3D blood vessel mapping of adult zebrafish using high frequency ultrasound ultrafast Doppler imaging

The zebrafish is an ideal vertebrate for studying developmental biology and genetics due to its external development and optical transparency in embryonic stages. Recently, with the rapid generation rate and genetic accessibility, adult zebrafish have been used to model tumorigenesis, angiogenesis, and regeneration in vivo. However, once the zebrafish has fully matured, especially wild-type lines, it develops stripes that run along the body and thus lose transparency. Owing to that, conventional optical imaging techniques will have difficulty for imaging the internal anatomy and vasculature. High frequency ultrasound imaging is one useful modality for overcoming this opacity problem. However, using conventional ultrasound Doppler imaging to depict the vascular imaging is limited by diffraction and frame-rates. Hence, the purpose of this study is to utilize ultrafast ultrasound with high frequency transducer to reconstruct the 3D blood mapping of adult zebrafish. A high-frequency transducer (operational frequency = 31.25 MHz, 256 elements) connected to a programmable ultrafast ultrasound system with a custom-built adapter was used in this study as an imaging platform. Experiments were carried out by 6–8 months old wild-type (AB line) adult zebrafish. The ultrafast imaging was performed from a five angles plane wave (from — 3° to 3° with 1. 5° incremental steps) at a total frame rate of 1000 fps. The linear array transducer was fixed on a 3-axis stepper motor stage and the transducer was placed at the left-right coordinates of the center of the fish, to obtain the ultrafast imaging across the dorsal side of zebrafish. Then, the acquisition data was compounded to improve signal-to-noise-ratio (SNR) and was processed with a band-pass spatiotemporal filter to extract the blood flow information for Doppler imaging. Following reconstruction, segmented images were skeletonized to produce the 3D blood map of dorsal side on zebrafish with high voxel resolution (19.68 μm × 24.64 μm × 50 μm). The result shows a strong potential for depicting the blood mapping of the adult zebrafish using ultrafast Doppler imaging, which would help for modeling adult zebrafish on tumorigenesis and tissue regeneration via ultrasound images in vivo.