Extracellular vesicles (EVs) are highly specialized nanoscale assemblies that deliver complex biological cargos to mediate intercellular communication. EVs are heterogeneous, and characterization of this heterogeneity is paramount to understanding EV biogenesis and activity, as well as to associating them with biological responses and pathologies. Traditional approaches to studying EV composition generally lack the resolution and/or sensitivity to characterize individual EVs, and therefore the assessment of EV heterogeneity has remained challenging. We have recently developed an atomic force microscope IR spectroscopy (AFM-IR) approach to probe the structural composition of single EVs with nanoscale resolution. Here, we provide a step-by-step procedure for our approach and show its power to reveal heterogeneity across individual EVs, within the same population of EVs and between different EV populations. Our approach is label free and able to detect lipids, proteins and nucleic acids within individual EVs. After isolation of EVs from cell culture medium, the protocol involves incubation of the EV sample on a suitable substrate, setup of the AFM-IR instrument and collection of nano-IR spectra and nano-IR images. Data acquisition and analyses can be completed within 24 h, and require only a basic knowledge of spectroscopy and chemistry. We anticipate that new understanding of EV composition and structure through AFM-IR will contribute to our biological understanding of EV biology and could find application in disease diagnosis and the development of EV therapies.
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We acknowledge the University of Sydney for the SOAR Fellowship for W.C. We acknowledge K. Kjoller, Photothermal Spectroscopy Corporation, for consultation and technical support.
The authors declare no competing interests.
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Key references using this protocol
Kim, S. Y., Khanal, D., Tharkar, P., Kalionis, B. & Chrzanowski, W. Nanoscale Horiz. 3, 430–438 (2018): https://doi.org/10.1039/C8NH00048D
Khanal, D. et al. Anal. Chem. 88, 7530–7538 (2016): https://doi.org/10.1021/acs.analchem.6b00665
Khanal, D. et al. Part. Part. Syst. Charact. 35, 1700409 (2018): https://doi.org/10.1002/ppsc.201700409
Integrated supplementary information
Supplementary Figure 1 Troubleshooting: examples of possible errors that can occur if critical steps are not properly implemented in this protocol.
(a) Image of an EV sample on ZnSe prism with crystals formed from the drying of the sample with residual phosphate buffer saline (PBS); (b) Atomic force microscopy (AFM) height image of an EV sample without proper immobilization, causing dragging during imaging; (c) AFM height image and corresponding AFM-IR spectra of EV sample, where laser was misaligned and had to be re-optimized before correct acquisition of the spectra.
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Kim, S.Y., Khanal, D., Kalionis, B. et al. High-fidelity probing of the structure and heterogeneity of extracellular vesicles by resonance-enhanced atomic force microscopy infrared spectroscopy. Nat Protoc 14, 576–593 (2019). https://doi.org/10.1038/s41596-018-0109-3
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