Gas vesicles (GVs) are a unique class of gas-filled protein nanostructures that 
are detectable at subnanomolar concentrations and whose physical properties 
allow them to serve as highly sensitive imaging agents for ultrasound and MRI. 
Here we provide a protocol for isolating GVs 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 MRI. GVs can be isolated from natural cyanobacterial and 
haloarchaeal host organisms or from Escherichia coli expressing a heterologous 
GV 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, whereas 
dynamic light scattering (DLS)and transmission electron microscopy (TEM) are 
used to determine nanoparticle size and morphology, respectively. GVs 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 
GV-bound and dissolved xenon-a technique currently implemented in vitro. Taking 
3-8 d to prepare, these genetically encodable nanostructures enable multimodal, 
noninvasive biological imaging with high sensitivity and potential for molecular 
targeting.