Project Summary/Abstract Our overarching goal is to aid anal cancer screening using a new, non-invasive imaging approach termed probe-based light sheet microscopy (pLSM). Incidence and mortality of anal cancer have been rapidly increasing: mortality increased by 5.7% per year between 2014 and 2020. The recent ANCHOR (Anal Cancer HSIL Outcomes Research) trial published in late 2022 showed that treatment of anal precancer (high-grade squamous intraepithelial lesion, HSIL) significantly reduces the risk of anal cancer. Therefore, it is expected that anal cancer screening in high-risk populations will become standard of care. However, high-resolution anoscopy, an integral component of anal cancer screening and treatment, has low-to-moderate diagnostic performance, and trained anoscopists are scarce due to a steep learning curve and no standardized training opportunities. In a recent Trailblazer R21 project, we developed a new optical microscopy approach termed scattering-based light sheet microscope (sLSM). While LSM is not new and has been widely used for imaging fluorescence- labelled samples in biology research, we optimized LSM for imaging animal and human tissues using the intrinsic scattering contrast for clinical applications. In our preliminary study, we imaged fresh anal biopsies (n = 110) ex vivo with a bench sLSM device and found that i) sLSM could clearly visualize the critical morphologic features pathologists use to diagnose anal squamous intraepithelial lesions and ii) could provide high diagnostic accuracy (overall accuracy = 87%; HSIL accuracy = 91%). In this 4-year R01 project, we will develop a new, probe-based light sheet microscopy (pLSM) device and evaluate its clinical utility for imaging human subjects in vivo. During anal cancer screening, the anoscopist directly places the pLSM device on the anal mucosa and examines the cellular details of the tissue. The pLSM images are analyzed by a trained clinician (pathologist or anoscopist) or by automate