Figure 8.
Figure 8. Pixel-by-pixel quantification of sensitized emission FRET between mGAT1XFP* and mGAT1XFP8. (A; left two panels) mGAT1CFP* fluorescence and mGAT1YFP* fluorescence (calibration bars, ACUs). In the third panel, ROIs were used to determine NFRET. The red-shaded area described the “peripheral” ROI, the green-shaded area corresponds to the “perinuclear” ROI, the black and green region with the red area describes the “intracellular ROI,” and the combined red, green, and black areas correspond to the whole cell ROI. The fourth panel displays the NFRET image of the same cell (color calibration bar, NFRET × 100). The color code in the NFRET image includes negative and positive signal amplitudes. Pixels with signal amplitude below threshold are shaded gray. Bars, 10 µm. (B) Box plots displaying NFRET for all pixels in each ROI of cells expressing mGAT1XFP*. 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 described for A (ROI), and the intracellular data are colored blue. (C) Table of results for mGAT1XFP* transfections displaying the mean of each Gaussian component of the summed fit of the distributions in D and the percentage of the pixels comprising each component. (D) Distributions of NFRET signal amplitude per pixel for each ROI (bin width, 0.02) from mGAT1XFP*-expressing cells. The individual components are shown as dashed lines, and the sum of the fit is shown as a solid line. (E) Representative fluorescence images of mGAT1XFP8-expressing N2a cells, the ROIs, and corresponding NFRET image. Calibration and color coding are as for A. (F) Box plots displaying NFRET for all pixels in each ROI of cells expressing mGAT1XFP8. Color coding and data are represented as in B. (G) Table of results for mGAT1XFP8 transfections displaying the mean of each Gaussian component of the summed fit of the distributions in H and the percentage of the pixels comprising each component. (H) Distributions of NFRET signal amplitude per pixel for each ROI (bin width, 0.02) from mGAT1XFP8-expressing cells. The individual components are shown as dashed lines, and their sum is shown as a solid line.

Pixel-by-pixel quantification of sensitized emission FRET between mGAT1XFP* and mGAT1XFP8. (A; left two panels) mGAT1CFP* fluorescence and mGAT1YFP* fluorescence (calibration bars, ACUs). In the third panel, ROIs were used to determine NFRET. The red-shaded area described the “peripheral” ROI, the green-shaded area corresponds to the “perinuclear” ROI, the black and green region with the red area describes the “intracellular ROI,” and the combined red, green, and black areas correspond to the whole cell ROI. The fourth panel displays the NFRET image of the same cell (color calibration bar, NFRET × 100). The color code in the NFRET image includes negative and positive signal amplitudes. Pixels with signal amplitude below threshold are shaded gray. Bars, 10 µm. (B) Box plots displaying NFRET for all pixels in each ROI of cells expressing mGAT1XFP*. 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 described for A (ROI), and the intracellular data are colored blue. (C) Table of results for mGAT1XFP* transfections displaying the mean of each Gaussian component of the summed fit of the distributions in D and the percentage of the pixels comprising each component. (D) Distributions of NFRET signal amplitude per pixel for each ROI (bin width, 0.02) from mGAT1XFP*-expressing cells. The individual components are shown as dashed lines, and the sum of the fit is shown as a solid line. (E) Representative fluorescence images of mGAT1XFP8-expressing N2a cells, the ROIs, and corresponding NFRET image. Calibration and color coding are as for A. (F) Box plots displaying NFRET for all pixels in each ROI of cells expressing mGAT1XFP8. Color coding and data are represented as in B. (G) Table of results for mGAT1XFP8 transfections displaying the mean of each Gaussian component of the summed fit of the distributions in H and the percentage of the pixels comprising each component. (H) Distributions of NFRET signal amplitude per pixel for each ROI (bin width, 0.02) from mGAT1XFP8-expressing cells. The individual components are shown as dashed lines, and their sum is shown as a solid line.

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