Photoactivatable Carbo‐ and Silicon‐Rhodamines and Their Application in MINFLUX Nanoscopy

Abstract

New photoactivatable fluorescent dyes (rhodamine, carbo‐ and silicon‐rhodamines [SiR]) with emission ranging from green to far red have been prepared, and their photophysical properties studied. The photocleavable 2‐nitrobenzyloxycarbonyl unit with an alpha‐carboxyl group as a branching point and additional functionality was attached to a polycyclic and lipophilic fluorescent dye. The photoactivatable probes having the HaloTagTM amine (O2) ligand bound with a dye core were obtained and applied for live‐cell staining in stable cell lines incorporating Vimentin (VIM) or Nuclear Pore Complex Protein NUP96 fused with the HaloTag. The probes were applied in 2D (VIM, NUP96) and 3D (VIM) MINFLUX nanoscopy, as well as in superresolution fluorescence microscopy with single fluorophore activation (VIM, live‐cell labeling). Images of VIM and NUPs labeled with different dyes were acquired and their apparent dimensions and shapes assessed on a lower single‐digit nanometer scale. Applicability and performance of the photoactivatable dye derivatives were evaluated in terms of photoactivation rate, labeling and detection efficiency, number of detected photons per molecule and other parameters related to MINFLUX nanoscopy.

No-wash fluorogenic labeling of proteins for reversible photoswitching in live cells

“…The use of photoswitchable fluorescent molecules (PSFMs) for tracking cellular dynamics 1 and visualizing cellular components with high spatial resolution using super-resolution imaging 2–6 has greatly increased in recent years. While photoactivatable fluorophores irreversibly photoswitch from non-fluorescent to fluorescent states upon light irradiation, 7–12 PSFMs exhibit reversible fluorescence switching upon light irradiation allowing each PSFM to be localized multiple times. This is useful for live-cell studies where molecular dynamics can be visualized over long periods.…”