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This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1552-4922/suppmat .

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cytoa20551-PaaretalFigureS1.tif908K Supplementary Figure 1: Setup calibration factor G For determination of setup calibration factor G donor and sensitized emission images were recorded before and after acceptor photobleaching of the construct C-Y. For a given emission filter set the ratio between increase in donor intensity (ΔF DD ) and decrease in sensitized emission intensity (ΔF c ) is constant. The slope in the plot is equivalent to G=2.0 (R 2 =0.95).
cytoa20551-PaaretalFigureS2.tif634K Supplementary Figure 2: Histograms of FRET-efficiencies and expression (FDD). 374 Jurkat T cells from 4 images taken within one minute using fast TDI based imaging enable statistical analysis of FRET constructs. The distribution of FRET efficiencies recorded on cells that express the construct N65-C-Y show a single peak, indicating a homogeneous population (A) and a broad distribution of expression levels (B).
cytoa20551-PaaretalFigureS3.tif2304K Supplementary Figure 3: Influence of confocal spot size on detection of membrane signals black line: concentration of fluorophores (volume concentration in mol/m 3 ). The volume concentration of fluorophore in the membrane (centered at 0nm) was assumed to be 100-fold higher than in the cytosol (<0nm), red lines: fluorescence intensities for varying width of confocal spots (σ =50nm, 100nm, 150nm), dashed black lines indicate the used gaussian profiles for calculating the fluorescence intensities.

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