Decoding cellular processes requires visualization of the spatial distribution and dynamic interactions of biomolecules. It is therefore not surprising that innovations in imaging technologies have facilitated advances in biomedical research. The advent of super‐resolution imaging technologies has empowered biomedical researchers with the ability to answer long‐standing questions about cellular processes at an entirely new level. Fluorescent probes greatly enhance the specificity and resolution of super‐resolution imaging experiments. Here, we introduce key super‐resolution imaging technologies, with a brief discussion on single‐molecule localization microscopy (SMLM). We evaluate the chemistry and photochemical mechanisms of fluorescent probes employed in SMLM. This Review provides guidance on the identification and adoption of fluorescent probes in single molecule localization microscopy to inspire the design of next‐generation fluorescent probes amenable to single‐molecule imaging.

A β-galactosidase activatable fluorescent turn-on theranostic Gal-CGem exhibits gemcitabine release specifically to the β-galactosidase overexpressed hepatic carcinoma cells. The cytotoxicity of Gal-CGem in cancer cells is achieved through apoptotic cell...

Decoding cellular processes requires visualization of the spatial distribution and dynamic interactions of biomolecules. It is therefore not surprising that innovations in imaging technologies have facilitated advances in biomedical research. The advent of super‐resolution imaging technologies has empowered biomedical researchers with the ability to answer long‐standing questions about cellular processes at an entirely new level. Fluorescent probes greatly enhance the specificity and resolution of super‐resolution imaging experiments. Here, we introduce key super‐resolution imaging technologies, with a brief discussion on single‐molecule localization microscopy (SMLM). We evaluate the chemistry and photochemical mechanisms of fluorescent probes employed in SMLM. This Review provides guidance on the identification and adoption of fluorescent probes in single molecule localization microscopy to inspire the design of next‐generation fluorescent probes amenable to single‐molecule imaging.