Compact lens-based dual-channel adaptive optics scanning laser ophthalmoscopy for in-vivo three-dimensional retinal imaging in mice

Abstract

Adaptive optics (AO) enables cellular-resolution retinal imaging, yet mirror-based AOSLO systems are constrained by off-axis aberrations that restrict high-quality imaging to narrow fields of view, limiting in vivo studies of dynamic, large-scale retinal processes. Clinical translation of regenerative cell therapy to neurologic disease is hampered by attrition of donor neurons following transplantation. We hypothesized that early innate neuroinflammatory responses to retinal ganglion cell (RGC) transplantation underlie early death of donor cells and that next-generation imaging technologies would provide evidence for microglial attack of grafted neurons. We present a compact refractive lens-based AOSLO system that achieves two-color fluorescence imaging across up to a 16{degrees} field of view in mouse retina. Dual-wavelength excitation (488 nm and 552 nm) enables visualization of two fluorescence labels, while AO correction improves axial resolution and depth fidelity, allowing robust separation of structures through anatomical layers in retina. Using this platform, we performed 3D time-lapse imaging of microglia and longitudinal imaging in an optic nerve crush model, revealing layer-dependent differences in microglial motility, early activation signatures, and large-scale redistribution longitudinally. The system enabled widefield visualization of injury-associated vascular changes and spatial coupling between microglia and vasculature. Finally, depth-resolved two-color imaging captured immune responses to intravitreally transplanted RGCs, including host-cell recruitment, rapid neurite retraction following local immune-cell contact, and microglial phagocytosis of donor RGCs. Together, these results demonstrate that refractive AOSLO enables in vivo observations of microvascular organization, neuroimmune dynamics, injury responses, and transplanted-cell behavior with spatiotemporal resolution. Our data also suggests that modulation of microglial reactivity may improve outcomes of RGC transplantation. Significance StatementWide-field, depth-resolved imaging is essential for understanding how different cell types interact across a large retinal area, yet existing AOSLO systems retain limited imaging fields due to a conventional optical design using reflective spherical mirrors. Our refractive large-FOV AOSLO overcomes this limitation, enabling simultaneous two-color, diffraction-limited 3D imaging across an up to 16{degrees} field in vivo. This platform reveals previously inaccessible biological phenomena--including layer-specific microglial dynamics after optic nerve injury, microvascular remodeling, and rapid microglial rejection of transplanted RGCs--providing critical insight into neuroimmune behavior and retinal repair mechanisms at single-cell and subcellular resolution.