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High-fidelity probing of the structure and heterogeneity of extracellular vesicles by resonance-enhanced atomic force microscopy infrared spectroscopy


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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Fig. 1: Workflow of AFM-IR spectroscopy for EVs.
Fig. 2: System optimization before the collection of nano-IR spectra.
Fig. 3: Collection of AFM-IR spectra from DMSC23 EVs collected from DMSC23 cells cultured in hypoxia.
Fig. 4: AFM-IR images with corresponding average spectra for subpopulations of EVs isolated from CMSC29 and DMSC23 cells.
Fig. 5: Collection of AFM-IR spectra from multiple points on individual EVs.

Data availability

All data generated or analyzed during this study are included in this published article and its Supplementary Information.


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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.

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Authors and Affiliations



W.C. conceived, designed and oversaw the project; S.Y.K. co-designed experiments and performed the isolation of EVs; D.K. performed the AFM-IR; and B.K. isolated and developed the stem cell lines. S.Y.K., D.K., B.K. and W.C. wrote the manuscript.

Corresponding author

Correspondence to Wojciech Chrzanowski.

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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):

Khanal, D. et al. Anal. Chem. 88, 7530–7538 (2016):

Khanal, D. et al. Part. Part. Syst. Charact. 35, 1700409 (2018):

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).

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