Because of the opacity of living tissue, in vivo PIV has only been conducted on a limited basis in small transparent vessels in rodents,25,26 chicken embryos,27–29 and zebrafish embryos.2,30 These studies have either injected tracer particles25,28–30 or used the red blood cells themselves as the tracer particles.2,26,27 Hove et al. applied PIV analysis to the heart using bright field imaging, using the red blood cells as tracers.2 This method produces images in which the signal from the blood cells is contained within the same gray scale as other objects that are not of interest, for instance the heart wall. Because of the nature of PIV, both stationary and moving objects will contribute to the cross correlation and thus will impact the final velocity measurement. Hove et al. identified the presence of the heart wall and superficial tissue in their images as interfering with accurate velocity calculation, suggesting their velocity measurements represented an underestimation of the true value.2 Lu et al. removed the impact of the wall by utilizing defocusing particle tracking velocimetry (PTV) on fluorescent tracer particles injected into the bloodstream, measuring velocity in three dimensions.30 The use of fluorescent particles allows for the optical filtering of the contribution of the heart wall from the velocity calculation improving the accuracy of the flow measurements. However, as PTV requires the distance between tracer particles to be larger than their displacement, there is a severe limitation in the spatial resolution of the technique, which provides only sparse instantaneous vector fields. Other studies in the zebrafish have utilized high-speed confocal or selective plane illumination microscopy of fluorescently labeled samples, together with the synchronization of confocal slices, to form dynamic 3-D models31 for examining the pumping mechanism of the embryonic heart,1 and investigating valve formation.3,32 Despite the significant findings of these studies, the requirement for fluorescent imaging reduces the temporal resolution because of the longer integration times required.