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

Adaptive optics (AO) has been instrumental in ophthalmic imaging, by correcting wavefront aberrations in ocular optics and achieving diffraction-limited resolution. Current state-of-the-art AO retinal imaging systems use mirror-based optics to avoid surface reflection and chromatic aberrations, requiring a large system footprint with long focal length spherical mirrors. Here we report a compact refractive lens-based AO scanning laser ophthalmoscopy (SLO) system with simultaneous dual-channel fluorescence imaging capacity in mouse retina. The optical layout fits on a 2’x2’ optical breadboard and the whole system is constructed on a mobile 3’x4’ optical table. We show that the 3D image resolutions are significantly improved with AO correction, particularly in the z-axis (2x improvement compared to without AO, approaching diffraction-limited resolution). The optical design enables survey of a relatively large retinal area, up to 20º field of view, as well as high magnification AO imaging. Simultaneous imaging with 488nm and 561nm laser lines was evaluated using dual-channel AOSLO in CX3CR1-GFP transgenic mice expressing EGFP in microglia, undergoing rhodamine angiography. We performed dynamic high-resolution 3D imaging of microglial morphology every 5 mins for one hour and longitudinally over 3 weeks, demonstrating microglial activation and translocation over short and long time periods in an optic nerve crush model. This lens-based compact AOSLO offers a versatile and compact design for retinal fluorescence imaging in mice.