Figure 4

(A) Original data sometimes suffered from dimly visualized vessels (blue arrowhead) and had a double-peak cross-vessel intensity distribution (red arrowhead), which was particularly pronounced in bigger vessels. (B) Application of General Filtering (GF) failed to enhance dim vessels. (C) Application of Tubular Filtering (TF) successfully enhanced dim vessels and extracted vessels with double-peak intensity distributions as one segment (red arrowhead). (D) Assessment of the cross-vessel intensity distribution showed that the original double-peak distribution (black line; averages of n = 12 embryos) was successfully converted into a single-peak when scale reached vessel size (e.g., 9.3604 or 10.6848), while larger scales were found to reduce central intensity and blur vessel edges (e.g., 15.359 and above). This was further validated by visual investigation (E,F) where the TF response is shown in green overlaid onto the original image in red.