Figure 3

Determination of random FRET. Osteosarcoma cells U-2OS were transfected with plasmids expressing the fluorophores ECFP- and EYFP only. 48 h later cells were scanned for fluorescence and random FRET was determined. Panel A: ECFP fluorescence after donor excitation; panel B: EYFP acceptor fluorescence after acceptor excitation; panel C: non-specific energy transfer fluorescence encoded in pseudo colors and panel E: corresponding frequency distribution of FRET efficiencies; panel D: non-specific energy transfer fluorescence encoded in pseudo colors after cutting of efficiency values below a certain threshold and panel F: corresponding frequency distribution of FRET efficiencies; panel H: resulting non-specific, random FRET curve after weighing FRET efficiencies for their relative frequency.