Project Summary Intraoperative consultations (IOC, often refers to frozen-section histopathology during surgery) significantly improve the outcome of many oncological surgeries. However, even though this procedure generates results much faster than traditional formalin-fixed paraffin-embedded (FFPE) histopathology, it is still lengthy for time- sensitive surgeries such as head and neck tumor resections. Surgeries often need to be paused during the IOC, leading to an expensive waste of operating room time and resources. Because IOC usually requires a fast turnaround, only a few slices can be prepared and evaluated at a time. Miscommunication between surgeons and pathologists leads to sampling errors. Freezing artifacts result in degraded histology quality. These limitations increase the risk of missing important histopathology information in the slice, resulting in false- negative margins which affects the success rate and long-term outcomes of cancer surgeries. Thus, there is a need for faster and more reliable intraoperative histopathology evaluation. To address these limitations, we propose to develop an alternative histology framework for intraoperative histopathology assessment using a combination of optical clearing and 3D imaging technologies developed in our lab. Our proposed product will provide 3D histology information of intraoperative specimens with a faster and simpler workflow than the incumbent solution. The product comprises three major components: 1) a rapid tissue processing pipeline that makes tissue transparent with a few washes, 2) a user-friendly imaging system that enables real-time 3D microscopy imaging of the transparent whole-mount clinical specimens, 3) A software user interface that translates the 3D data into 2D images with pathologist-familiar color contrast and assists the pathologist in interpreting the data. 3D virtual histology has been proposed as an alternative to slice-based histopathology to provide more accurate diagnoses. However, current 3D virtual histopathology techniques often have high complexity and long turnaround time. Our preliminary customer discovery suggested it was highly challenging for histology labs to incorporate these new techniques into the existing well-established workflow due to high capital cost and complexity. By simplifying the sample preparation into 2-3 fluid washes and providing a plug- and-play 3D imaging device that requires minimal calibration and maintenance, our product will enable 3D virtual histopathology that is easier, faster, and more accurate than the traditional methods.