期刊
NATURE PROTOCOLS
卷 12, 期 10, 页码 2050-2080出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2017.081
关键词
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资金
- National Institutes of Health [R01-EB018975]
- DARPA [W911NF-14-1-0111]
- CIHR [FDN148367]
- HFSP [RGP0050/2016]
- NSF [1144469]
- NSERC
- Caltech Summer Undergraduate Research Fellowship
- HFSP Cross-Disciplinary Postdoctoral Fellowship
- Heritage Medical Research Institute
- Burroughs Wellcome Fund
- Pew Charitable Trust
- Sontag Foundation
- David and Lucile Packard Foundation
- Michael J. Fox Foundation for Parkinson's Research [12549]
- Koselleck Grant by the German Research Foundation (DFG) [SCHR 995/5-1]
- Beckman Foundation
- Gordon and Betty Moore Foundation
- Agouron Institute
- HHMI
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 (DLSLS) and transmission electron microscopy (TETEM) 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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