Characterizing dark state kinetics and single molecule fluorescence of FusionRed and FusionRed-MQ at low irradiances
Srijit Mukherjee, Connor Thomas, Ryan N. Wilson, Emma M. Simmerman, Sheng‐Ting Hung, Ralph Jimenez
Abstract
), which were previously unexplored experimental conditions. We characterized the kinetics of dark state transitions in these two FPs by using single molecule blinking and ensemble photobleaching experiments bridged with a dark state kinetic model. We find that at low irradiances, the recovery process to the ground state is minimally light-driven and FusionRed-MQ has a 1.3-fold longer ground state recovery time indicating a conformationally restricted dark-state chromophore in comparison to FusionRed. Our studies indicate that the brighter FusionRed-MQ variant exhibits higher dark state conversion rates with longer ground state recovery lifetimes, thus it is potentially a better candidate for SMLM applications than its progenitor FusionRed.