Multiplexed Ultrasound Imaging Using Spectral Analysis on Gas Vesicles

Current advances in ultrasound imaging techniques combined with the next generation contrast agents such as gas vesicles (GV) revolutionize the visualization of biological tissues with spatiotemporal precision. In optics, fluorescent proteins enable understanding of molecular and cellular functions in biological systems due to their multiplexed imaging capability. Here, a panel of GVs is investigated using mid-band fit (MBF) spectral imaging to realize multiplexed ultrasound imaging to uniquely visualize locations of different types of stationary GVs. The MBF spectral imaging technique demonstrates that stationary clustered GVs are efficiently localized and distinguished from unclustered GVs in agarose gel phantom and 3D vessel structures are visualized in ex vivo mouse liver specimens. Mouse macrophages serve as carriers of clustered and unclustered GVs and multiplexing beacons to report cells' spatial locations by emitting distinct spectral signals. 2D MBF spectral images are reconstructed, and pixels in these images are classified depending on MBF values by comparing predetermined filters that predict the existence of cells with clustered and unclustered GVs. This pseudo-coloring scheme clearly distinguishes the locations of two classes of cells like pseudo-color images in fluorescence microscopy.