# Pushing Optical Resolution to the Few-Nanometer Scale via dSTORM Imaging of Expanded Specimen–Gel Composites

## TL;DR

This paper introduces a new method combining expansion microscopy and dSTORM to achieve ultra-high optical resolution imaging of cellular structures.

## Contribution

The novel plTREx-dSTORM workflow enables ten-fold expansion of specimens for sub-10 nm resolution imaging.

## Key findings

- plTREx-dSTORM improves mechanical stability and fluorescence signal density in expanded specimens.
- The workflow prevents gel shrinkage during dSTORM imaging.
- The method bridges the resolution gap between optical and electron microscopy.

## Abstract

Direct stochastic optical reconstruction microscopy (dSTORM) circumvents the diffraction limit of light, emerging as a powerful superresolution technique for visualizing subcellular structures with a nanoscale resolution of 10–20 nm. Yet achieving ultrastructural resolution using dSTORM alone remains challenging, despite its advantage of requiring only minimal modifications to the imaging setup and sample preparation compared to conventional fluorescence microscopy. A recent advancement that integrates expansion microscopy (ExM), which embeds specimens in a swellable polymer gel, with dSTORM holds promise for attaining imaging resolutions below 10 nm. The combined resolution, however, is governed by the expansion factor of samples, and prior studies have primarily focused on integrations involving approximately 4-fold gel expansion, as dSTORM imaging of high-fold-expanded specimens is still technically demanding. Here, we propose a pragmatic expansion strategy—post-labeling ten-fold robust expansion microscopy (plTREx)—and outline a workflow to facilitate its compatibility with dSTORM, collectively termed plTREx-dSTORM. Specifically, this workflow enhances the mechanical stability of the expansion hydrogel and improves fluorescence signal density across both widefield and dSTORM imaging platforms. Furthermore, we optimize the re-embedding protocol to integrate hydrogel expansion with dSTORM while preventing gel shrinkage. Together, plTREx-dSTORM enables highly refined imaging capable of ultrastructural interpretation of cellular proteins, effectively bridging the resolution gap between electron microscopy and optical microscopy.