Pixel-by-pixel quantification of sensitized emission FRET between mGAT1XFP45. (A; from left to right) mGAT1CFP45 fluorescence and mGAT1YFP45 fluorescence (calibration bars, ACUs), ROIs used to determine NFRET (color coding as in Fig. 8), and the NFRET image (color calibration bar, NFRET × 100) of the same cell. Pixels with amplitude below threshold are shaded gray. Bars, 10 µm. (B) Box plots displaying NFRET for all pixels in each ROI of cells expressing mGAT1XFP45. The box, whiskers, and other data points are represented as in Fig. 7 B. Box plots for each ROI are colored according to the code in A (ROI), and the intracellular data are colored blue. (C) Table of results for mGAT1XFP45 transfections displaying the mean of each Gaussian component of the summed fit of the distributions in Fig. 8 D and the percentage of the pixels that comprise each component. (D) Distributions of NFRET signal amplitude per pixel for each ROI (bin width, 0.02) from mGAT1XFP45-expressing cells. The individual components and their sum are shown as dashed and solid lines, respectively.