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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 3D-particle
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.
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