The emergence of hybrid super-resolution microscopy

Abstract Super-resolution microscopy (SRM) has transformed biological imaging, yet many techniques remain constrained by limited accessibility. Expansion Microscopy (ExM) and fluorescence fluctuation-based super-resolution microscopy (FF-SRM) methods provide complementary strategies for achieving nanoscale resolution compatible with diffraction-limited microscopes. While ExM physically magnifies biological structures through isotropic hydrogel expansion, FF-SRM computationally extracts high-frequency information from temporal intensity fluctuations. When combined, these approaches enable hybrid super-resolution imaging capable of resolving structures down to single-digit nanometer. Recent developments, including ExSRRF, Ex-FEAST, Magnify-SOFI, and ONE microscopy, demonstrate how integrating ExM with FF-SRM can deliver high-fidelity nanoscale imaging in both cells and clinical tissues. These hybrid methods broaden the reach of SRM, support applications ranging from clinical pathology to multiplexed molecular profiling and offer a powerful route towards scalable and clinically relevant nanoscale analysis.