Summary The clinical gold-standard method for interrogating tissue specimens, slide-based (2D) histopathology, is based on centuries-old technologies with many inherent limitations. Recent technological advances have demonstrated the feasibility of achieving high-throughput slide-free 3D histology of biopsy and surgical specimens. In comparison to conventional slide-based histology, nondestructive 3D histology has the potential to provide a transformative improvement in diagnostic pathology performance for a number of reasons: (1) vastly greater (>100X) sampling of tissue specimens, (2) volumetric imaging of 3D cell distributions and tissue structures that are prognostic and predictive, (3) nondestructive imaging, which allows valuable biopsy specimens to be used for downstream biomarker assessment, and (4) a simplified process with cost benefits for healthcare institutions and payers. In recent years, we have developed a technology, open-top light-sheet (OTLS) microscopy, to enable high-throughput nondestructive 3D histology of ex vivo specimens. Our first generations of OTLS microscopes and imaging protocols demonstrated the ability to reliably image a variety of optically cleared clinical tissue specimens (surgical excisions and biopsies) in a nondestructive manner that does not interfere with conventional pathology methods. Here, we propose to develop a multi-resolution hybrid OTLS microscope (Aim 1), based on a novel non-orthogonal dual-objective (NODO) architecture, which will be superior in every regard to our previous systems, including resolution (and range of resolutions), imaging depth, and compatibility with nearly all clearing/labeling protocols and sample-holder materials (insensitivity to refractive-index mismatch). Furthermore, we will develop innovative pre-imaging methods to automate and standardize the tissue-labeling and clearing process for a robust fluorescent analog of H&E staining (Aim 2). Finally, we will develop post- imaging technologies for image-guided macro-dissection of thick tissues, which we will show has the ability to significantly improve the sensitivity of genomic assays (Aim 3). Collectively, our project aims are designed to extend current 2D pathology workflows into 3D to minimize clinical-adoption barriers. A rapid translational pathway exists through a 3D-pathology-services company (Lightspeed Microscopy Inc.) that has licensed our entire 3D pathology IP portfolio. As part of this larger translational effort, clinical studies are ongoing in our labs, along with development of AI-analysis methods for clinical decision-support (i.e. prognostication and prediction of treatment response). The instrumentation platform developed in this project will directly support a number of future disease-focused clinical studies to demonstrate the value of 3D pathology for the precision treatment of diverse conditions such as kidney disease, neurodegenerative diseases, and various forms of cancer.

Relevance to public health We are developing a suite of technologies to facilitate the clinical adoption of nondestructive 3D pathology. By developing automated tissue-preparation systems, a novel multi-scale 3D microscopy device, and volumetric macro-dissection apparatus, this project will enable future studies to demonstrate the value of 3D pathology for patient diagnosis, prognostication, and personalized treatments.