Speaker
Description
Direct stochastic optical reconstruction microscopy (dSTORM) overcomes the diffraction limit of light, enabling nanoscale visualization (~10–20 nm) of subcellular structures. Yet, achieving true ultrastructural resolution with dSTORM alone remains challenging. Recent advances combining dSTORM with expansion microscopy (ExM)—which physically enlarges specimens via a swellable polymer hydrogel—provide a route to imaging below 10 nm, with effective resolution largely determined by the expansion factor. Most previous studies have focused on ~4-fold expansion, as imaging highly expanded specimens is technically demanding. Here, we present a practical imaging strategy for dSTORM of expanded mammalian centrioles, pushing light microscopy toward molecular-level resolution and enabling unprecedented mapping of protein organization within intact cells. Using this workflow, we resolve ultrastructural higher-order protein complexes in centrioles. Furthermore, we introduce buffer-exchanged STORM (beSTORM), a method that distinguishes individual molecules independently of spectral properties by exploiting blinking behaviors modulated by buffer conditions. Simple buffer exchanges enable spectrum-unlimited multi-target dSTORM with minimal crosstalk. Integration with ExM further extends its capability to resolve multiple proteins at the molecular level within a single emission channel, free from chromatic aberration. Together, beSTORM provides a versatile and compatible platform for integration with other imaging techniques, offering a powerful approach for highly multiplexed nanoscopy and nanoscale exploration of complex biological systems.
