Long-Term Super-Resolution Imaging through Torsional Angle Inversed Rhodamines

Cells continually change their protein arrangements, yet the molecular fine details are obscured by the short-lived deficiency of fluorophore blinking for super-resolution imaging. Herein, we propose a hybrid crowding strategy leveraging the geometric electron effect from bridging atom substitution 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 steric repulsion to enhance ring-opening energetic barriers. Our crowded rhodamines demonstrated decreased recruiting rates and extended survival lifetimes at the single-molecule level, validating the decreased self-blinking kinetics from the crowding strategy. Accordingly, our silicon-substituted rhodamine enabled persistent molecular localization imaging of various suborganelle proteins to state-of-the-art time (0.5 h) in living cells, with versatile capabilities for three-dimensional and dual-color imaging. We envision our crowding strategy sets the stage for prolonging super-resolution imaging through structural engineering.