Cy3 photoprotection mediated by Ni2+ for extended single-molecule imaging: old tricks for new techniques

The photostability of reporter fluorophores in single-molecule fluorescence imaging is of paramount importance, as it dictates the amount of relevant information that may be acquired before photobleaching occurs. Quenchers of triplet excited states are thus required to minimize blinking and sensitization of singlet oxygen. Through a combination of single-molecule studies and ensemble mechanistic studies including laser flash photolysis and time-resolved fluorescence, we demonstrate herein that Ni(2+) provides a much desired physical route (chemically inert) to quench the triplet excited state of Cy3, the most ubiquitous green emissive dye utilized in single-molecule studies.