Protocol for 3D sub-10-nm multicolor expansion STED nanoscopy using small-molecule organic probes

Expansion microscopy (ExM) physically enlarges specimens, enhancing resolution. Here, we present a protocol for integrating small-molecule organic probes (SMOPs) and adaptive optics (AO) into expansion stimulated emission depletion (ExSTED) microscopy to achieve sub-10-nm resolution at depths of 10-50 μm in biological specimens. We describe steps for probe design and synthesis, cell labeling, multicolor staining, and cell expansion. We detail procedures for AO-corrected stimulated emission depletion (STED) imaging workflow. This protocol applies to high-resolution 3D imaging of cellular and tissue structures. For complete details on the use and execution of this protocol, please refer to Han et al.¹ and Tu et al.².