Photoactivation of silicon rhodamines via a light-induced protonation

Abstract

Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore’s outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

HaloFlippers: A General Tool for the Fluorescence Imaging of Precisely Localized Membrane Tension Changes in Living Cells

“…Since the introduction of HaloTag technology, 48 the strategy has found many applications. Examples include protein labeling with synthetic ligands 48 , 51 , 52 or fluorescent dyes to study biological processes, such as redox signaling, 53 , 54 cell dynamics, 55 − 58 and protein degradation, 59 or to detect specific ions, 60 − 65 the viscosity, 22 , 66 and the membrane potential 18 , 23 inside compartments of living organisms. They were also used as covalent long-lived tethers for protein nanomechanics.…”

HaloTag Engineering for Enhanced Fluorogenicity and Kinetics with a Styrylpyridium Dye

“…However, we were hopeful that the improved reaction rate and fluorogenicity with the HaloTag7 variants might make this system suitable for imaging in other cellular locations. Both HaloTag7 and the variants were tagged with a localization target for actin (Lifeact) [16] . The cells transfected with HaloTag7, HT‐SP4 , and HT‐SP5 were compared upon incubation with JF549 (Figures 5 and S7) or F1 (Figures 5 and S8).…”

Oxime as a general photocage for the design of visible light photo-activatable fluorophores

“…In recent years, super-resolution microscopy techniques have been well developed for biological imaging that overcomes the diffraction limit for light microscopy. 84,85 Photoactivatable or photoswitchable uorophores have been widely applied to the development of SMLM techniques, 7,8,36,47,86 such as photoactivated localization microscopy (PALM) 17 and stochastic optical reconstruction microscopy (STORM). 18 The excellent photoactivation properties of BAD-Oxm in solution and living cells make it an attractive probe for use in PALM.…”

“…13,[42][43][44][45][46] Quite recently, a novel light-induced protonation strategy has been employed to prepare a photoactivatable silicon rhodamine derivative. 47 The utility of this uorophore was successfully demonstrated in live-cell single molecule localization microscopy (SMLM) imaging. However, these strategies are limited to certain uorophore scaffolds.…”