(A) The integration of light-sheet and light-field microscopy captures the contracting myocardium (cmlc2:GFP) and traveling blood cells (gata1a:dsRed) at 200 volumes per second. The light field-generated blood cell images (upper illustration) are synchronized with the light sheet-generated myocardial cross-section images (lower illustration). (B) GFP-labeled cardiomyocyte light chain (cmlc2) and dsRed-labeled blood cells (gata1a) are simultaneously visualized in a 3-D reconstructed embryonic heart. The red arrows indicate the direction of blood flow. A: atrium; V: ventricle. (C & D) A time sequence of a cardiac cycle is illustrated in the coronal (C) and sagittal (D) plane, respectively. During diastole (light-blue bar), the blood cells traverse the atrioventricular canal (AVC) into ventricle. During systole, the blood cells travel through the outflow tract (OFT).

(A) Images of the myocardium from 2 different time points are overlaid to demonstrate the myocardial displacement. The arrow indicates the direction of displacement. (B) Deformable image registration (DIR) is used to infer the myocardial displacement between frames. Each vector indicates the direction and magnitude of the displacement velocity as color-coded by the magnitude (μm/s) in the corresponding voxel. Intracardiac blood cells (red) and flow direction (white arrow) are superimposed with the vector field. (C) A representative heatmap depicts the segmental variations in the magnitude of the myocardial velocity. (D) Particle tracking velocity follows the trajectories of the blood cells during the cardiac cycles. V: ventricle; OFT: outflow tract; AVC: atrioventricular canal. (E) The vector field represents the direction and speed of the blood cells. Dissected myocardium (green) and flow direction (white arrow) are superimposed with the vector field. (F) The velocity vector fields for myocardial displacement and the traveling blood cells are merged.

(A) The trajectory of the blood cells is depicted as the flow centerline, starting from the AV canal (dark colors) to the outflow tract (light colors). (B) The myocardium is divided into 4 segments to assess regional variations in the vector field. (C) The velocity of individual blood cells is projected onto the flow centerline, and the mean flow velocity is plotted as a function of time. Myocardial velocity in each segment is quantified as the magnitude-weighted mean vector for each segment, as plotted in the color line. The black dotted line demonstrates the average velocity magnitude of the entire ventricle. (D) Six representative time points (from t₁ to t₆) of the heatmaps are presented for the myocardium (upper panels, color-coded in vector magnitude) and vector maps (lower panels). Red arrows indicate the rotational direction of the mean vector for each segment.

(A) Blood flow velocity is depicted during a cardiac cycle. To compare among different heart rates, we normalize the time to one cardiac cycle for each zebrafish embryo. (B) The ventricle is divided into 4 segments. Arrows indicate the flow direction. The dotted line delineates the atrium. (C) The tracing of the myocardial velocity is normalized to one cardiac cycle in each segment. (D) The mean of relative displacement velocity is depicted in each segment during systole and diastole. The relative velocity in each segment is compared as the ratio of the segmental velocity to the mean of global velocity during a cardiac cycle. Kruskal-Wallis test and Dunn’s multiple comparisons test are performed in GraphPad Prism (** p≤0.01; *** p≤0.001, n = 7).