Abstract
Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.
Highlights
Over the last decade, the development of adaptive optics (AO) instruments has made it possible to routinely image the human retina in vivo at cellular resolution [1]
Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle
We describe in detail a reflective afocal Adaptive optics scanning laser ophthalmoscopy (AO-SLO) retinal imaging system designed for high resolution retinal imaging in mice
Summary
The development of adaptive optics (AO) instruments has made it possible to routinely image the human retina in vivo at cellular resolution [1]. AO imaging of the mouse retina has been delayed by the challenge of designing a system for an eye ten-fold smaller than that of the human, and by the availability of highly developed ex vivo histochemical retinal imaging methods. Such methods cannot report the properties and functions of living tissue, and are relatively expensive inasmuch as they require cohorts of experimental and control animals, often for each of a number of time points in a study. The mouse eye has an AO-corrected numerical aperture of ~0.5, exceeding that of the human eye by ~2.5-fold [4, 11], affording exquisite submicron resolution
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