Single-molecule photophysics of dark quenchers as non-fluorescent FRET acceptors

Dark quencher chromophores are interesting alternatives to common single-molecule FRET acceptors. Due to their short excited state lifetime, they should be less prone to complex photophysics and bleaching. We find, however, that for common enzymatic oxygen scavenging systems and photoprotection strategies - the gold standard of single-molecule measurements - the quenchers BBQ650 and BHQ-2 induce frequent blinking of the donor molecule. They switch in a photoinduced process to what we identify as a radical anion state and back. We further make use of the broad absorption spectrum for selective bleaching of the quenchers in order to photoactivate the fluorescence of initially completely quenched dye molecules. This represents a general strategy to turn fluorescent dyes into photoactivatable probes. The quenchers BBQ650 and BHQ-2 exhibit significant redox blinking under common single-molecule measurement conditions. Non-fluorescent FRET acceptors can further be used to convert any dye into a photoactivatable probe.