One step 4× and 12× 3D-ExM enables robust super-resolution microscopy of nanoscale cellular structures.

Super-resolution microscopy has become an indispensable tool across diverse research fields, offering unprecedented insights into biological architectures with nanometer scale resolution. Compared with traditional nanometer-scale imaging methods such as electron microscopy, super-resolution microscopy offers several advantages, including the simultaneous labeling of multiple target biomolecules with high specificity and simpler sample preparation, making it accessible to most researchers. In this study, we introduce two optimized methods of super-resolution imaging: 4-fold and 12-fold 3D-isotropic and preserved Expansion Microscopy (4× and 12× 3D-ExM). 3D-ExM is a straightforward expansion microscopy technique featuring a single-step process, providing robust and reproducible 3D isotropic expansion for both 2D and 3D cell culture models. With standard confocal microscopy, 12× 3D-ExM achieves a lateral resolution of <30 nm, enabling the visualization of nanoscale structures, including chromosomes, kinetochores, nuclear pore complexes, and Epstein-Barr virus particles. These results demonstrate that 3D-ExM provides cost-effective and user-friendly super-resolution microscopy, making it highly suitable for a wide range of cell biology research, including studies on cellular and chromatin architectures.