Expansion Microscopy: High-Resolution Fluorescent Imaging with a Conventional Microscope

Expansion microscopy (ExM) is an innovative super-resolution fluorescence microscopy technique that physically enlarges biological specimens, enabling imaging beyond the diffraction limit using conventional systems such as confocal microscopes. By embedding samples in a swellable hydrogel matrix, ExM allows isotropic expansion of the sample, enhancing spatial resolution without requiring specialized nanoscopy equipment. Advanced variants, such as Ultrastructure Expansion Microscopy (U-ExM), preserve fine cellular details, including organelles and macromolecular assemblies. The observation of specific cellular structures or organelles depends significantly on the method of fixation applied to the sample, notably through the use of aldehyde-based chemical crosslinkers and protein precipitation with cold methanol. Recently, integrating cryo-fixation -- a gold-standard fixation technique -- with the U-ExM method has enabled nanoscale observations of a wide range of biological samples, from cultured mammalian cells to whole organisms, in their near-native state. Expansion microscopy is a powerful tool for studying biological structures across scales, from isolated organelles to entire tissues. When combined with cryo-fixation, it provides unprecedented insights into cellular architecture and dynamic processes while minimizing artifacts associated with traditional fixation methods. These developments position ExM as a versatile framework for high-resolution, near-native biological imaging. The present study outlines a detailed protocol for cryo-fixation coupled with expansion microscopy of samples from various origins. This protocol highlights critical steps, including sample fixation, polymer formation, expansion, immunostaining, and imaging, providing the necessary guidance to implement this method in any laboratory.