Spontaneously Blinkogenic Probe for Wash‐Free Single‐Molecule Localization‐Based Super‐Resolution Imaging in Living Cells

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 "R_DC" 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 R_DC 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.