Tetrahedral expansion gel enables superior preservation of molecular structure for super-resolution imaging

Abstract

Expansion microscopy (ExM) can increase the resolution of sub-diffraction microscopy methods such as stochastic optical reconstruction microscopy (STORM). However, it remains unknown to what extent ExM preserves structural features on length scales below 30 nm, in particular when using standard polyacrylamide (PA) based gels assembled through free-radical chemistry. Here we directly measure structural preservation in PA gels, by using STORM to image DNA origami reference samples with precisely spaced labeling sites. We find that embedding in PA gels resulted in random displacements of labeled sites with a standard deviation of ~16 nm. In contrast, we find that embedding in tetra-gel, a gel that does not depend on free-radical chain-growth polymerization, preserves labeled sites with a standard deviation of less than 5 nm. By combining tetra-gel expansion microscopy with STORM, we are able to resolve 11-nm structural features without the loss in accuracy seen with PA gels. Our findings thus describe a strategy for using ExM to improve the resolution of super-resolution microscopy without incurring the loss in accuracy associated with PA gel embedding.