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

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.

N-Cyanorhodamines: cell-permeant, photostable and bathochromically shifted analogues of fluoresceins.

Fluorescein and its analogues have found only limited use in biological imaging because of the poor photostability and cell membrane impermeability of their O-unprotected forms. Herein, we report rationally designed N-cyanorhodamines as orange- to red-emitting, photostable and cell-permeant fluorescent labels negatively charged at physiological pH values and thus devoid of off-targeting artifacts often observed for cationic fluorophores. In combination with well-established fluorescent labels, self-labelling protein (HaloTag, SNAP-tag) ligands derived from N-cyanorhodamines permit up to four-colour confocal and super-resolution STED imaging in living cells.