High-Resolution Ultrasound Molecular Imaging With Incremental Burst Sequence: In Vitro and In Vivo Validation

Ultrasound localization microscopy (ULM) enables super-resolution ultrasound (SRUS) imaging of microvasculature, while ultrasound molecular imaging (USMI) characterizes molecular signatures using microbubbles (MBs) targeted to specific biomarkers. Although the colocalization of SRUS and USMI has been demonstrated previously, USMI resolution is limited by ultrasound diffraction-based effects and does not match the super-resolved microvasculature. This study introduces the incremental burst sequence (IBS) method to induce the population of polydisperse targeted MBs to burst progressively, achieving MB spatial separation and enabling high-resolution USMI (HR-USMI) localization. The IBS method employs interleaved imaging and bursting pulses, with transmit voltages of bursting pulses incrementally increased to produce a gradual rise in the acoustic pressure. IBS is first validated optically in vitro using a cellulose tubing phantom, and MB remaining count during IBS is measured. Thereafter, in vivo validation is performed in a murine tumor model, and the intratumoral targeted MB signal intensity is measured during IBS. Furthermore, high-frame-rate data for SRUS and IBS data for HR-USMI are acquired from a single bolus injection of MBs to generate composite images with high-resolution molecular signatures superimposed on the tumor microvasculature. Both in vitro and in vivo results validate the technical feasibility of the proposed IBS method. In addition, we demonstrate that higher bursting pulse repetitions lead to a faster disruption of the MB population during IBS. Finally, HR-USMI signals localized within a $50\times 50~\mu $ m grid are aligned with microvessels resolved better than $100~\mu $ m, presenting a combination of molecular signatures and anatomical structures at fine resolution.