(E-170) Multiplexed Ultrasound Imaging Using Spectral Analysis on Gas Vesicles

Introduction:: A great deal of medical treatment takes place at a cellular level and in places difficult to reach with traditional imaging. Without a way to track or monitor a particular therapy upon delivery, the effectiveness is temporarily unknown, and the next steps are delayed. High-resolution ultrasound and genetically engineered gas vesicles (GV) advance the visualization of smaller objects at depth. GVs can be isolated from a variety of bacteria and archaea. Because GVs are a gas pocket surrounded by a thin protein shell, they can be used as contrast agents or as a transport system for imaging or drug delivery. Here, we have developed a multiplexed imaging approach to visualize two types of GVs using mid-band fit (MBF) and pseudo-coloring. The goal was imaging in deep tissue and with at least micrometer level resolution.

Materials and Methods:: Three types of GVs were isolated from Anabaena flos-aquae (AnaGV), Bacillus Megaterium (MegaGV), and Serratia sp. ATCC 39006 (SerratiaGV) (Fig 1B). We found that SerratiaGVs made a cluster of GVs (CLUS) which had larger size compared to unclustered SerratiaGVs (UN), AnaGV, and MegaGV (Fig. 1B). The experiment was conducted in an agarose gel phantom and in ex vivo mouse liver specimens (Fig. 1A). MBF images were then reconstructed and classified using a predetermined filter. A pseudo-coloring scheme was used to distinguish different GVs obtained by developed 130 MHz ultrasonic transducers (Fig. 1C-D).

Results, Conclusions, and Discussions:: MBF-based multiplexed imaging approach was able to distinguish CLUS and unclustered GVs including AnaGV, MegaGV, and UN due to their shape variations at a wavelength of 130 MHz. Each type had a different length and size, which produced distinct echo spectrums (Fig. 1B-C). A comparison was made between MBF, and the slope of spectral analysis at micrometer-level resolution. The locations of CLUS and non-clustered GVs were efficiently visualized by the proposed approach (Fig. 1C). Using GVs in the cytoplasm of a cell is recommended as it: 1) increases the size of the imaging target and 2) stays intact longer without being cleared by the host immune system. This study suggests that GVs may be used as diagnostic contrast agents due to their multiplexed imaging capability. As high resolution with deep penetration depth is vital when considering the application of this study, a next step would be to develop a technique that allowed for in vivo deep tissue imaging while optimizing ultrasound frequency.

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