Figure 4. Representative laser scanning microscopy images of FRET acceptor photobleaching of HeLa cells co-transfected with GFP-VIM and RFP-WTαB. 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-WTαB is 9.5%, much greater than the negative control of untagged GFP and RFP. The increase of GFP fluorescence intensity is converted to pseudocolor (right panel) that displays variations of pixel gray scales with color.