Pixel-by-pixel quantification of sensitized emission FRET between mGAT1⁵⁷⁷XFP⁵⁷⁸CT. (A; from left to right) mGAT1⁵⁷⁷CFP⁵⁷⁸CT fluorescence and mGAT1⁵⁷⁷YFP⁵⁷⁸CT 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 mGAT1⁵⁷⁷XFP⁵⁷⁸CT. 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 mGAT1⁵⁷⁷XFP⁵⁷⁸CT transfections displaying the mean of each Gaussian component of the summed fit of the distributions in 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 mGAT1⁵⁷⁷XFP⁵⁷⁸CT-expressing cells. The individual components and the sum of the fit are shown as dashed and solid lines, respectively.