Recruiting 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 pK_cycling of <6 is recognized as the criterion for self-blinking, yet a few rhodamines matching the standard fail for super-resolution reconstruction. To resolve this controversy, we constructed two classic rhodamines (pK_cycling < 6) and four sulfonamide rhodamines with three exhibited exceptional larger pK_cycling characteristics (6.91-7.34). A kinetic study uncovered slow equilibrium rates, and limited switch numbers resulted in the reconstruction failure of some rhodamines. From the kinetic disparity, a recruiting rate was first abstracted to reveal the natural switching frequency of spirocycling equilibrium. The new parameter independent from applying a 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 sulfonamide rhodamines enabled live-cell super-resolution imaging of various organelles through Halo-tag technology. It is determined that the recruiting rate would be a practical indicator of self-blinking and imaging performance.