TIRF and WF imaging of Ca2+ signals was accomplished using a custom-built system, based around an Olympus IX50 microscope equipped with either an Olympus 10× objective [numerical aperture (NA), 0.4] for WF microscopy or an Olympus 60× objective (NA, 1.45) for TIRF mode. Fluorescence images were acquired with an Evolve electron multiplying charge-coupled device (EMCCD) camera (Photometrics), using 488-nm laser fluorescence excitation and a 510 long-pass emission filter. WF images were captured from a 278 × 243 pixel field (1 pixel = 2.2 μm) at a rate of 10 frames s−1. TIRF images were captured using 2 × 2 pixel binning for a final field of 70 × 35 pixels (1 pixel = 0.53 μm) at a rate of ~333 frames s−1. Where indicated, some TIRF imaging was performed with a larger field of view (100 × 100 pixels or 128 × 128 pixels) at ~151 or ~125 frames s−1, respectively. To photorelease i-IP3, UV light from a xenon arc lamp was filtered through a 350- to 400-nm band-pass filter and introduced by a UV-reflecting dichroic in the light path to uniformly illuminate the field of view. The amount of i-IP3 released was controlled by varying the flash duration, set by an electronically controlled shutter (UniBlitz). All image data were streamed to computer memory using MetaMorph v7.7 (Universal Imaging/Molecular Devices) and stored on hard disk for offline analysis. WF ratiometric imaging of cells loaded with fura-2 was performed with a Nikon Eclipse Ti microscope equipped with a 40× objective and a Hamamatsu EMCCD camera. Light from an arc lamp was filtered through 340- and 380-nm filters and collected through a 510-nm filter. Alternating frames with 340- and 380-nm excitation (400- and 100-ms exposure times, respectively) were captured using NIS Elements software (Nikon) and stored on hard disk for offline analysis.

Lattice light-sheet imaging was performed using a custom-built system as described (71). Images were acquired with an Andor Zyla 4.2 sCMOS camera from a single, diagonal light-sheet slice (512 × 256 pixels; 1 pixel = 0.11 μm) at 250 frames s−1. Ca2+ puffs were imaged in Cal-520– and EGTA-loaded cells for several seconds after photorelease of i-IP3 by a flash from a 405-nm laser diode, using 473-nm laser fluorescence excitation and a 510- to 560-nm band-pass emission filter. A 562-nm laser and 590-nm long-pass filter were then used to image the plasma membrane stained with Deep Red Dye. The locations of puff were marked by visual inspection and overlaid on the plasma membrane image to classify puffs as near membrane (within ~2 μm) or arising deeper within the cell.

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