Multiplexed ultrasound imaging using clustered gas vesicles and spectral imaging

Abstract

Current advances in ultrasound imaging techniques including super-resolution ultrasound imaging allows us to visualize microvasculature in biological specimens using microbubbles. However, microbubbles diffuse in blood stream limiting imaging acquisition and frame subtraction scheme of super-resolution ultrasound imaging cannot improve spatial resolution without moving microbubbles. Fluorescent proteins revolutionized to understand molecular and cellular functions in biological systems. Here, we devised a panel of gas vesicles to realize multiplexed ultrasound imaging to uniquely visualize locations of different species of gas vesicles. Mid-band fit spectral imaging technique demonstrated that stationary gas vesicles were efficiently localized in gel phantom and murine liver specimens by visualizing three-dimensional vessel structures. Clustered and unclustered gas vesicles were phagocytosed by murine macrophages to serve as carriers and beacons for the proposed multiplexed and single cell level imaging technique. The spatial distribution of macrophages containing clustered and unclustered gas vesicles were reconstructed by mid-band fit spectral imaging with pseudo-coloring scheme.