Quantifying vessel orientation as a function of depth at embryonic day 13. (a) Heart surface created from DAPI images with region-of-interest (ROI) indicated with black box. (b) Mean helical angle (red line, left y-axis) calculated at each depth with a moving window (+ /− 15 μm), with the standard uncertainty as error bars. The concentration parameter κ was obtained from a von Mises fit performed at each depth (black dashed line, right y-axis). Layers of interest indicated with grey/white shaded regions. Helical angle of 0° indicated with a dashed red line. (c) Vessel images with color coding for helical angle at increasing depths (panels 1–5, depth range indicated in b). White arrows indicate large vessels. (d) Helical angle distribution matched to above panels in (c) (same color scale used). (e) Transmural cross-section of vessels from the epicardium (Epi) to the endocardium (Endo). Black tick marks separate the five regions in depth shown in (b). One representative heart shown with n = 4 hearts for ED13. Images rendered in Amira with angles calculated in MATLAB.