Comparison of calcium sparks in WT and MO preparations. (A) Confocal images of fluo-4 AM–loaded myocytes isolated, the same day, from 72 hpf WT and MO-injected larvae. The bathing solution included 0.3 mM caffeine to stimulate SR calcium release. (Left) X-Y images. The bright puncta in these images are not Ca²⁺ sparks but localized regions of high resting fluorescence that persist with repeated scanning. These bright puncta may arise from fluo-4 molecules contained within a small organelle with elevated free [Ca²⁺] (Hollingworth et al., 2001). (Middle and right) X-T images from longitudinal line scans of the myocytes. The horizontal banding pattern in the X-Y and X-T images likely arises from binding of the dye to sarcomeric structures, here spaced at distances close to 1.85 µm. Ca²⁺ sparks are the brief, spatially localized increases in fluorescence (bright orange) that are clearly seen in the WT X-T images; they vary in frequency from cell to cell. The bottom right panel shows sparks from one of the few sparking MO cells. (B, left) Mean spark frequency measured in 42 WT and 39 morphant cells from four separate experiments of each type. (Right) The distribution of spark frequency in the WT and morphant cells. For frequencies above 0, the symbols are plotted at the center frequency of bin widths of 0.1 sarcomere⁻¹ s⁻¹. In the case of the morphant cells, sparks were sufficiently rare that the mean value of the frequencies in the first bin is 0.0065 ± 0.0028 sarcomere⁻¹ s⁻¹ (n = 8; ±SEM), considerably less than the position of the central frequency (0.05 sarcomere⁻¹ s⁻¹).