High-fold Homogeneous Expansion Microscopy Reveals Ultrastructural Centrioles

Super-resolution microscopy has transformed cellular imaging by enabling nanoscopic visualization of biomolecules. Expansion microscopy (ExM), which physically enlarges biological specimens, offers a complementary, optics-independent route to super-resolution. However, existing single-round high-fold ExM approaches have rarely been validated for their ability to preserve ultrastructural uniformity. Here, we present high-fold homogeneous expansion microscopy (hiHomoExM), a single-round ExM technique that achieves uniform ∼8-9x expansion while preserving cellular ultrastructure across diverse targets, including centrioles, nuclear pore complexes, and other organelles. hiHomoExM streamlines sample preparation and supports postexpansion labeling, allowing for both high labeling density and structural preservation. Furthermore, we extend the capability of hiHomoExM by integrating it into an iterative expansion framework to achieve even higher spatial resolution. Notably, coupling hiHomoEx with single-molecule localization microscopy (hiHomoEx-dSTORM) enables the resolution of previously elusive centriole ultrastructures, including the organization of CEP44, the microtubule-associated pattern of CCDC77, and the canonical 9-fold symmetry of SAS6. Together, hiHomoExM and hiHomoEx-dSTORM provide a robust and accessible platform for ∼2 nm-resolution characterization, bridging physical and optical super-resolution to advance cellular structural analysis.