Automated identification and recording from puncta. (A and B) TIRF images of Orai1-GCaMP6f (A) and mCherry-STIM1 (B) from a transfected HEK 293A cell during whole-cell recording at +20 mV resting membrane potential (average of 10 frames). Green arrowheads indicate regions of diffuse Orai1-GCaMP6f fluorescence in corners and along edges of the TIRF footprint that lack STIM1-Orai1 puncta. (C and D) Cropped and enlarged portions of TIRF images corresponding to the yellow squares shown in A and B, respectively. (E) Puncta automatically identified by the program TrackMate, indicated by pink circles superimposed upon a local background-subtracted version of D. (F) Plot of puncta center for each of 50 consecutive frames for Orai1-GCaMP6f (green dots) and mCherry-STIM1 (red dots). Note that Orai1-GCaMP6f and mCherry-STIM1 positions are tightly clustered and clearly associated with the same punctum. (G) Corresponding SRRF computational superresolution image corresponding to D. (H and I) TIRF images of Orai1-GCaMP6f (H) and mCherry-STIM1 (I) from another transfected HEK 293A cell during whole-cell recording at +20 mV resting membrane potential (average of 10 frames). (J and K) Cropped, enlarged, and counterclockwise-rotated portions of TIRF images corresponding to the yellow rectangles shown in H and I, respectively. (L) Corresponding map of Orai1-GCaMP6f plateau ΔF/F0 for the region shown in J. Arrowheads in J–L indicate two puncta. (M and N) Corresponding graphs of ΔF/F0 for puncta 1 (black line) and puncta 2 (gray line) during a 600-ms test pulse to −100 mV measured using automated (M) and manually (N) positioned ROIs. Note the different magnitudes of ΔF/F0 for puncta 1 and 2. Scale bar in A is 10 µm (applies to B, H, and I), and the scale bar in C is 2 µm (applies to D–G and J–L). A–G correspond to cell C, and H–N correspond to cell D (see Fig. S3).