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Jesse B Schallek, Aby Joseph, Andres Guevara-Torres; Label Free Imaging of Ganglion Cells in the Living Mouse Eye. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
The majority of neurons in the mammalian retina are transparent, providing a transmissive path for photons to reach photoreceptors. This poses a formidable challenge to image these cells without added contrast agents. Recent advances in adaptive optics scanning ophthalmoscopy (AOSLO) such as offset pinhole and split-detection have optimized contrast based on scatter, thus rendering translucent cells such as horizontal cells, photoreceptor somas and inner segments visible (Chui et al. 2012, Scoles et al. 2014, Guevara-Torres et al. 2015). We deploy these strategies to demonstrate the first label free imaging of cells within the ganglion cell layer of living mice.
Anesthetized C57BL/6J and Thy-1 YFP transgenic mice were imaged with an AOSLO in split-detector configuration described previously (Guevara-Torres et al. 2015). Endogenous cell contrast was optimized in this mode using 796 nm light. A second imaging channel simultaneously captured fluorescence (488nm excitation, 534/30 emission) from Thy-1 YFP mice that express sparsely labeled ganglion cells. Split-detector images were overlaid with simultaneously imaged fluorescent ganglion cells.
A dense organization of cells was observed with label free contrast when focused below the nerve fiber layer (figure 1). Putative ganglion cells and displaced amacrine cells were densely packed and ranged in size from 9-27 microns. In addition to identified somas, a subset of images showed sub-cellular organelles within the cells (figure 1,left). These cells came into best focus at the ganglion cell layer, but were not visible at different focal planes. Thy-1 YFP mice showed sparse labeling of ganglion cells where both somas and neurites were strongly labeled (figure 1,right) similar to Geng et al 2012. When overlaid with split-detection images at the same plane of focus, sparsely labeled cells colocalized with the somas identified in split-detection validating that the structures observed represent ganglion cell layer somas.
We have demonstrated the ability to image ganglion cells without the use of added contrast agents. Transgenic mice with fluorescent ganglion cells showed colocalized cell bodies confirming at least a subset of the imaged cells represent ganglion cells. Further refinements of the non-confocal imaging methods may be used to track cell loss associated with diseases that impact the inner retina such as glaucoma in the human population.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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