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.

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This research was supported by the National Institutes of Health (R01-EB018975 to M.G.S.), DARPA (W911NF-14-1-0111 to M.G.S.), CIHR (grant no. FDN148367 to F.S.F.) and HFSP (RGP0050/2016 to M.G.S. and L.S.). A.L. is supported by an NSF graduate research fellowship (no. 1144469). A.F. is supported by an NSERC graduate fellowship. S.P.N. is supported by a Caltech Summer Undergraduate Research Fellowship. D. Maresca is supported by an HFSP Cross-Disciplinary Postdoctoral Fellowship. Research in the Shapiro laboratory is also supported by the Heritage Medical Research Institute, the Burroughs Wellcome Fund, the Pew Charitable Trust, the Sontag Foundation and the David and Lucile Packard Foundation. Research in the Schröder laboratory is also supported by the Michael J. Fox Foundation for Parkinson's Research (no. 12549) and a Koselleck Grant by the German Research Foundation (DFG; project SCHR 995/5-1). We acknowledge the Jensen Lab and the Beckman Resource Center for Electron Microscopy (EM) at the California Institute of Technology for technical guidance and for allowing us to use their resources. The EM facility is funded by the Beckman Foundation, the Gordon and Betty Moore Foundation, the Agouron Institute and the HHMI.

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Author notes

    • Anupama Lakshmanan
    • , George J Lu
    • , Arash Farhadi
    •  & Suchita P Nety

    These authors contributed equally to this work.


  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.

    • Anupama Lakshmanan
    •  & Arash Farhadi
  2. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA.

    • George J Lu
    • , Suchita P Nety
    • , Audrey Lee-Gosselin
    • , David Maresca
    • , Raymond W Bourdeau
    • , Dina Malounda
    •  & Mikhail G Shapiro
  3. Molecular Imaging, Department of Structural Biology, Leibniz-Forschungsinstitute für Molekulare Pharmakologie, Berlin, Germany.

    • Martin Kunth
    • , Christopher Witte
    •  & Leif Schröder
  4. Sunnybrook Research Institute, University of Toronto, Toronto, Ontario, Canada.

    • Melissa Yin
    • , Judy Yan
    •  & F Stuart Foster
  5. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

    • F Stuart Foster


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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. Maresca, D. Malounda, R.W.B. and M.G.S developed methods for GV production, functionalization, and characterization, as well as for 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 interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mikhail G Shapiro.

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