Figure 5. Representative laser scanning microscopy images of FRET acceptor photobleaching of HeLa cells co-transfected with GFP-VIM and RFP-R120G αB. The pair of constructs was co-transfected into HeLa cells. After culture, laser scanning microscopy (LSM) images were taken before and after photobleaching of the acceptor for 45 s with a 543 nm laser beam. A decrease of red fluorescence and increase of green fluorescence were observed. The transfer efficiency was calculated with the equation: E=1 – F_GFP-min/F_GFP-max. The efficiency for this cell that co-transfected with GFP-VIM and RFP-R120G αB is 19.5%, twofold greater than the GFP-VIM and RFP-WTαB. The increase of GFP fluorescence intensity is converted to pseudocolor (right panel) that displays variations of pixel gray scales with color.