Abstract

Single‐molecule localization super‐resolution fluorescence imaging relies on the fluorescence ON/OFF switching of fluorescent probes to break the diffraction limit. However, the unreacted or nonspecifically bound probes cause non‐targeted ON/OFF switching, resulting in substantial fluorescence background that significantly reduces localization precision and accuracy. Here, we report a blinkogenic probe HM‐DS655‐Halo that remains blinking OFF until it binds to HaloTag, thereby triggering its self‐blinking activity and enabling its application in direct SMLM imaging in living cells without wash‐out steps. We employed the ratio of the duty cycle before and after self‐blinking activation, termed as the parameter "RDC" to characterize blinkogenicity. The covalent binding to HaloTag induces HM‐DS655‐Halo to transition from a fluorescent OFF state to a fluorescence blinking state. This transition also leads to a change in the RDC value, which is calculated to be 12, ensuring super blinkogenicity to effectively suppress background signals in living cells. HM‐DS655‐Halo was successfully applied in dynamic SMLM imaging of diverse intracellular sub‐structures with minimal background noise, including mitochondrial fission and contact, cell migration, and pseudopod growth.