Abstract 17025: Investigating Retinal Neurovascular Interactions via Multi-View Light-Sheet Microscopy

Introduction: Vascular retinopathy is a leading cause of preventable blindness in diabetes and retinopathy of prematurity. While most studies focus on individual neural or vascular structures, there remains a knowledge gap in understanding the retinal neurovascular interactions after injury and during repair (Fig. 1A). We seek to demonstrate 3-D neurovascular interactions with a multi-view light-sheet imaging system.

Methods and Results: Intact 3-D retinas from 2-month-old mice were fixed and permeabilized followed by the optical clearance (CytoVista). Retinal vessels were stained with biotinylated isolectin B4 for microvasculature and Alexa Fluor 488-conjugated streptavidin (Fig. 1B-C) for labeling, while bipolar cells were stained with anti-PKCα (rabbit host) for neurons and Alexa Fluor Plus 555-conjugated goat anti-rabbit IgG for labeling. Refractive index matching was performed to 1.53. Multi-view light-sheet imaging was performed at 5 different angles followed by 3-D registration and reconstruction. The point spread function (PSF) demonstrates a spatial resolution of less than 5 μm (Fig. 1B, inlet). This enabled 3-D co-registration of the vasculature (Fig. 1D, in green) in relation to the bipolar cells (Fig. 1D, in red) for visualization.

Conclusion: We revealed the 3-D neuro-microvascular network providing both deep tissue penetration and high spatial resolution for investigating hyperoxia and diabetes-associated vascular retinopathy.