We demonstrate the ability to exploit in-plane resolution of ~ 15 nm and axial resolution of ~ 35 nm by combining X10 Expansion Microscopy with Airyscan 3D imaging.
Expansion Microscopy is the newest of super-resolution imaging methods which allows finer details of samples to be visualised with relatively conventional fluorescence imaging techniques by physically expanding the sample. This is achieved by embedding the samples in an acrylamide hydrogel matrix, crosslinking the fluorescent probes, enzymatically clearing the sample and then osmotically swelling the hydrogel (this paper demonstrates ~ 1000-fold volume expansion).
The enhanced three-dimensional resolution achieved by combining this with Airyscan microscopy (hence, the name Enhanced Expansion Microscopy, or EExM) is better suited than conventional implementations of localisation microscopies, particularly for imaging cell interiors and ultrastructures in cell types with 3D complexity, more so than some of the more popular super-resolution techniques. We demonstrate this by imaging cytoskeletal alpha-actinin lattices and RyR nanodomains in ventricular cardiomyocytes. We go a step further to show how this single-channel resolution can reveal dispersed RyR array structures and the altered single-channel phosphorylation patterns which coincide with the fatal heart pathology – right ventricular failure. To better-understand the functional implications, we have teamed up with Dr Michael Colman (http://physicsoftheheart.com/) to simulate the local calcium signalling events based on the experimentally-mapped RyRs.
This is the first research paper for Tom Sheard, and marks the first home-publication for the Nanoscale Microscopy Group together with a multi-lateral collaboration. Well done, everyone! This work was funded by the MRC DiMeN and Wellcome Trust Seed Award.
Access the paper at https://pubs.acs.org/doi/10.1021/acsnano.8b08742