Bioorthogonal Caging-Group-Free Photoactivatable Probes for Minimal-Linkage-Error Nanoscopy

[Image: see text] Here we describe highly compact, click compatible, and photoactivatable dyes for super-resolution fluorescence microscopy (nanoscopy). By combining the photoactivatable xanthone (PaX) core with a tetrazine group, we achieve minimally sized and highly sensitive molecular dyads for the selective labeling of unnatural amino acids introduced by genetic code expansion. We exploit the excited state quenching properties of the tetrazine group to attenuate the photoactivation rates of the PaX, and further reduce the overall fluorescence emission of the photogenerated fluorophore, providing two mechanisms of selectivity to reduce the off-target signal. Coupled with MINFLUX nanoscopy, we employ our dyads in the minimal-linkage-error imaging of vimentin filaments, demonstrating molecular-scale precision in fluorophore positioning.