Fig. 3. Detection of FRET at the plasma membrane by acceptor photobleaching. Hek293T cells were transfected with an expression vector encoding gp130/id-C/YFP. 48 hours after transfection cells were fixed and FRET between CFP and YFP was measured using a confocal microscope. (A) YFP (yellow) and CFP (blue) fluorescence of a cell expressing gp130/id-C/YFP selected for qualitative FRET detection is shown on the left. Rows of ten consecutive images represent the fluorescence intensities of YFP and CFP in a plasma membrane region of the selected cell in false colour mode showing highest and lowest fluorescence intensities in yellow and black, respectively. Within the rectangular ROI, YFP is bleached after recording of image number 5. (B) For quantitative FRET analysis, fluorescence intensities of CFP (blue) and YFP (yellow) during a time series of ten images in a ROI at the plasma membrane of a Hek293T cell expressing gp130/id-C/YFP was recorded. YFP was bleached after image 5. Fluorescence of CFP and YFP was recorded simultaneously as described in Materials and Methods. CFP fluorescence intensities of images 5 and 6 were used for quantification of FRET efficiency. (C) Comparison of individual FRET efficiencies at the plasma membrane of cells expressing gp130/id-C/YFP (red squares) or gp130/id-CFP (blue diamonds). The FRET efficiencies are given as a function of the average fluorescence intensity of CFP and YFP. A different cell was selected for each FRET measurement. (D) Mean FRET efficiencies (±s.d.) at the plasma membrane of cells expressing gp130/id-CFP (white bar) or gp130/id-C/YFP (grey bar). Gp130/id-CFP is termed `negative control' and gp130/id-C/YFP `positive control' for further evaluations. Bar, 10 µm.