Deformation Mapping and Correction of Expansion Micrographs

Expansion microscopy, in which fixed biological samples are physically enlarged through their crosslinking to and subsequent expansion of a superabsorbent hydrogel, enables super-resolution imaging using standard microscopes. Although the process is generally assumed to be spatially isotropic, preserving relative intracellular geometry, we find that intracellular structures are often distorted in complex and unpredictable ways during expansion. To address this, we developed a localization microscopy–based protocol that quantifies and corrects expansion-induced intracellular deformations using biocompatible fiducial markers as pre- and post-expansion landmarks. We use this method to map deformation fields across different cell types and show that these distortions can preclude reliable measurement of ultrastructure using uncorrected expansion microscopy. Finally, we analyze how variations in hydrogel formulation and crosslinking chemistry influence the degree and character of deformation. Together, these results establish the first quantitative, three-dimensional correction framework for expansion microscopy, enabling accurate structural measurements across diverse biological contexts.