Dimethylacrylamide-based gels for expansion microscopy across different solvents

Expansion microscopy (ExM) enables super-resolution visualization using standard light microscopes. Recent developments have explored dimethylacrylamide (DMAA) to improve gel robustness, but compatibility with organic solvents remains a challenge. This study presents a novel hydrogel formulation based solely on DMAA that omits acrylamide (AA) and sodium acrylate (SA): This formulation achieves superior mechanical properties and is compatible with a range of solvents, including ethanol, isopropanol, and acetone. Using eosin-stained tropoelastin fibers as a model, we demonstrated that the gel preserves structural integrity and achieves tunable linear expansion factors of 2.0 ± 0.1-fold in water and ethanol, 1.9 ± 0.1-fold in isopropanol, and 1.6 ± 0.1-fold in acetone. We validated the protocol using dense murine aortic tissue, achieving a 2.0-fold expansion, which successfully resolved fine architectural details that were unresolvable in the native tissue at the same magnification. Furthermore, we found that expanding ethanol-based eosin-stained samples improves imaging contrast compared to the aqueous protocol. By enabling high-fidelity imaging of biological samples in diverse solvent environments, this DMAA-based gel system substantially broadens the applicability of ExM, opening new possibilities for integrating it with complex labeling workflows requiring organic solvents and enabling future correlative microscopy studies across multiple imaging platforms.