Preparation and Noninvasive Imaging of Biogenic Gas Vesicle Nanostructures

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 Correspondence should be addressed to MGS: mikhail@caltech.edu, Phone: 626-395-8588, 1200 E. California Blvd, MC 210-41, Pasadena, CA 91125. AUTHOR CONTRIBUTIONS: A.L., G.J.L., A.F., S.P.N. and M.G.S. conceived the manuscript layout and coordinated its writing. A.L., G.J.L., A.F., S.P.N., A.L-G., D.M., D.M., R.W.B. and M.G.S developed methods for GV production, functionalization, characterization and ultrasound imaging. M.Y., J.Y. and F.S.F. contributed methods for in vivo ultrasound imaging of GVs. M.K., C.W., L.S. and M.G.S. contributed methods for hyperpolarized xenon MRI of GVs. All authors contributed to writing the manuscript. A.L. compiled and edited the protocol and G.J.L. assembled the figures. COMPETING FINANCIAL INTERESTS: The authors declare no competing financial interests. HHS Public Access Author manuscript Nat Protoc. Author manuscript; available in PMC 2018 October 13. Published in final edited form as: Nat Protoc. 2017 October ; 12(10): 2050–2080. doi:10.1038/nprot.2017.081. A uhor M anscript