly sensitive, non-invasive capability for (1) early detection, diagnosis, and monitoring of oral dysplasia and malignancy; (2) screening high-risk populations; and (3) early evaluation of treatment response is urgently needed. The proposed work combines innovative optical in vivo technologies to image and characterize pathological oral changes at three levels: macroscopic, vascular, and cellular. Our multimodal approach will include Optical Coherence Tomography (OCT) multi-photon excited fluorescence microscopy and second harmonic generated imaging (MPM). Previous studies using this combined approach provide strong support for this approach, but lack of a combined in vivo probe has prevented comprehensive meaningful in vivo and clinical testing. Using animal oral carcinogenesis studies, multimodal imaging capability will be optimized and evaluated using the conventional histopathological gold standard, augmented by specific immunohistochemistry (IHC). We will use the micro-endoscopic OCT and muitiphoton tomography (MPT) developed in LAMMP to extend this studies in human subjects with leukoplakic and malignant oral lesions to define diagnostic capability. This work also will advance our understanding of the sequential and spatial processes involved in oral carcinogenesis. The specific hypotheses to be tested are: 1. High resolution in vivo F-OCT can map dysplasia-, malignancy-related epithelial, subepithelial, and vascular change. 2. In vivo MPT can image tissue extracellular matrix, cellular, vascular, and microtumor presence. 3. Improved diagnostic capability will be achieved by combining F-OCT and MPT data. 4. Specific biomarkers for angiogenesis and extracellular matrix remodeling (VEGF, HIF-1¿, uPA, PAI-1, MMP1,2,9) will target events detected by multimodal imaging during development of premalignancy and malignancy. To test these hypotheses, we proposed following specific aims: 1. F-OCT will be used to image and quantify specific epithelial, subepithelial and vascular changes that correspond with specific histopathological stages in the progression health-dysplasia-malignancy. Based on the techniques and diagnostic benchmarks for the imaging data established in animal studies, F-OCT's diagnostic sensitivity and specificity will be determined in 84 human subjects with healthy, leukoplakic, or malignant oral tissue. Diagnostic capability will be compared with conventional clinical and histopathological diagnosis. OCT-based evaluation of criteria such as epithelial keratinization, thickening, proliferation and invasion; rete pegs; epithelial stratification; basal hyperplasia; and presence of basement membrane will be benchmarked against specific stages of premalignancy and malignancy as defined by histopathology (existing gold standard), In addition, Doppler OCT based quantification of vessel density and blood flow at specific locations will identify the relationship between histopathological stages of dysplasia, malignancy vs. vascularization and blood flow. 2. MPT will be used to define specific events at a cellular level that correspond with specific histopathological stages in carcinogenesis. Based on the imaging techniques and diagnostic benchmarks established in animal studies, diagnostic sensitivity and specificity of MPT will be determined in 84 human subjects with healthy, leukoplakic, or malignant oral tissue. MPT's diagnostic capability will be compared with conventional clinical and histopathological diagnosis. Using MPT, changes in the extracellular matrix, specifically, collagen presence, fiber length/organization, matrix structure, cellular exudates, vascularization, and microtumors -- will be benchmarked against specific stages of premalignancy and malignancy, as defined by the histopathological gold standard. In addition, 3. F-OCT and MPT data will be combined to evaluate for improved diagnostic capability using this approach. 4. To advance our understanding at a macroscopic, vascular, and cellular level of the sequential and spatially-resolved processes involved in oral carcinogenesis, specific biomarker expression, combined with imaging data and histopathological staging, will provide a comprehensive, spatially-resolved, time- and sequence-sensitive map of specific components of the carcinogenesis process within the oral epithelial and subepithelial tissues.