Figure 1.

Photophysical processes in Cy and Alexa fluorophores are strongly power-dependent. (A) Blinking observed in the fluorescence trajectories of single Cy (left) and Alexa (right) dye pairs linked to a 12-basepair DNA under oxygen scavenging conditions and without the presence of triplet state quenchers (0.65 kW/cm²). (B) Corresponding smFRET time traces for Cy (left) and Alexa (right) dyes showing dwells in FRET and dark states. FRET is calculated from the relationship:$E_{\text{FRET}}=\frac{I_{\text{donor}}}{I_{\text{donor}}+I_{\text{acceptor}}}$. Idealization of smFRET time traces (red line) using hidden Markov modeling procedures is used to estimate the kinetics of switching between zero and nonzero FRET states. (C) Total t_on (circles; red) decreases with increasing laser intensities for Cy (left) and Alexa (right) dye pairs. The photobleaching rate of the donor fluorophore (dotted green line) is shown for reference to indicate that trends in Total t_on are largely independent of donor lifetime.