Local subcellular Ca2+ microdomains were identified on a frame-by-frame basis and defined as small, compact, connected pixel sets (minimum pixel set size: 6 pixels; maximum pixel set size: 20 pixels; minimum pixel set circularity of 0.5, with circularity defined as [4π × area of pixel set]/[perimeter of pixel set]2) with high [Ca2+]I values compared to the mean [Ca2+]i value in the cell. To distinguish between background noise and Ca2+ microdomains, cell-free homogeneous Ca2+-EGTA buffer ([Ca2+] = 225 nM) containing Fluo4 and Fura Red (8) was analyzed. After processing the cell-free images as described for single-cell images, no noise-induced, fictive Ca2+ microdomains were detected for Ca2+ levels of more than 112.5 nM above the cell-free buffer mean Ca2+ level. Hence, for Ca2+ microdomain detection in cell images, all pixel [Ca2+]i values of the microdomain had to be at least ∆[Ca2+]i = 112.5 nM higher than the frame-specific mean [Ca2+]i of the considered cell. Reported local Ca2+ signal values refer to maximum [Ca2+]i values for the signal pixel set. Data are shown as Ca2+ microdomains per cell and frame.

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