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

Here we describehighly compact, click compatible, and photoactivatabledyes for super-resolution fluorescence microscopy (nanoscopy). Bycombining the photoactivatable xanthone (PaX) core with a tetrazinegroup, we achieve minimally sized and highly sensitive molecular dyadsfor the selective labeling of unnatural amino acids introduced bygenetic code expansion. We exploit the excited state quenching propertiesof the tetrazine group to attenuate the photoactivation rates of thePaX, and further reduce the overall fluorescence emission of the photogeneratedfluorophore, providing two mechanisms of selectivity to reduce theoff-target signal. Coupled with MINFLUX nanoscopy, we employ our dyadsin the minimal-linkage-error imaging of vimentin filaments, demonstrating molecular-scale precision in fluorophore positioning. The quenching properties of tetrazine are used to controlthe photoswitching of fluorophores, yielding highly compact, clickcompatible, photoactivatable dyes for super-resolution microscopy.

Automated summary

Highly compact, click compatible, and photoactivatable dyes have been developed for super-resolution fluorescence microscopy. By combining the photoactivatable xanthone (PaX) core with a tetrazine group, minimally sized and highly sensitive molecular dyads were achieved for selective labeling of unnatural amino acids. The quenching properties of tetrazine were exploited to attenuate the photoactivation rates of PaX and reduce the overall fluorescence emission, providing selectivity to reduce off-target signal. These dyads were used in MINFLUX nanoscopy to demonstrate molecular-scale precision in fluorophore positioning.