Zhang, X.; Zheng, Y.; Yang, L.; Ye, Z.; Xiao, Y.

Life continually changes its protein arrangements, yet the molecular ultradetails are covered by the short-lived deficiency of fluorophore blinking for super-resolution imaging. Herein, we proposed a crowding strategy to conserve the self-blinking events for prolonging the imaging time. We engineered sulfonamide rhodamines through atom-radii expansion (O-C-Si), rationally reversing xanthene intersection and creating stacking to enhance ring-opening energetical barriers. Our stacked rhodamines demonstrated decreased recruiting rates and extended survival lifetimes at single-molecule level, validating the decreased self-blinking kinetics from stacking strategy. Accordingly, our silicon-substituted rhodamine enabled persistent molecular localization imaging of various sub-organelle proteins to state-of-art time (0.5 h) in living cells, with versatile capabilities for three-dimensional and dual-color imaging. We envision our crowding strategy sets a new stage for prolongating super-resolution imaging through structural engineering.