Years-long stability of X10 expansion microscopy hydrogels.

Expansion microscopy (ExM) is developing rapidly to explore biological structures, both towards nanoscale imaging and the wider-scale analysis of cells and tissues. ExM is based on embedding samples in swellable hydrogels, which then expand to enable superior microscopy performance with conventional microscopes. However, imaging precision is limited by the stability of the hydrogels, which are typically regarded as less robust than conventional samples. Here, we show that hydrogels based on N, N-dimethylacrylamide (DMAA) remain stable for several years, without a perceivable loss of structure. No special measures of stabilization are necessary for the gel matrix, but post-fixation is required to maintain the integrity of antibody labeling, when post-expansion labeling procedures are employed. Otherwise, antibodies slowly lose epitope binding over years, corrupting both signal intensity and resolution. We conclude that ExM hydrogels are substantially more robust than previously envisioned, enhancing signal intensity and enabling years-long performance, with minimal care. Moreover, this paves the avenue to re-imaging, inter-lab sample exchange, and retrospective analyses, which could be employed in medical applications ranging from diagnostic validation and biomarker discovery to comparative studies across time points or cohorts. This approach extends ExM from a single-use technique to a platform compatible with sustained experimental workflows.