Self-Healing Dye-Conjugated Nanobody Probe for Super-Resolution Imaging with Enhanced Photon Budget

Super-resolution microscopy imaging requires target-specific probes that deliver a high photon budget while minimizing perturbation to the protein of interest. Here, we report a nanobody probe generated by site-specific conjugation of a photostabilizer-fluorophore construct to an anti-GFP single-domain nanobody. This conjugate enhances the photon budget of the fluorophore by suppressing triplet-state populations via intramolecular triplet energy transfer, while simultaneously preserving nanomolar affinity for targeting GFP-tagged proteins. Compared to unmodified counterparts, the self-healing dye-conjugated nanobody exhibits a 38-83% excitation-power-dependent increase in solution molecular brightness and markedly enhanced single-molecule photophysics, yielding 6-fold and 42-fold increases in cumulative photon output under ambient and oxygen-scavenging conditions, respectively. When applied to GFP-tagged Nup96 protein in mammalian cells, the self-healing dye-conjugated nanobody proves significant advantage in single-molecule localization microscopy, where the improved photon budget and photoswitching kinetics directly improve localization precision. These results establish the self-healing dye-conjugated nanobody as a robust, ready-to-use probe for high-performance single-molecule localization-based super-resolution imaging.