A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization

Abstract

Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with ‘self-healing’ properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer–dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard.

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

“…5B, right). Although improved organic dyes have been developed (76, 77), dye photobleaching remains the main constraint on the maximal observation time and temporal resolution (78). Analysis of burst (freely diffusing) or time-binned (immobilized) data allows identification of distinct conformational subpopulations, their FRET efficiency, and, in favorable cases, their interconversion rates.…”

On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores

“…These are generated by either addition of the photostabilizer to the imaging buffer at millimolar concentrations 2,3,10,13 to allow diffusional intermolecular quenching (Figure 1a), or via covalent linkage, i.e., to create high local concentrations of the photostabilizer (Figure 1b). [14][15][16][17][18][19][20] Figure 1. Methods to create collisions between photostabilizer and reactive fluorophore state (here triplet-state T1) using intermolecular diffusional quenching (a) or intramolecular processes (b).…”

Effects of donor and acceptor's fluorescence lifetimes on the method of applying Förster resonance energy transfer in STED microscopy

“…All these factors compromise the achievable resolution due to the poor signal-to-noise ratio. To alleviate these problems, using the frequency dependent detection with a lock-in-amplifier (Ronzitti et al, 2013) or the measures to improve photobleaching resistance under high intensity of STED laser by introducing photostabiliserdye conjugates (van der Velde et al, 2016) have recently been reported.…”