Abstract
Purpose :
The retinal pigment epithelium (RPE) is a monolayer of pigmented cells critical for sight; however, to date, no results have shown RPE structures using super-resolution microscopy in whole-mount tissue samples. We sought to examine this feasibility issue using our single-molecule localization microscopy (SMLM) technology to image RPE tissue at the molecular scale.
Methods :
Adult albino C57BL/6 mice were used to acquire RPE tissue samples, isolating the RPE monolayer from the retina before being cut into four sections to increase the number of samples for imaging. After dissection, the RPE tissue was fixed in PBS containing 4% polyformaldehyde (PFA) and then permeabilized via 1% Triton x-100 in PBS for 30 minutes. From here, samples were treated to different staining protocols optimized for different antibodies to image the following cellular structures: ZO-1 tight junction proteins, f-actin filaments, beta-tubulin, mitochondria, and histone-H4 within the nucleus. After staining, RPE samples were then flat mounted to a microscope slide with the apical side of the tissue facing the covering glass upwards. Samples were imaged using our custom-built SMLM system, recording 10,000 frames at an exposure time of 10ms. We used ThunderSTORM, a known imageJ application used for processing super-resolution images, to reconstruct our super-resolution images, reporting successful imaging of all structures.
Results :
We successfully imaged all five reported structures, as shown in Figure 1. These images showcase marked improvement in resolution compared to epifluorescence imaging of the same structures. An important feature revealed by SMLM was the separation between tight-junction proteins and f-actin filaments, successfully demonstrating how f-actin filaments form around the space where tight junctions connect cells.
Conclusions :
Our results demonstrate the first successful imaging of RPE tissue using SMLM. We imaged multiple intracell structures, including mitochondria, histones, and ZO-1 tight junctions. This work lays the foundation for further investigations of the RPE using SMLM toward AMD and other blinding diseases.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.