Label-Free Imaging and Histo-Optical Evaluation of Head and Neck Cancers with Multiphoton Autofluorescence Microscopy

Simple Summary Efforts to identify lesions of head and neck cancers with high malignant potential are important to improve patient outcomes, as 5-year survival rates remain low due to late detection. Optical imaging approaches that provide direct cellular and structural atypia could be helpful in detection and pathology guidance. This study evaluates the method of multiphoton autofluorescence microscopy (MPAM) for its ability to reveal atypia associated with neoplasia in resected tumor samples without the need for exogenous dyes or tissue sectioning, showing a correlation with the corresponding histology. Depth-resolved label-free optical imaging by the method of multiphoton autofluorescence microscopy (MPAM) may offer new ways to examine cellular and extracellular atypia associated with epithelial squamous cell carcinoma (SCC). MPAM was evaluated for its ability to identify cellular and microstructural atypia in head and neck tissues from resected discarded tumor tissue. Three-dimensional image volumes were obtained from tissues from the floor of the mouth, tongue, and larynx, and were then processed for histology. MPAM micrographs were evaluated for qualitative metrics of cell atypia and quantitative measures associated with nuclear pleomorphism. Statistical analyses correlated MPAM endpoints with histological grade from each imaged site. Cellular overcrowding, discohesion, anisonucleosis, and multinucleated cells, as observed through MPAM, were found to be statistically associated with dysplasia and SCC grading, but not in histologically benign regions. A quantitative measure of the coefficient of variance in nuclear size in SCC and dysplasia was statistically elevated above histologically benign regions. MPAM also allowed for the identification of cellular heterogeneity across transitional areas and other features, such as inflammatory infiltrates. In the future, MPAM could be evaluated for the non-invasive detection of neoplasia, possibly as an adjunct to traditional conventional examination and biopsy.