Fluorescence microscopy is a key technique for visualizing cellular organization, but its resolution is limited to ~200 nm by diffraction, restricting access to nanoscale structures. Super-resolution methods such as Single Molecule Localization Microscopy (SMLM) overcome this limit, achieving nanometer precision, but require specialized equipment and expertise, limiting their accessibility.

Expansion microscopy provides an alternative approach by physically enlarging biological samples, enabling nanoscale resolution using standard microscopes. Ultrastructure Expansion Microscopy (U-ExM) introduced a robust 4-fold expansion method, enabling discoveries such as the conserved conoid in Plasmodium and detailed mapping of centriole assembly. However, existing expansion protocols still fall short of SMLM-level precision.

Here, we developed iterative U-ExM (iU-ExM), achieving 15–26-fold expansion in cells and tissues. This enables visualization of nanoscale architectures, including nuclear pore complex symmetry and the structure of the Toxoplasma gondii conoid, using widely accessible imaging tools.