Justification for postprocessing of data. (A) Images of a bleb on a HeLa cell expressing lifeact-GFP, with and without the application of a threshold to remove noise. In the absence of a threshold, the total fluorescence intensity (red lines) closely follows the length of the bleb (blue lines). Applying a threshold disrupts this correlation. Image labels correspond to seconds after bleb initiation. Data represent the analysis of a single bleb. Bar, 2.5 µM. (B) The change in total lifeact-GFP intensity along the periphery of a single bleb (shown in A) over time, with (right) and without (left) the use of a threshold value to exclude noise. (C) Each colored line in the plots represents a single bleb on the same cell (nbleb = 4), with (right) and without (left) normalization. The black dotted line represents the mean in each case. (D) The heat map represents the lifeact intensity recorded at every boundary point over the course of a video in one particular cell. It is evident that the signal intensity diminishes over extended time periods. AU, arbitrary unit.
