Abstract
Purpose :
To develop a high-throughput method for retinal whole-mount imaging and analysis that would allow for capture of images throughout the entire depth of the retina at a resolution that accommodates tracing of retinal ganglion cell dendrites, identification of individual filled cells, and determination of retinal orientation based on a marker protein.
Methods :
Mouse retinal whole-mounts that were used for intracellular retinal ganglion cell electrophysiology were fixed in 2% paraformaldehyde at 4oC for 24 hrs. Retinas were immunolabeled for nonphosphorylated neurofilament H (SMI-32, 1:1,000; BioLegend), choline acetyltransferase (ChAT, 1:500; Millipore), and S-cone opsin (1:500; Millipore). Retinas were first blocked in 5% normal donkey serum for 2 hours, incubated in primary antibodies for 3 days at 4oC, incubated with appropriate secondary antibodies (1:200; Jackson ImmunoResearch Laboratories, Inc.) and coverslipped with Fluoromount G (Southern Biotech, Birmingham, AL). A Nikon STORM super-resolution fluorescent microscope was used to obtain micrographs of whole retinal flat-mounts en montage via the Vanderbilt University Medical Center Cell Imaging Shared Resource. Images were obtained every 2.5 microns over a total z-stack depth of 50 microns to ensure RGC dendritic arbors and S-cone opsin (used for retinal orientation) were captured. Image z-stacks were combined to create an all-in-focus image using the Extended Depth of Focus (EDF) plug-in in Nikon Elements.
Results :
Average time to image a mouse whole-mount retina using the STORM microscope was 50.9 ± 2.7 minutes. Image processing (EDF and montage generation) took an additional 20 to 30 minutes. The same imaging on a FV-1000 inverted confocal microscope takes 56.0 ± 6.9 minutes. Image processing for the confocal files takes an additional hour as montages are generated manually. Images from the STORM microscope can be used to trace retinal ganglion cell dendrites by hand or using a user-defined macro.
Conclusions :
The benefits of STORM imaging for retinal whole-mounts include shorter image-capture and processing times, image processing steps that are automated, and higher resolution images that allow for outcome measurements such as dendritic length, dendritic area, distance from optic disc.
This abstract was presented at the 2019 ARVO Imaging in the Eye Conference, held in Vancouver, Canada, April 26-27, 2019.