Kate Grieve1, Olivier Thouvenin2, Abhishek Sengupta3, Vincent M Borderie1, Michel Paques1. 1. Quinze Vingts National Ophthalmology Hospital, Paris, France 2Vision Institute, UPMC Université Paris 06, Paris, France. 2. Institut Langevin, Paris, France. 3. Vision Institute, UPMC Université Paris 06, Paris, France.
Abstract
PURPOSE: To interpret full-field optical coherence tomography (FFOCT) images of ex vivo retina. METHODS: Flatmounted retinas of human, primate, pig, sheep, rat, and mouse were imaged using FFOCT. To identify retinal ganglion and amacrine cells, fixed samples immunolabeled against Tuj1 and Brn3a or live samples labeled in vitro with green fluorescent protein (GFP) were analyzed by combining FFOCT, fluorescence confocal microscopy (FCM), and fluorescence-FFOCT. To investigate postmortem tissue changes, time series were acquired over 48 hours and on fresh versus fixed tissue. RESULTS: With FFOCT, cell types and features such as nerve fiber bundles and RGC somas were resolved without use of contrast agents at 1-μm xyz resolution. Cell somas in the ganglion cell layer (GCL) in large mammals appeared predominantly bright with dark contours, while in rodents, GCL somas appeared dark with bright contours. RGC axon to soma junctions could be traced in the three-dimensional (3D) image stacks. Time series revealed undulation of retinal tissue samples over 48 hours, though no degradation of individual cells was detected, while paraformaldehyde fixation caused increased scattering and shrinkage. CONCLUSIONS: Full-field OCT reveals micrometric morphologic detail in the retina without the use of contrast agents. We observed interspecies differences in optical properties of GCL somas. Fixation significantly alters retinal transparency hence reducing the visibility of microscopic features.
PURPOSE: To interpret full-field optical coherence tomography (FFOCT) images of ex vivo retina. METHODS: Flatmounted retinas of human, primate, pig, sheep, rat, and mouse were imaged using FFOCT. To identify retinal ganglion and amacrine cells, fixed samples immunolabeled against Tuj1 and Brn3a or live samples labeled in vitro with green fluorescent protein (GFP) were analyzed by combining FFOCT, fluorescence confocal microscopy (FCM), and fluorescence-FFOCT. To investigate postmortem tissue changes, time series were acquired over 48 hours and on fresh versus fixed tissue. RESULTS: With FFOCT, cell types and features such as nerve fiber bundles and RGC somas were resolved without use of contrast agents at 1-μm xyz resolution. Cell somas in the ganglion cell layer (GCL) in large mammals appeared predominantly bright with dark contours, while in rodents, GCL somas appeared dark with bright contours. RGC axon to soma junctions could be traced in the three-dimensional (3D) image stacks. Time series revealed undulation of retinal tissue samples over 48 hours, though no degradation of individual cells was detected, while paraformaldehyde fixation caused increased scattering and shrinkage. CONCLUSIONS: Full-field OCT reveals micrometric morphologic detail in the retina without the use of contrast agents. We observed interspecies differences in optical properties of GCL somas. Fixation significantly alters retinal transparency hence reducing the visibility of microscopic features.
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