Fig. 2: Single- and multiple-release Ca2+ sparks.
TIRF imaging of cells loaded with Cal520-AM revealed two types of Ca2+ sparks. a, Single-release Ca2+ spark. Representative raw recordings of a spark time course are presented above DS images. The centroid of the Ca2+ signal for each frame is indicated by a red dot, with the surrounding white circle estimating localization uncertainty within the 300-nm diffraction limit. Contours indicate fitting dimensions corresponding to five levels between the 70th signal percentile and peak spark amplitude. The overall spark time course is indicated in the lower panel, with new Ca2+ released since the previous frame indicated as an orange bar, and the duration of spark detection shown in gray. 0 ms on the time axis is set as the point of earliest spark detection (>2 s.d. above background). b, Multiple-release Ca2+ spark. In a subset of recorded sparks, multiple releases were observed, separated by at least one frame where no Ca2+ release occurred. c, Comparison of properties of single- and multirelease sparks. From left to right: radius of gyration (as a measure of centroid movement), amplitude, TTP, FDHM and DS ΔF/F0. Data are presented as mean ± s.e.m. Differences between groups were tested with two-tailed linear mixed models with Tukey post hoc correction for multiple comparisons. Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001. P values: radius of gyration = 1.11 × 10–5, DS amplitude = 0.0601, amplitude = 0.0395, TTP < 2 × 10–16, FDHM = 7.5 × 10–15. For n values, see Source data. d, Simulation of 1D line-scans reveals mischaracterization of multirelease sparks. Slices were made along various axes in the vertical (V) or horizontal (H) orientation across a region with multiple releases (2D image shows pixel-wise maximal intensity during the spark). Corresponding line-scans and measured spark time courses are illustrated.
