Quantifying cardiac nuclei orientation as a function of depth at embryonic day 9. (a) Frontal section of the heart created from DAPI images with region-of-interest (ROI) indicated with the black box. Image colorized in FIJI. (b) Mean cardiac nuclei orientation μ calculated at each depth z (black line) + /− standard uncertainty u with five representative points selected at different depths. Black dashed line indicates 0° (horizontal). (c) Heart ventricle diagram showing the depth axis direction into the left ventricular wall. (d) Five DAPI images corresponding to points in b showing individual cardiac nuclei at different orientations (images used for analysis are 360 × 360 μm). (e) Polar plot showing the corresponding angular distribution of the 2D Fourier transform spectrum (black dots), the von Mises fit (black line), and the mean cardiomyocyte angle (red line) for each depth shown in (d). One representative heart shown with n = 4 hearts for ED9.