Fig. 3: Correlation of single- and multiple-release Ca2+ sparks to RyR clusters.
a, Super-resolution PALM imaging of RyRs at the cell surface (left) with enlargement of the indicated region and adaptive thresholding. Correlative Ca2+ imaging revealed superimposition of a Ca2+ spark centroid (red dot, localization uncertainty is indicated by a white circle) to an RyR cluster (right, upper panels). DS analyses revealed only a single Ca2+ release pulse at 4 ms, and no further release at 8 ms (lower right panels). b, For multiple-release Ca2+ sparks, DS improved mapping to RyR cluster origins, and revealed ‘traveling’ of Ca2+ release between adjacent clusters. In the presented example, distinct release events occurred at 4 ms and 8 ms, which summated to generate a large Ca2+ spark. c, Histogram showing distance from the centroid of each DS release event to the nearest RyR cluster. Dotted line indicates the expected localization accuracy of 300 nm, used as a cutoff for subsequent correlation analysis. n = 4 animals, 11 cells, 359 sparks, 608 frames. d, Left, visualization of the jSR using background Cal520 fluorescence (red), together with RyR positions (white) and Ca2+ release events (white crosses). Multiple-release sparks were observed to remain within the same jSR (middle) or travel to distant jSR (right). Experiments in a, b and d were repeated independently in cells from four hearts with similar results.
