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