Figure 2.

Normalizing the relative proximity ratio using calculated η_A/D. (A) Measured emission spectra of Cy3 (red) and Cy5 (blue) normalized to the emission maxima. The transmitted spectrum for each dye was calculated from the emission spectrum and the transmission spectrum for each element in the optical path and the camera response curve. Calculated transmission is shown for optical path 1 (solid line), optical path 2 (dashed line), and optical path 3 (shaded line). (B) Normalized relative proximity ratios for DNA under optical path 1 (solid), optical path 2 (shaded), and optical path 3 (open). E_PR was adjusted using the theoretical η_A/D, calculated as shown in panel A. Panel shows the mean value with the error bars indicating the width from a Gaussian fit. (C) FRET efficiencies for DNA recorded under optical path 1 (black) and optical path 3 (white) normalized using calculated γ^Empirical values specific to the indicated sample and filter set. Mean FRET efficiencies are shown with error bars indicating the width from a Gaussian fit.