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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]

The visualization of self‐assembled structure and dynamics at the molecular level has become a powerful method to understand structure–function relationships of self‐assembly. Herein, we in situ real‐time imaged the dynamic process of benzyl‐naphthalimide dyes at the nanoscale and inspected their internal structure with minimum 2.8 nm localization accuracy through single‐molecule localization microscopy (SMLM) imaging. We monitored the growth process of three different assemblies in situ, which possessed highly heterogeneous dynamics with different shapes and growth rates. Furthermore, diverse growth rates were also found at different sites in the same assembly. These results highlight the application of super‐resolution microscopy techniques for real‐time visualization of internal assembled structure and dynamics in situ. [ABSTRACT FROM AUTHOR]

Long‐term super‐resolution imaging appears to be increasingly important for unraveling organelle dynamics at the nanoscale, but is challenging due to the need for highly photostable and environment‐sensitive fluorescent probes. Here, we report a self‐blinking fluorophore that achieved 12 nm spatial resolution and 20 ms time resolution under acidic lysosomal conditions. This fluorophore was successfully applied in super‐resolution imaging of lysosomal dynamics over 40 min. The pH dependence of the dye during blinking made the fluorophore sensitive to lysosomal pH. This probe enables simultaneous dynamic and pH recognition of all lysosomes in the entire cell at the single‐lysosome‐resolved level, which allowed us to resolve whole‐cell lysosome subpopulations based on lysosomal distribution, size, and luminal pH. We also observed a variety of lysosome movement trajectories and different types of interactions modes between lysosomes. [ABSTRACT FROM AUTHOR]

Although super‐resolution imaging offers an opportunity to visualize cellular structures and organelles at the nanoscale level, cellular heterogeneity and unpredictability still pose a significant challenge in the dynamic imaging of live cells. It is thus vital to develop better‐performing and more photostable probes for long‐term super‐resolution imaging. Herein, we report a probe, LD‐FG, for imaging lipid droplet (LD) dynamics using structured illumination microscopy (SIM). LD‐FG allows wash‐free imaging of LDs, owing to a hydrogen‐bond sensitive fluorogenic response. The replacement of photobleached LD‐FG by intact probe molecules outside the LDs ensures the long‐time stability of the fluorescence imaging. With this buffering fluorogenic probe, fast and unpredictable dynamic processes of LDs can be visualized. Using this probe, two LD coalescence modes were discovered. The dynamic imaging also allowed us to propose a new model of LD maturation during adipocyte differentiation, i.e. a fast LD coalescence followed by a slow ripening step. The excellent performance of LD‐FG makes the buffer strategy an effective method for designing fluorescent probes for cell dynamic imaging. [ABSTRACT FROM AUTHOR]

Subcellular pH plays a key role to modulate protein structures or specific active sites to impact diverse biological processes. The heterogeneity and high dynamics of intracellular pH require probes that can detect pH in the vicinity of the protein and have a wide pH response range. Here, we reported a protein proximity ligation fluorescent probe for super-resolution imaging of subcellular pH dynamics. Relying on SNAP-tag protein labeling technology, the pH-sensitive fluorophore BG-NDM based on photo-induced electron transfer mechanism was localized to the adjacent positions of the target protein in different subcellular organelles. The equilibrium between the non-fluorescent aggregate and the fluorogenic monomer enabled BG-NDM to have the ability of wash-free super-resolution imaging in live cells. And the wide pH response ranges from pH 6 to pH 12 made the probe have the ability of recognizing pH dynamics. The pH changes around functional proteins during mitophagy, cellular alkalosis and acidosis were revealed. Through long-term super-resolution imaging, we could further record the pH dynamics around mitochondrial proteins COX8A during mitochondrial contacts, fusion and fission at the resolution of a single mitochondria. [Display omitted] • Probe BG-NDM was used to super-resolution imaging of subcellular pH dynamics. • SNAP-tag labeling ensures that BG-NDM detects the pH near the protein of interest. • The aggregate/monomer equilibrium endowed BG-NDM wash-free imaging in living cells. • Mitochondrial pH dynamics was recorded with Long-term SIM imaging. [ABSTRACT FROM AUTHOR]

Herein, we reported a simple, fast, and quantitative theoretical descriptor ΔGC‐O that allows accurate predictions of a wide range of spontaneously blinking rhodamines. ΔGC‐O denotes the Gibbs free energy differences between the closed and open forms of rhodamines and has a good linear relationship with experimental pKcycl values. This correlation affords an effective guide for the quantitative designs of spontaneously blinking rhodamines and eliminates trial‐and‐error. We have validated the predictive power of ΔGC‐O via the development of two spontaneously blinking rhodamines of different colors and enhanced brightness. We also demonstrated their super‐resolution imaging utilities in dynamic live‐cell imaging. We expect that ΔGC‐O will greatly facilitate the efficient creations of spontaneously blinking fluorophores and aid the advancements of super‐resolution bioimaging techniques. [ABSTRACT FROM AUTHOR]

Super-resolution fluorescence microscopy has emerged as a powerful tool for studying mitochondrial dynamics in living cells. However, the lack of photostable and chemstable probe makes long-term super-resolution imaging of mitochondria still a challenging work. Herein, we reported a 4-azetidinyl-naphthliamide derived SNAP-tag probe AN-BG exhibiting excellent fluorogenicity and photostability for long-term super-resolution imaging of mitochondrial dynamics. The azetidinyl group and naphthalimide fluorophore are in a flat conformation which can effectively suppress twisted intramolecular charge transfer and then effectively improve the brightness and photostability. This planarized molecular structure is conducive to the formation of fluorescence-quenched J -aggregates, and the protein labeling process will depolymerize the probes and restore fluorescence. Fluorescent labeling mitochondrial inner membrane proteins via SNAP tags overcomes the shortcomings that variations in mitochondrial inner membrane potential will release probes attached to mitochondria by electrostatic interactions. Therefore, AN-BG realized the stable labeling of mitochondria and the long-term imaging of mitochondrial dynamics under super-resolution microscopy. Image 1 • A naphthliamide-derived fluorogenic probe for SNAP-tag was developed. • Fluorescent labeling of mitochondria was achieved via SNAP-tag fused proteins. • Long-term super-resolution imaging of mitochondrial dynamics was achieved. • The dynamic mitochondrial fusion and fission were imaged with fluorescent nanoscopy. • Two-color SIM imaging revealed multiple modes of action between mitochondria and lysosomes. [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]