Figure 5

Typical recording of a FRET measurement. Osteosarcoma cells U-2OS were transfected with plasmids expressing the donor molecule ECFP-HIF-1α and the acceptor EYFP-HIF-1β. 48 h later cells were scanned for fluorescence and FRET was determined. Panel A: ECFP-HIF-1α donor fluorescence after donor excitation; panel B: EYFP-HIF-1β acceptor fluorescence after acceptor excitation; panel C: overlay of images in panels A and B; panel D: areas of co-localization (from panel C), very low signal (in blue) or overexposed areas (in red) and areas with FRET (from panel E); panel E: EYFP-HIF-1β acceptor fluorescence after donor excitation = FRET fluorescence intensity encoded in grey values as indicated by the bar scale; panel F: FRET efficiency encoded in grey values as indicated by the bar scale.