Fig. 7.
Frequency responses of bleedthrough corrected EEM channels for a three-color FRET mixture with incomplete FRET complexes and free fluorophores. (a) Cy5 EEM channel ê33. The lifetime of the final acceptor Cy5 remained unchanged at τCy5 = 1.8 ± 0.1 ns. (b) Cy3-Cy5 FRET channel ê23. The lifetime of quenched Cy3 was measured as τCy3-quench = 0.8 ± 0.1 ns. (c) Cy3 EEM channel ê22. A double exponential fit found the percentage of quenched Cy3 is PCy3 = 80 ± 5%. (d) Fluorescein-Cy3 FRET channel ê12. The quenched fluorescein lifetime was measured as τfluo-quench = 1.2 ± 0.1 ns. (e) Fluorescein EEM channel ê11. The percentage of quenched fluorescein was measured as Pfluo = 85 ± 2%. (f) Fluorescein-Cy5 FRET channel ê13. This channel contained signal from the two-step FRET (fluorescein-Cy3 then Cy3-Cy5). The measured frequency response was overlaid with the theoretical two-step FRET model, which was a product of individual frequency responses of quenched fluorescein, quenched Cy3 and Cy5. Fitted curves of quenched/unquenched donor and acceptor response were plotted for reference. Error bars represent standard deviations of multiple measurements with 1 ms integration time.