Pixel-by-pixel quantification of sensitized emission FRET within α4YFPβ2CFP nAChRs. (A; from left to right) Panels display an N2a cell coexpressing β2CFP and α4YFP nAChR subunits with nonfluorescent wild-type mGAT1 (calibration bars, ACUs). The whole cell ROI in which FRET was quantified is displayed in black in the third panel. The fourth panel displays the NFRET image (calibration bar, NFRET × 100). Pixels with signal amplitude below threshold are shaded gray. Bars, 10 µM. (B) Box plot displaying the range of NFRET detected from α4YFPβ2CFP-coexpressing N2a cells. The box, whiskers, and other data points are represented as in Fig. 7 B. The mean and median NFRET amplitudes for all pixels in each ROI are displayed in Table I. (C) Distribution of NFRET signal amplitudes per pixel (bin width, 0.02). The histogram was fit to two Gaussian distributions. The individual components are shown as dashed lines, and the sum of the fit is shown as a solid line. The inset table reports the mean NFRET amplitude of each component and the percentage of the total pixels that comprise each component.