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Caption : fig. s1 spline fitting of the identified filopodia and extraction of the integrated intensities. (a) section of a confocal fluorescent image of a pc12 cell expressing the i-bar of irsp53 tagged with egfp, where the focus is at the surface of the glass slide. the red line shows the spline fit for each identified filopodium. in blue and green the perpendicular segments to the spline function used to extract the underling intensity. the green segments correspond to the segments in (b). (b) examples of the intensity profile for three perpendicular segments with r2 < 0.9. (c) intensity profile of a segment passing the exclusion criteria with an r2 > 0.9 and corresponding gaussian fit.
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Caption : fig. s2 extraction of the integrated intensities of the plasma membrane. (a) cell cross-section imaged ~5 μm above the coverslip for the membrane channel. the outline of the pm determined by the watershed algorithm is fitted with a spline function (red line). the intensity profiles extracted from perpendicular cuts are included (blue) or excluded (cyan) based on the fwhm. (b) example of the intensity profile from one of the included perpendicular segments fitted with a gaussian function. the gaussian fitting allows to estimate the fwhm. the yellow colored points are the 5 points used to estimate the intensity in the protein channel.
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Caption : fig. s3 fitting tether ratios to obtain the conversion factor. (a) image of a tether pulled from a hek293 cell using optical trapped streptavidin-coated beads. (b) histogram of i(membrane) (light green) and the propagated errors (dark green) of the extracted segments from 8 individual cells. the black continuous line indicates lognormal fit of the ratio between the integrated intensities of the tether and the pm.
File Name : figures4_lzwcompression.tif
Caption : fig. s4 data points selection based on the fwhm. (a) confocal fluorescence image of a pc12 cell expressing the i-bar domain of irsp53 tagged with egfp, where the focus is at the surface of the glass slide. each color line represents an identified filopodia. (b) single cell measurements of normalized i-bar density plotted against the filopdia radius. in (a) and (b) all data points are included with no filter on the fwhm. (c) and (d) show the same graphs as in (a) and (b) excluding the data points with a fwhm above 280 nm . examples of excluded parts of filopodia are shown with black circles on (c).