FIG. 4.

FRET analysis of 65-87-EGFP-TMRE localization. (A) Using a Leica AOBS-SP2 confocal system, EGFP and TMRE emission spectra were recorded by wavelength scanning (lambda scan) between 500 and 660 nm with a 5-nm detection window. Fluorescence intensity plots show significant overlap between EGFP emission spectra and TMRE excitation spectra (green filled histogram) and sufficient separation between the TMRE excitation and emission spectra. (B) Spot bleaching (area marked in red) of the acceptor was performed as described in Material and Methods. (C) The ratio images of donor-acceptor fluorescence before (upper panel) and after (lower panel) photobleaching are indicated together with the look-up table on the left side of the figure. Notice that the only change is in the region of photobleaching, which demonstrates a large increase in donor signal. (D) Confocal images of the donor acquired using a 488-nm excitation line and emission between 500 and 550 nm obtained pre- and postbleaching reveal dequenching of the donor. Using a 568-nm excitation line and emission between 580 and 620 nm, sequential scan images of the acceptor were also acquired pre- and postbleaching; these reveal effective bleaching of the acceptor. (E) These panels depict the average pixel intensity profiles over time of the bleached region, with the images pre- and postbleaching marked by arrows. Notice the increase in intensity of EGFP (donor; upper graph) signal that occurs concomitantly with the bleaching-induced decrease in the TMRE (acceptor; bottom graph) signal.