An optimized method for delivering flow tracer particles to intravital fluid environments in the developing zebrafish

Growing evidence suggests that intravital flow-structure interactions are critical morphogens for normal embryonic development and disease progression, but fluid mechanical studies aimed at investigating these interactions have been limited in their ability to visualize and quantify fluid flow. In this study, we describe a protocol for injecting small (≤1.0 μm) tracer particles into fluid beds of the larval zebrafish to facilitate microscale fluid mechanical analyses. The microinjection apparatus and associated borosilicate pipette design, typically blunt-tipped with a 2-4 micron tip O.D., yielded highly linear (r(2)=0.99) in vitro bolus ejection volumes. The physical characteristics of the tracer particles were optimized for efficient particle delivery. Seeding densities suitable for quantitative blood flow mapping (≥50 thousand tracers per fish) were routinely achieved and had no adverse effects on zebrafish physiology or long-term survivorship. The data and methods reported here will prove valuable for a broad range of in vivo imaging technologies [e.g., particle-tracking velocimetry, μ-Doppler, digital particle image velocimetry (DPIV), and 4-dimensional-DPIV] which rely on tracer particles to visualize and quantify fluid flow in the developing zebrafish.