Improving the Photostability of Red- and Green-Emissive Single-Molecule Fluorophores via Ni²⁺ Mediated Excited Triplet-State Quenching

Abstract

Methods to improve the photostability/photon output of fluorophores without compromising their signal stability are of paramount importance in single-molecule fluorescence (SMF) imaging applications. We show herein that Ni provides a suitable photostabilizing agent for three green-emissive (Cy3, ATTO532, Alexa532) and three red-emissive (Cy5, Alexa647, ATTO647N) fluorophores, four of which are regularly utilized in SMF studies. Ni works via photophysical quenching of the triplet excited state eliminating the potential for reactive intermediates being formed. Measurements of survival time, average intensity, and mean number of photons collected for the six fluorophores show that Ni increased their photostability 10- to 45-fold, comparable to photochemically based systems, without compromising the signal intensity or stability. Comparative studies with existing photostabilizing strategies enabled us to score different photochemical and photophysical stabilizing systems, based on their intended application. The realization that Ni allowed achieving a significant increase in photon output both for green- and red-emissive fluorophores positions Ni as a widely applicable tool to mitigate photobleaching, most suitable for multicolor single-molecule fluorescence studies.

Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores

“…Although ATTO 647N was capable of outcompeting Cy5-4S-AC in the presence of oxygen in terms of total time ON at low illumination intensities ( SI Appendix , Fig. S12 B and Supplementary Methods ), the use of ATTO 647N presents significant drawbacks in practical experimental settings, including ROS generation ( 43 ), nonspecific binding ( 71 ), and idiosyncratic deviations in photon flux ( 7 , 72 ) ( SI Appendix , Fig. S13 ).…”

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

“…1a) with buffer cocktails is the most flexible approach to stabilize various classes of organic fluorophores in different biochemical environments. [1][2][3][9][10][11][12][13][14][15][26][27][28][29][30] This approach is widely applied in fluorescence microscopy and the antioxidant TX, 13 COT [9][10][11] or combinations of redox-active compounds (''ROXS'' 2 ) have become the additives of choice.…”

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

“…Currently, intermolecular photostabilization ( Figure 1a) with buffer cocktails is the most effective way to stabilize various classes of organic fluorophores in different biochemical environments. 21,22,23 This approach is widely accepted as the gold standard for photostabilization and the antioxidant TX 10 , COT 7,13 or combinations of redox-active compounds ("ROXS" 2 ) have become the additives of choice. 24 Intramolecular photostabilization, a technique already established in the 1980s 25,26 (Figure 1b), does not require buffer additives.…”