Overview of bleb detection and analysis. (A) The ADAPT user interface, illustrating the preview segmentation of a vaccinia-infected HeLa cell expressing GFP (Cyto Channel) and MLC2-RFP (Sig Channel). The green lines delineate the region over which fluorescence intensities will be quantified. The yellow circles indicate detected curvature extrema. The red line outlines the segmented cell. (B) Curvature is evaluated at each point on the cell boundary as the angle (θ) subtended by vectors (v₁ and v₂) to two points n pixels away in each direction. When all curvature values are superimposed on the cell boundary, bleb necks are identifiable as sharp concavities (in red) on a generally convex curve (green). (C) Blebs are detected as local maxima in the velocity map. (D) Blebs are tracked using local extrema in curvature as “anchor points.” The yellow lines denote the reference for the kymograph in Fig. 7 A. Image labels show seconds after bleb initiation. The white lines delineate the region over which fluorescence intensities will be quantified. (E) The mean membrane velocity, mean fluorescence intensity of the cortical component of interest (MLC2-RFP), and the length of boundary between the two anchor points for a single bleb (shown in D). The dotted line in the plots indicates the onset of retraction, and image labels correspond to seconds after bleb initiation. AU, arbitrary unit. Bars, 5 µM.