FRET efficiency dependence on the donor-acceptor distance along the DNA. Values obtained by ixFLIM global analysis (cyan), ixFLIM spectral fit (red), donor lifetime FLIM (black) and by calculation (green dashed line) are compared between themselves and with values from literature, ref. 38 (purple). To illustrate the sensitivity to geometry, the dependence for a hypothetical isotropic molecular orientation is shown as well (orange). The error bars represent the standard deviation, obtained either directly from the standard error of the fit (ixFLIM spectra, red), or from the fit error of the FRET rates using error propagation (ixFLIM GA and FLIM); for the FLIM data the error bars reach 25% of the values (see Table 1 ) and were omitted for clarity.
