In recent years, fluorescence image-guided surgery (FIGS) with contrast provided by 5-aminolevulinic acid (5- ALA)-induced PpIX fluorescence has been demonstrated to improve surgical outcomes for glioma patients. However, there are several limitations to these wide-field imaging methods. For example, it is difficult to accurately determine a surgical margin based on subtle variations in fluorescence intensity in glioma tissues, which are diffuse and lack a distinct transition between tumor and normal. Furthermore, wide-field (low- resolution) approaches, such as FIGS and MRI, provide pixel intensities that represent an average value from many cells, resulting in a diminished ability to detect the sparse tumor cell populations at the margins of diffuse gliomas. This problem is exacerbated in low-grade gliomas, where 5-ALA-induced PpIX fluorescence is only generated in rare proliferating cell populations and is undetectable via wide-field FIGS. Recently, Dr. Nader Sanai, a Co-Investigator on this project, has shown that intraoperative cellular-resolution confocal microscopy can be used to visualize the sparse fluorescent cells in low-grade glioma patients treated with 5-ALA. The implications of this finding are highly significant since reported rates of gross-total resection (GTR) have been relatively low for low-grade gliomas (14% to 46%), suggesting a need for improved image-guidance techniques. Here, we propose to optimize a hand-held intraoperative confocal microscope, previously developed by the PI, to visualize low-grade glioma cells that express 5-ALA-induced PpIX, and to demonstrate that this real-time quantitative in vivo imaging technique correlates with MRI and invasive histopathology for margin assessment. This project brings together a proven team of collaborators, including Co-Investigator Olav Solgaard at Stanford University, to develop an optimized optical-sectioning microscope that incorporates a highly robust MicroElectroMechanical System (MEMS) scanner and an achromatic (wavelength-agile) optical design recently published by the PI's lab. Preclinical ex vivo and in vivo imaging will be performed with a highly realistic and infiltrative at model of glioma to demonstrate the ability to correlate the density of cells expressing PpIX fluorescence vs. actual tumor-cell density and histological grade. Clinical studies will leverage the existing expertise of Co-Investigator Nader Sanai, at Barrow Neurological Institute (BNI), with both wide-field FIGS and intraoperative confocal microscopy of PpIX fluorescence in glioma patients. Based on established and approved protocols at BNI, an exploratory clinical study will be performed to correlate the density of PpIX-labeled cells with histopathology, pre-operative MRI (intraoperative neuronavigation), and post- operative MRI. The results of these preliminary studies will justify future clinical investigations to assess patient outcomes such as volumetric extent of resection, overall survival, and neurological morbidity.