Extended Data Table 1 FRET quantifications displayed as intensity based relative to: the positive control (upper rows), the maximal FRET state (DA) (middle rows) and the estimated Förster resonance energy transfer (E) (lower rows)

From: In vivo FRET–FLIM reveals cell-type-specific protein interactions in Arabidopsis roots

  1. Upper rows (light purple): Intensity-based FRET fraction relative to NLS:YFP:RFP. The relative intensity contribution of the FRET state was determined using triple lifetime unmixing of the phasors in the polar plot: the FRET phasor, the auto-fluorescence phasor (at ≈1.93 ns) and the control phasor (at ≈3.21 ns) in Fig. 1. For each transcription factor pair and cell type, the fractional intensity contribution of the FRET state relative to the control state was calculated and normalized to that of the average of the positive control NLS:YFP:RFP. By using this approach, the contribution of the auto-fluorescence component is removed from the FRET analysis. CI represents the 95% confidence interval and was calculated using statistical bootstrapping of the control and sample phasor clouds. Graphically, the calculation can be understood by extrapolating the line connecting the white filled diamond marker and the coloured filled circle marker in the polar plot and finding the intercept with the top dashed line of the triangular shape in the polar plot indicated in Fig. 1. The percentages in the table correspond to the distance between the filled triangle markers and this intercept point relative to that of the positive control.
  2. Middle rows (light orange): intensity-based FRET fraction with respect to the maximal FRET state (DA). The lifetime of the FRET state was estimated to be around 1.93 ns, which corresponded to a maximum possible FRET value of Emax = 40%, this assuming an estimated donor life time of 3.21 ns. The relative intensity contribution of the FRET state was determined using triple lifetime unmixing of the phasors in the polar plot, thus removing the auto-fluorescence component from the FRET analysis. Graphically, the percentages in the table correspond to the distance between the filled triangle markers and the intercept point relative to the length of the top side of the triangular shape in Fig. 1.
  3. Lower rows (light blue): estimated Förster resonance energy transfer (E), for the different transcription factor pair combinations and cell types. The E-value can be directly calculated using the formula E = αDA × Emax.
  4. When the confidence interval does not contain zero (black numbers), it is likely, or at 95% confidence that the estimated values are higher than zero and FRET occurs.