Probing cilia-driven flow in living embryos using femtosecond laser ablation and fast imaging

Embryonic development strictly depends on fluid dynamics. As a consequence, understanding biological fluid dynamic is essential since it is unclear how flow affects development. For example, the specification of the left-right axis in vertebrates depends on fluid flow where beating cilia generate a directional flow necessary for breaking the embryonic symmetry in the so-called left-right organizer. To investigate flow dynamics in vivo proper labeling methods necessitate approaches that are compatible with both normal biology and in vivo imaging. In this study, we describe a strategy for labeling and analyzing microscopic fluid flows in vivo that meets this challenge. We developed an all-optical approach based on three steps. First we used sub-cellular femtosecond laser ablation to generate fluorescent micro-debris to label the flow. The non-linear effect used in this technique allows a high spatial confinement and a low invasiveness, thus permitting the targeting of sub-cellular regions deep inside the embryo. Then, we used fast confocal imaging and 3Dparticle tracking were used to image and quantify the seeded flow. This approach was used to investigate the flow generated within zebrafish left-right organizer, a micrometer scale ciliated vesicle located deep inside the embryo and involved in breaking left-right embryonic symmetry. We mapped the velocity field within the vesicle and surrounding a single beating cilium, and showed that this method can address the dynamics of cilia-driven flows at multiple length scales. We could validate the flow features as predicted from previous simulations. Such detailed descriptions of fluid movements will be valuable in unraveling the relationships between cilia-driven flow and signal transduction. More generally, this all-optical approach opens new opportunities for investigating microscopic flow in living tissues.