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.
Highlights
Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy
The first analog of this class of fluorophores was serendipitously found during the attempted synthesis of a silicon rhodamine (SiR) derivative bearing an alkyl chain in place of the aromatic substituent at the 9 position of the xanthene scaffold (Fig. 1a)
Noteworthy is the quantitative nature of the photoconversion of PA-SiR, which becomes apparent when following the conversion of PA-SiR to 3 by nuclear magnetic resonance (NMR)
Summary
Noteworthy is the quantitative nature of the photoconversion of PA-SiR, which becomes apparent when following the conversion of PA-SiR to 3 by nuclear magnetic resonance (NMR) These experiments revealed that the photoactivation is reversible on a time scale of days (Fig. 1f and Supplementary Fig. 5). Carbopyronine and fluorescein derivatives with such an exocyclic double bond have been described (see Supplementary Fig. 3 for an overview of related structures and reactions), but have not been reported to undergo light-induced protonation. In the absence of structural information on HaloTag labeled with PA-SiR-Halo we cannot provide more detailed insights on the nature of these interactions This apparent fluorogenicity of the probe should prove beneficial for live-cell imaging as unconjugated PA-SiRHalo is not fluorescent, which increases the signal-to-background ratio.
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