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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]

Genetically encoded light-up RNA aptamers have become powerful tools for visualizing RNAs in cells. However, fluorogenic dye/aptamer pairs used for super-resolution RNA imaging are still rare. Herein, we reported an environment-sensitive dye Nap which can light up its target RNA aptamer NapRA for aptamer-based RNA imaging with confocal and structured illumination microscopy (SIM). Nap was designed to be amphiphilic to assemble into nano-particles in water associated with the quenched fluorescence by the aggregation effects. NapRA was obtained from SELEX technology with a 12-round Nap -based selection and has a high binding affinity to Nap of K d = 3.142 nM. The super affinity disintegrated the dye aggregate and resulted in a turn-on fluorescent signal. The light-up feature combined with high photostability made Nap / NapRA a useful system capable of super-resolution imaging of the expression of RNA in bacteria. This work represents the first application of dye aggregation-disaggregation for aptamer-based RNA imaging. [Display omitted] • A novel fluorogenic dye-aptamer pair was developed for RNA imaging. • Aggregation-disaggregation property of dye Nap was used to light up aptamer. • Aptamers targeting Nap of K d = 3.142 nM was screened by SELEX. • Super-resolution imaging of bacteria mRNA was achieved. [ABSTRACT FROM AUTHOR]