Dissecting the hierarchy of IP₃-mediated Ca²⁺ signaling from global cellular oscillations to single-channel events. (A) Fluorescence traces illustrate global Ca²⁺ oscillations evoked by a strong (800-ms) UV photolysis flash in an SH-SY5Y cell loaded with fluo-4 and ciIP₃. (B) Local Ca²⁺ puffs evoked by weaker (100-ms flash) photorelease of i-IP₃ in another cell. (C) Image shows a Ca²⁺ puff (pseudocolored in green) captured at the time of peak amplitude from a single video frame and superimposed on a monochrome image of resting fluo-4 fluorescence in an SH-SY5Y cell. The trace shows a profile of Ca²⁺-dependent fluorescence measured along the dotted line passing through the center of the puff. (D and E) Ca²⁺ puffs evoked, respectively, by strong and weak photolysis flashes equivalent to those in A and B after intracellular loading with EGTA. Global responses were abolished, and the traces show local signals measured from small regions of interest centered over puff sites. (F) Image shows a Ca²⁺ puff and corresponding fluorescence profile as in C, but recorded in the presence of EGTA. (G) Comparison of representative puffs recorded in SH-SY5Y cells after photorelease of iIP₃ using wide-field fluorescence microscopy (gray trace), and using TIRF microscopy together with EGTA loading (black trace). Both traces show fluorescence ratio changes (ΔF/F₀) averaged within 1 × 1–µm regions of interest centered on puff sites. (H) Examples of sites that displayed exclusively single-channel blip activity. (I) Inset shows a Ca²⁺ signal from a 1-µm² region of interest, showing five discrete amplitude levels during a single puff. The histogram shows the distributions of event and step-level amplitudes derived from measurements at 87 sites in 20 cells, revealing “quantal” components at integer multiples of the single-channel level. A–F are adapted from Smith et al. (2009a), and G–I are from Smith and Parker (2009).