Pixel-by-pixel quantification of sensitized emission FRET between mGAT1CFP8 and α4YFPβ2 nAChRs. A negative control experiment. (A; from left to right) mGAT1CFP8 fluorescence and α4YFP nAChR subunit fluorescence unmixed from an N2a cell coexpressing mGAT1CFP8, α4YFP nAChR subunit, and wild-type nonfluorescent β2 nAChR subunit (calibration bars in arbitrary calibration units [ACUs]). ROIs were used to determine FRET. The red-shaded area described the “peripheral” ROI, and the combined red and black areas correspond to the whole cell ROI. 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 plots displaying the range of NFRET detected from these negative control data. The box highlights the IQR (Q1–Q3), the center line in the box indicates the median, and the closed square symbol represents the mean. The whiskers' ends represent the boundaries of the lower and upper inner fences (1.5 × IQR). The × marks the first and 99th percentiles. The half-shaded diamond symbols indicate the absolute maximum and minimum data point in each set. The plots from the whole cell and peripheral ROIs are colored black and red, respectively, corresponding to the ROI color codes in A. The mean and median NFRET amplitudes for all pixels in each ROI are displayed in Table I. (C) Histograms displaying the distribution of pixel NFRET amplitudes for each condition (bin width, 0.02). Distributions for each ROI were fit to two Gaussian components. The individual components are shown as dashed lines, and the sum of the fit is shown as a solid line. The tables in each panel report the means of each component and the percentage of the pixels comprising each component.