Turn-on Rate Determines the Blinking Propensity of Rhodamine Fluorophores for Super-Resolution Imaging

Live-cell single-molecule localization microscopy has advanced with the development of self-blinking rhodamines. A pKcycling of <6 is recognized as the criterion for self-blinking, yet partial rhodamines matching the standard fail for super-resolution reconstruction. To resolve this controversy, we constructed two typical self-blinking rhodamines (pKcycling = 5.67, 5.35) and a tetramethylsulfonamide rhodamine with unfit pKcycling characteristic (7.00). Kinetic study uncovered slow equilibrium rates and limited blink numbers resulted in the reconstruction failure of partial rhodamines. From the kinetic disparity, a turn-on rate was abstracted to reveal the natural blinking frequency. The new parameter independent from applying laser satisfactorily explained the imaging failure, efficacious for determining the propensity of self-blinking from a kinetic perspective. Following the prediction from this parameter, the tetramethylsulfonamide rhodamine enabled live-cell super-resolution imaging of various organelles through Halo-tag technology. It is convinced that the turn-on rate would be a practical indicator of self-blinking and imaging performance.