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Andres Guevara-Torres, David R Williams, Jesse B Schallek; . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4371. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Despite the micron-level resolution of adaptive optics scanning light ophthalmoscopy (AOSLO), many cell classes in the mammalian retina are challenging to image because they provide weak endogenous optical contrast. Here we use split-detector imaging (Scoles et al. , 2014) to enhance the contrast of cell boundaries, rendering them visible in high-resolution imaging.
Anesthetized C57BL/6 mice were imaged with an AOSLO using near infrared light. The detection arm of the AOSLO was modified by placing a knife edge at the retinal image plane to direct the left and right half of the point spread function into two photomultiplier tubes (PMTs). Simultaneously captured signals from each PMT were subtracted to generate a differential contrast image. AO was used for defocus control and the vascular layers were used to confirm axial position.
We resolved a variety of retinal cells that are invisible with conventional confocal AOSLO without contrast agents. We observed a monolayer of photoreceptor distal processes that showed a Yellot’s ring peak at 23 cycles/degree (figure 1a). This corresponds to a photoreceptor density of 477,000 cells/mm2, consistent with previous reports of photoreceptor density in the mouse. When focusing slightly more vitread, we could distinguish a multilayer arrangement of photoreceptor somata within the outer nuclear layer (figure 1b). Above this, at the boundary of the outer plexiform layer, we resolved a sparse mosaic of cells (figure 1c) with a diameter of 11 ± 2 μm (mean ± SD). This mosaic of cells counted by 4 individuals showed a density of 1250 ± 270 cells/mm2 (mean ± SD, 0.25 mm2 analyzed between 10-25 degrees from the optic disc), which is consistent with previous histological reports of horizontal cell density in mouse and more than an order of magnitude different than any other neural cell class in the retina.
This approach reveals a variety of cell structures that are hidden when imaged by confocal systems. The capability to image horizontal cells and the photoreceptor somata enables quantification of these cell classes over the course of disease. Optimizing this contrast method in the mouse may instruct best strategies for imaging transparent cells in the human retina.
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