Three-dimensional Super-Resolution Ultrasound Imaging of Chicken Embryos - A Validation Framework for Analysis of Microvascular Morphology

The purpose of this present study was to improve the quantification of microvascular networks depicted in three-dimensional (3D) super-resolution ultrasound (SR-US) images and compare results with matched brightfield microscopy and B-mode ultrasound (US) images. Standard contrast-enhanced US (CEUS) images were collected using a high-frequency US scanner (Vevo 3100, FUJIFILM VisualSonics Inc) equipped with an MX250 linear array transducer. Using a developing chicken embryo as our model system, US imaging was performed after administration of a custom microbubble (MB) contrast agent. Guided by stereo microscopy, MBs were introduced into a perfused blood vessel by microinjection with a glass capillary needle. Volume data was collected by mechanically scanning the US transducer throughout a tissue volume-of-interest (VOI) in $90\ \mu\mathrm{m}$ step increments. CEUS images were collected at each increment and stored as in-phase/quadrature (IQ) data ( $N=2000$ at 152 frames per sec). SR-US images were created for each cross-sectional plane using established data processing methods, and all were then used to form a final 3D volume for subsequent quantification of morphological features. Vessel diameter quantifications from 3D SR-US data exhibited an average error of 1.9% when compared with microscopy images, whereas measures from B-mode US images had an average error of 75.3%. Overall, 3D SR-US images clearly depicted the microvascular network of the developing chicken embryo and measurements of microvascular morphology achieved better accuracy compared to traditional B-mode US.