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 pKcycling 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 (pKcycling < 6) and four sulfonamide rhodamines with three exhibited exceptional larger pKcycling 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.

Automated summary

The development of self-blinking rhodamines has advanced live-cell single-molecule localization microscopy. Despite the criterion of a pKcycling of <6 for self-blinking, some rhodamines fail for super resolution reconstruction. To resolve this controversial issue, two classic rhodamines (pKcycling <6) and four sulfonamide rhodamines were constructed. A kinetic study revealed slow equilibrium rates and limited switch numbers resulting in the reconstruction failure of some rhodamines. By abstracting a recruiting rate from the kinetic disparity, the natural switching frequency of spirocycling equilibrium was revealed, providing a new parameter to explain the imaging failure and determine the propensity of self-blinking from a kinetic perspective. Based on this parameter, sulfonamide rhodamines enabled live-cell super-resolution imaging of various organelles through Halo-tag technology.