Search

Single‐molecule localization microscopy (SMLM) has found extensive applications in various fields of biology and chemistry. As a vital component of SMLM, fluorophores play an essential role in obtaining super‐resolution fluorescence images. Recent research on spontaneously blinking fluorophores has greatly simplified the experimental setups and extended the imaging duration of SMLM. To support this crucial development, this review provides a comprehensive overview of the development of spontaneously blinking rhodamines from 2014 to 2023, as well as the key mechanistic aspects of intramolecular spirocyclization reactions. We hope that by offering insightful design guidelines, this review will contribute to accelerating the advancement of super‐resolution imaging technologies. [ABSTRACT FROM AUTHOR]

[Display omitted] • Spirolactam based pH probes (RLH A-C) were built with Rhodamine 6G, Rhodamine B and Rhodamine 101 to discuss the tuning effect of rhodamine fluorophore. • The p K a values and observed rate constant k obs of RLH A-C were determined and found to negatively correlated with the calculated Gibbs free energy differences ΔG C-O and ΔG TS respectively. • The p K a values and observed rate constant k obs of RLH A-C determined the potential application in intracellular pH detection. Ring-opening reaction of rhodamine spirolactam has been widely applied to construct fluorescent probes. The fluorescence properties of the probe were finely tuned for specific purpose through changing the rhodamine fluorophore. However, the influence on response range and kinetic parameters of the probe during the change has been seldom discussed. Herein, we took pH detection as an example and constructed spirolactam based probes (RLH A-C) with Rhodamine 6G, Rhodamine B and Rhodamine 101. The p K a values and observed rate constant k obs of RLH A-C were determined and found to negatively correlated with the calculated Gibbs free energy differences ΔG C-O and ΔG TS respectively. The potential applications of RLH A-C in imaging acidic microenvironment were also investigated in cells. We expect the comparison of rhodamine fluorophores will facilitate the quantitative optimization of rhodamine spirolactam based fluorescent probes. [ABSTRACT FROM AUTHOR]