ONE microscopy.

The introduction of expansion microscopy (ExM), a decade ago, marked a shift in super-resolution imaging, by physically separating fluorophores to bypass the diffraction limit. Numerous ExM developments have extended the method's reach since, yet molecular-scale resolution remained inaccessible. We recently developed one-step nanoscale ExM, which combines ExM with fluctuation-based super-resolution analysis to enable the direct visualization of individual protein shapes, using conventional fluorescence microscopes, a capability that was previously limited to cryo-electron microscopy and averaging-based techniques. Here we provide detailed procedures for gel embedding, labeling, expansion, image acquisition and data analysis. We also introduce a stable, user-friendly software package for efficient fluctuation analysis. Although one-step nanoscale ExM is broadly applicable to a range of samples, including purified proteins, cells and tissues, its most distinctive contribution lies in making single-protein shape analysis accessible and reproducible. Overall, we provide a practical framework for protein imaging on conventional equipment.