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

Abstract

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 offtarget 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.

Bioorthogonal Reactions in Bioimaging

“…They combined a photoactivatable xanthone (PaX) core with a tetrazine unit to access a small-sized probe for the selective labeling of unnatural amino acids (BCN-Lys) introduced by genetic code expansion to proteins. The minimal bioorthogonal tag and the small probe allowed to take full advantage of the nanometer precision of MINFLUX and MINSTED [ 86 ]. Sequential switching of probes between an ON and an OFF state is a popular super-resolution method to distinguish adjacent fluorophores at molecular-scale proximities.…”

Picture Perfect Precision: Biorthogonal Photoactivatable Tools Achieve Imaging with Molecular-Scale Precision

“…In this issue of ACS Central Science , Hell and colleagues harness the synergistic power of two techniques awarded the Nobel Prize in chemistry–super-resolution microscopy (2014) and biorthogonal labeling (2022)to achieve optical imaging with molecular-scale precision. Their work describes the development of a family of probes containing both a fluorescence quenching tetrazine for bioorthogonal labeling and a photoactivatable xanthone, allowing for minimal-linkage-error, near-background-free imaging …”

“…Their work describes the development of a family of probes containing both a fluorescence quenching tetrazine for bioorthogonal labeling and a photoactivatable xanthone, allowing for minimal-linkage-error, near-background-free imaging. 1 …”

“…(f) PALM image taken with compounds 6 (left) and 11 (right). Reproduced with permission from ref ( 1 ). Copyright 2023 American Chemical Society.…”

“…(e) Structure of vimentin. Reproduced with permission from ref ( 1 ). Copyright 2023 American Chemical Society.…”