High resolution of primate and human photoreceptors' structure using Expansion microscopy.

Abstract

Purpose : Physiology of photoreceptors (PR) is closely related to their anatomy, as most dysfunctions are related to changes in PR morphology and in the distribution of molecular actors. For example, different morphological stages of degeneration have been described for cone. Therefore, fine assessment of such changes is needed to understand PR physiopathology, interpret in-vivo imaging or design new clinical strategies.
We here illustrated how Expansion microscopy (Chen et al. 2015) can provide super-resolution of PR anatomy and proteins distribution in the primate and human retina.

Methods : Perfused primate and post-fixed human retinas were immunolabelled and embedded in an acrylamide gel. Anchoring of fluorescent probes to the gel then allowed sample digestion while preserving fluorescent probes and their 3D arrangement. Changing the immersion media then caused sample's expansion with the associated gain in resolution. The sample was imaged using a confocal microscope. Examinations were performed on samples generated with this new technique and compared to classic preparations. 3D reconstructions were achieved with software analysis (Imaris).

Results : Expansion microscopy applied to the primate and human retina produced a 4.5 gain in magnification of the samples. We examined the distribution of proteins involved in phototransduction, cytoskeleton and its associated proteins, especially Usher proteins in both cones and rods. When Usher1 proteins appeared in the classic approach as a continuous ring around the outer segment, expansion microscopy enabled us to distinguish individual protocadherin15 bands between actin-containing calyceal processes and the nascent outer segment discs in cones, and the junction between periciliary membrane and outer segment in rods. Similarly, Usher2 proteins were observed as 6/7 spots between the cone cilium and the periciliary membrane with the same resolution as immunogold electron microscopy but with a 3D image in both cones and rods. Examination of postmortem human retinas by expansion microscopy allowed 3D reconstruction of pathological cones.

Conclusions : Expansion microscopy has allowed us to create fine new 3D models of the cells both in macaque and human retina, which will prove invaluable to compare to aging and pathological PR. This technique provides unprecedented resolution of retinal cell morphology and microanatomy to investigate cell function and physiopathology.

This is a 2020 ARVO Annual Meeting abstract.