Description
Understanding cellular functions in all their complexity can greatly benefit from spatially mapping of the diverse molecules within a cell using multi-target single-molecule localization microscopy (SMLM). Current developments primarily rely on fluorescent spectrum, lifetime, or cyclic staining, necessitating complex optical configurations, fluorophore identifications, or labeling designs. Consequently, there remains a need for a simple imaging platform. Here, we introduce buffer-exchanged STORM (beSTORM), a method that distinguishes between single molecules regardless of their spectral properties by leveraging their responsive blinking behaviors influenced by buffer conditions. Through simple buffer exchanges, beSTORM achieves spectrum-unlimited dual or four-target SMLM imaging with minimal crosstalk (<1%). Integration with expansion microscopy (ExM) extends its capability to resolve up to six proteins at the molecular level within a single emission color, free from chromatic aberration. beSTORM's simplicity and compatibility offer a versatile platform for seamless integration with other techniques, promising advancements in highly multiplexed nanoscopy for exploring complex biological systems with nanoscale precision.
