Multiplexed Nanoscopy\nvia Buffer Exchange

Understanding cellular\nfunctions, particularly in their\nintricate\ncomplexity, can greatly benefit from the spatial mapping of diverse\nmolecules through multitarget single-molecule localization microscopy\n(SMLM). Existing methodologies, primarily restricting the encoding\ndimensions to color and lifetime or requiring cyclic staining, often\ninvolve broad chromatic detection, specialized optical configurations,\nor sophisticated labeling techniques. Here, we propose a simple approach\ncalled buffer-exchange stochastic optical reconstruction microscopy\n(beSTORM), which introduces an additional dimension to differentiate\nbetween single molecules irrespective of their spectral properties.\nThis method leverages the distinguishable photoblinking responses\nto distinct buffer conditions, offering a straightforward yet effective\nmeans of fluorophore discrimination. Through buffer exchanges, beSTORM\nachieves multitarget SMLM imaging with minimal crosstalk. Direct integration\nwith expansion microscopy (ExM) demonstrates its capability to resolve\nup to six proteins at the molecular level within a single emission\ncolor without chromatic aberration. Overall, beSTORM presents a highly\ncompatible imaging platform, promising significant advancements in\nhighly multiplexed nanoscopy for exploring multiple targets in biological\nsystems with nanoscale precision.