Figure 3. Mapping simultaneous V_m and Ca²⁺ in the zebrafish heart in vivo. (A) Fluorescence of GCaMP5G in a fish expressing CaViar under the cmlc2 promoter (Supplementary Movie 1). (B) Single optical section of a zebrafish heart expressing CaViar at 4 dpf (Supplementary Movie 2). Cell membranes fluoresced in both the voltage channel (top) and the Ca²⁺ channel (bottom) as the AP propagated from the atrium to the ventricle. (C) Map of AP isochrones overlaid on a three-dimensional reconstruction of the heart. AP onsets were measured as time to reach 5% of maximum on the rising edge, with sub-frame timing achieved through spline interpolation. (D) Three-dimensional reconstruction of the electrical AP (Supplementary Movie 3). Scale bars in (A?D) 50 ?m.

Figure 4. Voltage and calcium transients of the atrium and ventricle in an embryonic zebrafish heart during development. Top: Fluorescence images of zebrafish hearts expressing CaViar showed transition of the heart tube into two chambers. Scale bars 50 ?m. Bottom: Dual-wavelength imaging of CaViar reported voltage (red), and calcium (blue) transients in the atrium (lighter shade) and ventricle (darker shade) at (A) 36 hpf, (B) 54 hpf, (C) 102 hpf. At 36 hpf, low expression of Arch(D95N) led to higher noise than at other time-points. Blue illumination intensity was minimized at all time-points to minimize photo-inactivation of blebbistatin. This occasionally led to increased noise in the GCaMP5G fluorescence (e.g., at 102 hpf).