PROJECT SUMMARY/ABSTRACT Glaucoma, a leading cause of blindness worldwide, is clinically treated by lowering intraocular pressure (IOP) which can delay or prevent vision loss. However, first-line standard-of-care treatments for primary open-angle glaucoma (POAG), the most common form, do not target the conventional outflow pathway (COP) where IOP is tightly regulated and pathology causes elevated IOP. Moreover, much of our knowledge about COP anatomy was derived from histological samples that are subject to artifacts from long-term glaucoma therapy that alters physiologic outflow. Unfortunately, these samples represent only a snapshot in time and space of the dynamic, spatially heterogeneous COP tissues (causing a “restricted field of view” problem) that is particularly worrisome because spatial heterogeneity is worse in glaucoma patients, and is thought to contribute to ocular hypertension. In our previous highly-productive funding period, we pioneered techniques using near-infrared (NIR) optical coherence tomography (OCT) to image the COP in living mice, known to be anatomically, physiologically, and pharmacologically very similar to the human COP. Further, we developed AI-driven software to automatically segment OCT images, observing functional changes in COP tissues in response to drugs, disease, and age. We then used these images to estimate stiffness/fibrosis of the trabecular meshwork (TM) in vivo. While we made tremendous progress, we suffered from the “restricted field of view” problem and less-than-desired spatial resolution. In the upcoming funding period, we will overcome these obstacles by using two exciting, cutting-edge imaging technologies: (i) robotically-positioned (r) visible light (vis) OCT (denoted “r-vis-OCT”), and (ii) optical coherence refraction tomography (OCRT), enabling ultra-high-resolution visualization and segmentation of the 3D, spatially heterogeneous architecture of COP tissues around the eye’s circumference. This integrated, whole- system view will allow us to understand the inherently spatially distributed physiology of IOP (dys)regulation. Our overarching hypothesis is that r-vis-OCT/OCRT can be used to accurately quantify spatial and temporal changes in conventional outflow function with age, disease, and drugs. To test this hypothesis, we propose 3 specific aims (SAs): Aim 1: Use r-vis-OCT in living mice to monitor dynamic, spatially-heterogeneous COP anatomic and functional responses to drugs and age. Aim 2: Use r-vis-OCT in mice to monitor dynamic, spatially- heterogeneous COP anatomy and function in 2 relevant disease models. Aim 3: Develop r-vis-OCRT for ultra- high-resolution monitoring of COP behavior in living mice. Successful completion of our aims will provide fundamental information about the dynamic processes that maintain conventional outflow in the face of transient challenges such as pigment showers, IOP spikes or changes in preferential flow pathways. Further, we will learn about the homeostat...