Correspondence between puncta ΔF/F0, A-index, and relative STIM1/Orai1 ratio. Maps of plateau ΔF/F0 (A–E), A-index (F–J), and relative STIM1/Orai1 ratio (K–O) for five HEK 293A cells cotransfected with Orai1-GCaMP6f and mCherry-STIM1 in response to a 600-ms test pulse to −100 mV in 2 mM extracellular Ca²⁺. White arrowheads indicate puncta with little or no change in fluorescence in the corners of the cell footprint, and orange arrowheads indicate puncta with and low A-indices. Note that puncta in all seven corners of the cell footprint in J exhibit slow activation of Orai1-GCaMP6f fluorescence. Scale bar in A is 20 µm (applies to B–O). A, F, and K correspond to cell A; B, G, and L to cell C; C, H, and M to cell D; D, I, and N to cell E; and E, J, and O to cell F (see Fig. S3).