Intramolecular photostabilization via triplet-state quenching: design principles to make organic fluorophores “self-healing”

Abstract

Covalent linkage of fluorophores and photostabilizers was recently revived as a strategy to make organic fluorophores "self-healing"via triplet-state quenching. Although Lüttke and co-workers pioneered this strategy already in the 1980s, the general design principles still remain elusive. In this contribution, we combine experiments and theory to understand what determines the photostabilization efficiency in dye-photostabilizer conjugates. Our results from single-molecule microscopy and molecular dynamics simulations of different Cy5-derivatives suggest that the distance and relative geometry between the fluorophore and photostabilizer are more important than the chemical nature of the photostabilizer, e.g. its redox potential, which is known to influence electron-transfer rates. We hypothesize that the efficiency of photostabilization scales directly with the contact rate of the fluorophore and photostabilizer. This study represents an important step in the understanding of the molecular mechanism of intramolecular photostabilization and can pave the way for further development of stable emitters for various applications.

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

“…The focus of previous work on self-healing fluorophores has been on the optimization of photostabilization efficiency, 18,20 the development of versatile bioconjugation strategies, 19 and to benchmark the method for different fluorophore types 19 or environmental conditions. 18 Here we conducted experiments in which photostabilizers were present in solution and linked to the fluorophore simultaneously ( Figure 2), in an attempt to determine whether inter-and intramolecular photostablization effects can be additive or synergistic.…”

“…In previous work, we also investigated the importance of stabilizer-fluorophore arrangement and linker length for self-healing processes. 18 Different linking chemistries or orientations of the photostabilizer resulted in different photostabilities. Our interpretation of the data in Figure 3 is further corroborated by experiments in which covalently linked NPA was arranged differently in space with respect to Cy5 ( Figure S29).…”

“…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).…”

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

“…1b). [18][19][20][21][22][23][24][25] Currently, intermolecular photostabilization ( Fig. 1a) with buffer cocktails is the most flexible approach to stabilize various classes of organic fluorophores in different biochemical environments.…”

Self-healing dyes for super-resolution microscopy

“…More recently, intramolecular triplet-state quenching [17][18][19] of synthetic organic dyes ("self-healing" [20][21][22] ) emerged as an alternative strategy to achieve high photostability 20,23,24 . This approach overcomes important caveats of adding photostabilizers to the imaging buffer 20,[23][24][25][26][27][28][29] . For example, these conditions are often incompatible with biological requirements of living cells.…”