Calcium Imaging of Intracellular Puffs

Cultured SH-SY5Y cells were loaded for imaging by incubation with membrane-permeant esters of Fluo-4 (1 μM, Invitrogen), EGTA (5 μM, Invitrogen), and caged i-IP₃ (ci-IP₃, 1 μM, SiChem, Bremen, Germany) in HBS, as described (4 (link)). Cells averaged about 40 μm in length along the cell body (Fig. S4A). Cytosolic Ca²⁺ changes were imaged using a TIRF microscope system (4 (link)) constructed around an Olympus IX 70 microscope with a 60× TIRF objective (NA, 1.45). Fluo-4 fluorescence was excited by 488 nm laser light within an evanescent field extending a few hundred nanometers into the cells, and emitted fluorescence (λ > 510 nm) was imaged at a resolution of 256 × 256 pixels (1 pixel = 0.266 μm) at an exposure time of 15 ms (~66 frames sec⁻¹) using the center quad of an Evolve 512 electron-multiplied CCD camera (Roper Scientific; Tucson, AZ). Image data were acquired as stack files using MetaMorph v7.7 (Universal Imaging/Molecular Devices; Sunnyvale, CA) and were analyzed offline to detect the locations of puff sites and measure puff latencies. The custom software used for analysis is described in (14 (link)), and is freely available on request to the authors of that paper. Measurements were exported to Microcal Origin V. 8.0 (OriginLab, Northampton, MA) for analysis and graphing. Unless otherwise noted, data are presented as means ± 1 S.E.M.