Orai1-GCaMP6f fluorescence responses to changes in membrane potential during whole-cell recording and Ca²⁺ perfusion after cell unroofing. (A and F) TIRF micrographs of cotransfected HEK 293A cells showing Orai1-GCaMP6f (A) and mCherry-STIM1 (F) before pipette break-in. The image intensities have been increased threefold with respect to B–E and G–J to facilitate comparison. (B, C, G, and H) TIRF images of Orai1-GCaMP6f (B) and mCherry-STIM1 (G) at +20 mV holding potential and during −100 mV test pulse (C and H). (D, E, I, and J) TIRF images of the same cell after mechanical unroofing showing Orai1-GCaMP6f and mCherry-STIM1 upon perfusion of solutions containing (D and I) and lacking (E and J) 2 mM Ca²⁺. Scale bar in J is 20 µm (applies to A–I). (K–M) Plots of whole-cell current (K), Orai1-GCaMP6f fluorescence (L), and mCherry-STIM1 (M) fluorescence in response to test pulses to −40 and −100 mV. Note the parallel changes in Orai1-GCaMP6f current and fluorescence during test pulses and the lack of change in mCherry-STIM1 fluorescence. (N) Comparison of Orai1-GCaMP6f fluorescence at +20 mV holding potential and upon −40 and −100 mV test pulses with Orai1-GCaMP6f fluorescence from the same, subsequently unroofed, cell perfused with solutions containing (2 Ca) and lacking (0 Ca) 2 mM Ca²⁺. Data are representative of six cells acquired over 4 d. Images and traces correspond to cell E (see Fig. S3).