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Fig. 3 Blood cell attraction towards the ISV follows two oscillatory phase-shifted forces. (A,B) Schematic representation (A) and in vivo live image (B) to detail the optical tweezing experiments undertaken to characterize blood cell motion near the ISV entry. Detection of the constrained blood cell motion within the optical tweezer reflects the effects of surrounding flow forces. The reconstruction of the displacement along the x and y axes allowed us to decipher the force applied to the cell (fx and fy). (C) In vivo, blood cells are trapped at different positions in the DA near and at the ISV entry, with dotted red circles representing the immobilizing focus of the optical tweezer in the same embryo (B,C). The corresponding motion patterns are shown in D-F. (D-F) Real-time blood cell displacement (represented by the signal from the quadrative detector, dV) is plotted as a function of time, illustrating the typical motion resulting from blood flow. When blood cells are positioned in the DA away from the ISV, the displacement along the x-axis is predominant, showing the effect of pulsatile flow in the DA (D). When positioned near the ISV entry, an additional motion in Y is observed that is always out of phase from the X direction (Δφ) (E). The attractive force is thus oscillating and is not fully coupled with DA flow. When positioned in the ISV, blood cell motion in X and Y directions are in phase again (F). (G) Phase-shift (Δφ) mapping at the ISV entry demonstrates the attractive force is correlated with the proximity to the ISV entry and shows the XY correlation decrease when closer to the ISV. (H) Force map in pN obtained after optical-tweezer measurement. The maps in G and H were obtained by analysing three different embryos.