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 Ni2+ 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. Ni2+ 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 Ni2+ 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 Ni2+ allowed achieving a significant increase in photon output both for green- and red-emissive fluorophores positions Ni2+ as a widely applicable tool to mitigate photobleaching, most suitable for multicolor single-molecule fluorescence studies.
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