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* The reported Toff for each variant is the lowest temperature at which fluorescence could be detected above noise. Tmax is the temperature at which fluorescence was maximal. Supplementary Table 2-Genetic constructs used in the study All plasmids were constructed using the pETDuet-1 backbone (EMD Biosciences) with the relevant thermal biosensor elements replacing multiple cloning sites 1 and 2.

Ultrasound is among the most widely used biomedical imaging modalities, but has limited ability to image specific molecular targets due to the lack of suitable nanoscale contrast agents. Gas vesicles – genetically encoded protein nanostructures isolated from buoyant photosynthetic microbes – have recently been identified as nanoscale reporters for ultrasound. Their unique physical properties give gas vesicles significant advantages over conventional microbubble contrast agents, including nanoscale dimensions and inherent physical stability. Furthermore, as a genetically encoded material, gas vesicles present the possibility that the nanoscale mechanical, acoustic and targeting properties of an imaging agent can be engineered at the level of its constituent proteins. Here, we demonstrate that genetic engineering of gas vesicles results in nanostructures with new mechanical, acoustic, surface and functional properties to enable harmonic, multiplexed and multimodal ultrasound imaging, as well as cell-specific molecular targeting. These results establish a biomolecular platform for the engineering of acoustic nanomaterials.

Gas vesicles are a unique class of gas-filled protein nanostructures whose physical properties allow them to serve as highly sensitive imaging agents for ultrasound and magnetic resonance imaging (MRI), detectable at sub-nanomolar concentrations. Here we provide a protocol for isolating gas vesicles from native and heterologous host organisms, functionalizing these nanostructures with moieties for targeting and fluorescence, characterizing their biophysical properties and imaging them using ultrasound and magnetic resonance imaging. Gas vesicles can be isolated from natural cyanobacterial and haloarchaeal host organisms or from E. coli expressing a heterologous gas vesicle gene cluster, and purified using buoyancy-assisted techniques. They can then be modified by replacing surface-bound proteins with engineered, heterologously expressed variants, or through chemical conjugation, resulting in altered mechanical, surface and targeting properties. Pressurized absorbance spectroscopy is used to characterize their mechanical properties, while dynamic light scattering and transmission electron microscopy are used to determine nanoparticle size and morphology, respectively. Gas vesicles can then be imaged with ultrasound in vitro and in vivo using pulse sequences optimized for their detection versus background. They can also be imaged with hyperpolarized xenon MRI using chemical exchange saturation transfer between gas vesicle-bound and dissolved xenon – a technique currently implemented in vitro. Taking 3–8 days to prepare, these genetically encodable nanostructures enable multi-modal, noninvasive biological imaging with high sensitivity and potential for molecular targeting.