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April 2nd, 2025
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Johns Hopkins University
bioengineering
biorxiv

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

Li, Z.Open in Google Scholar•Mary, S.Open in Google Scholar•Johnson, T. V.Open in Google Scholar•Yi, J.Open in Google Scholar

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 x 2 inch optical breadboard and the whole system is constructed on a mobile 3x4 inch optical table. We show that the 3D image resolutions achieved 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 20 degree field of view, as well as higher magnification AO imaging. Interestingly, resolution and signal improvements with AO are still observed over a larger field of view. 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.

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