Multiplexed Ultrasound Imaging Using Spectral Analysis on Gas Vesicles

Abstract

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.

Gas vesicle-blood interactions enhance ultrasound imaging contrast

“…Potential techniques include genetic functionalization of the GV surface 21 , ligand conjugation to serum-equilibrated GVs 42 , fusion of peptides to recruit specific serum proteins 43 , and adsorption of an artificial corona 44 . These strategies could also enable in vitro diagnostic applications, such as clustering-based detection in which GVs selectively enriched with specific proteins are combined with the corresponding antibodies, allowing for rapid optical and acoustic measurements via flotation and enhanced ultrasound backscatter 14,45 , respectively. Furthermore, the GV corona can aid in proteomics by reducing the dynamic range of protein concentrations in biological fluids, facilitating detection of lowabundance components 46 .…”

Gas Vesicle–Blood Interactions Enhance Ultrasound Imaging Contrast

“…Potential techniques include genetic functionalization of the GV surface, 22 wrapping with cell membranes, 48 ligand conjugation to serum-equilibrated GVs, 49 fusion of peptides to recruit specific serum proteins, 50 and adsorption of an artificial corona. 51 These strategies could also enable in vitro diagnostic applications, such as clustering-based detection in which GVs selectively enriched with specific proteins are combined with the corresponding antibodies, allowing for rapid optical and acoustic measurements via flotation and enhanced ultrasound backscatter, 15,52 concentrations in biological fluids, facilitating detection of lowabundance components. 53 GVs are advantageous for these applications due to their easy buoyancy-based isolation and use of structural proteins as internal concentration standards.…”