(a) Outline of the experimental setup. The light emitted from a white LED is projected on the sample (S) by a telecentric lens (TL). A telecentric objective (TO) creates the bright-field image of the sample on a CMOS camera. The sample is rotated along the vertical axis by a rotation stage (not shown). (b) Picture of a 5 dpf zebrafish. The acquisition region is indicated by the black rectangle. (c) 3D Reconstruction of the notochord and somites of the zebrafish (grey) obtained with OPT superimposed with the vasculature (red) obtained with Flow-OPT. Scale bar is 100 µm. (d–e) Close-up of the vasculature obtained with Flow- OPT (d) and SPIM (e). Flow-OPT visualizes only the patent vessels. Labeled vessels are: Caudal Vein (CV), Caudal Artery (CA), Vertebral Arteries (VTAs) and Dorsal Longitudinal Anastomotic Vessels (DLAVs).

Image processing in PIV analysis. (a) Raw brightfield images showing a 5 dpf zebrafish tail. A complete dataset is made up by 100 successive frames. (b) Contrast images obtained by subtracting the average intensity image to every brightfield image. They show blood cells as bright spots. (c) Segmented image of vessels superimposed in red color on a raw image. (d–e) Blood cell shift between one frame (d) and the successive one (e). ROIs are depicted as green rectangles. They are placed on two particular vessels, namely the caudal vein (blue vector, small shift) and the caudal artery (red vector, large shift). (f) Planar vector maps obtained with the PIV algorithm. Blue and red asterisks indicate the caudal vein and the caudal artery, respectively. Red arrows show the instantaneous velocity of blood cells. (g) Speed versus time profiles for two vectors chosen in correspondence of the asterisks in (f). The red curve corresponds to arterial speed profile and shows peaks that relate to the cardiac cycle phases. The blue one corresponds to venous speed profile and shows a more continuous trend, with only large oscillations slightly shifted in phase with respect to the aortic one. Scale bars are: (a–c, f) 50 µm; (d–e) 30 µm.