To enable analysis and comparison of Ca2+ microdomains for specific subcellular divisions (such as “close to point of activation/bead contact”) and cell groups, a so-called “dartboard projection” approach was introduced: First, a circular, dartboard-like template (with three annuli and individual annuli layers of same area) was matched onto the individual cell images using an automated template-to-cell matching by circle Hough transform (fig. S1). Then, on the basis of their spatial coordinates, the identified local Ca2+ signals were assigned to the corresponding template layers. The resulting dartboards were normalized with respect to the location of the bead contact zone by rotating the template. Last, the cell-specific segment-wise local Ca2+ signal information was aggregated and evaluated for the considered group. All Ca2+ analysis steps were implemented using Matlab.

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