Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Abstract

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.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

References

  1. Barile, L. & Vassalli, G. Exosomes: therapy delivery tools and biomarkers of diseases. Pharmacol. Ther. 174, 63–78 (2017).

    CAS  Article  Google Scholar 

  2. Tkach, M. & Théry, C. Communication by extracellular vesicles: where we are and where we need to go. Cell 164, 1226–1232 (2016).

    CAS  Article  Google Scholar 

  3. Iraci, N., Leonardi, T., Gessler, F., Vega, B. & Pluchino, S. Focus on extracellular vesicles: physiological role and signalling properties of extracellular membrane vesicles. Int. J. Mol. Sci. 17, 171 (2016).

    Article  Google Scholar 

  4. Uta, E. & Joanne, L. Analytical challenges of extracellular vesicle detection: a comparison of different techniques. Cytometry Part A 89, 123–134 (2016).

    Article  Google Scholar 

  5. Jørgensen, M. et al. Extracellular vesicle (EV) array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping. J. Extracell. Vesicles 2, 20920 (2013).

    Article  Google Scholar 

  6. Wang, X. et al. Unique molecular profile of exosomes derived from primary human proximal tubular epithelial cells under diseased conditions. J. Extracell. Vesicles 6, 1314073 (2017).

    Article  Google Scholar 

  7. Jakobsen, K. R. et al. Exosomal proteins as potential diagnostic markers in advanced non-small cell lung carcinoma. J. Extracell. Vesicles 4, 26659 (2015).

    Article  Google Scholar 

  8. Kowal, J. et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc. Natl. Acad. Sci. USA 113, E968–E977 (2016).

    CAS  Article  Google Scholar 

  9. De Toro, J., Herschlik, L., Waldner, C. & Mongini, C. Emerging roles of exosomes in normal and pathological conditions: new insights for diagnosis and therapeutic applications. Front. Immunol. 6, 203 (2015).

    Article  Google Scholar 

  10. Revenfeld, A. L. et al. Diagnostic and prognostic potential of extracellular vesicles in peripheral blood. Clin. Ther. 36, 830–846 (2014).

    Article  Google Scholar 

  11. Kim, S. Y., Khanal, D., Tharkar, P., Kalionis, B. & Chrzanowski, W. None of us is the same as all of us: resolving the heterogeneity of extracellular vesicles using single-vesicle, nanoscale characterization with resonance enhanced atomic force microscope infrared spectroscopy (AFM-IR). Nanoscale Horiz. 3, 430–438 (2018).

    CAS  Article  Google Scholar 

  12. Minciacchi, V. R., Zijlstra, A., Rubin, M. A. & Di Vizio, D. Extracellular vesicles for liquid biopsy in prostate cancer: where are we and where are we headed? Prostate Cancer Prostatic Dis. 20, 251–258 (2017).

    CAS  Article  Google Scholar 

  13. Armstrong, D. & Wildman, D. E. Extracellular vesicles and the promise of continuous liquid biopsies. J. Pathol. Transl. Med. 52, 1–8 (2018).

    Article  Google Scholar 

  14. Stanko, P., Altanerova, U., Jakubechova, J., Repiska, V. & Altaner, C. Dental mesenchymal stem/stromal cells and their exosomes. Stem Cells Int. 2018, 8973613 (2018).

    Article  Google Scholar 

  15. Zhu, Q., Heon, M., Zhao, Z. & He, M. Microfluidic engineering of exosomes: editing cellular messages for precision therapeutics. Lab Chip 18, 1690–1703 (2018).

  16. Ye, Z. et al. Methotrexate-loaded extracellular vesicles functionalized with therapeutic and targeted peptides for the treatment of glioblastoma multiforme. ACS Appl. Mater. Interfaces 10, 12341–12350 (2018).

    CAS  Article  Google Scholar 

  17. Yang, T. et al. Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio. Pharm. Res. 32, 2003–2014 (2015).

    CAS  Article  Google Scholar 

  18. Willms, E. et al. Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci. Rep. 6, 22519 (2016).

    CAS  Article  Google Scholar 

  19. Giebel, B. On the function and heterogeneity of extracellular vesicles. Ann. Transl. Med. 5, 150 (2017).

    Article  Google Scholar 

  20. Hammiche, A. et al. Photothermal FT-IR spectroscopy: a step towards FT-IR microscopy at a resolution better than the diffraction limit. Appl. Spectrosc. 53, 810–815 (1999).

    CAS  Article  Google Scholar 

  21. Anderson, M. S. Infrared spectroscopy with an atomic force microscope. Appl. Spectrosc. 54, 349–352 (2000).

    CAS  Article  Google Scholar 

  22. Dazzi, A., Prazeres, R., Glotin, F. & Ortega, J. M. Local infrared microspectroscopy with subwavelength spatial resolution with an atomic force microscope tip used as a photothermal sensor. Opt. Lett. 30, 2388–2390 (2005).

    CAS  Article  Google Scholar 

  23. Xiao, L. & Schultz, Z. D. Spectroscopic imaging at the nanoscale: technologies and recent applications. Anal. Chem. 90, 440–458 (2018).

    CAS  Article  Google Scholar 

  24. Dazzi, A. & Prater, C. B. AFM-IR: technology and applications in nanoscale infrared spectroscopy and chemical imaging. Chem. Rev. 117, 5146–5173 (2017).

    CAS  Article  Google Scholar 

  25. Centrone, A. Infrared imaging and spectroscopy beyond the diffraction limit. Annu. Rev. Anal. Chem. 8, 101–126 (2015).

    CAS  Article  Google Scholar 

  26. Hinrichs, K. & Shaykhutdinov, T. Polarization-dependent atomic force microscopy-infrared spectroscopy (AFM-IR): infrared nanopolarimetric analysis of structure and anisotropy of thin films and surfaces. Appl. Spectrosc. 72, 817–832 (2018).

    CAS  Article  Google Scholar 

  27. Khanal, D. et al. Biospectroscopy of nanodiamond-induced alterations in conformation of intra- and extracellular proteins: a nanoscale IR study. Anal. Chem. 88, 7530–7538 (2016).

    CAS  Article  Google Scholar 

  28. Quaroni, L., Pogoda, K., Wiltowska-Zuber, J. & Kwiatek, W. M. Mid-infrared spectroscopy and microscopy of subcellular structures in eukaryotic cells with atomic force microscopy-infrared spectroscopy. RSC Adv. 8, 2786–2794 (2018).

    CAS  Article  Google Scholar 

  29. Dazzi, A., Glotin, F. & Carminati, R. Theory of infrared nanospectroscopy by photothermal induced resonance. J. Appl. Phys. 107, 124519 (2010).

    Article  Google Scholar 

  30. Qin, S. Q. et al. Establishment and characterization of fetal and maternal mesenchymal stem/stromal cell lines from the human term placenta. Placenta 39, 134–146 (2016).

    CAS  Article  Google Scholar 

  31. Kusuma, G. D. et al. Mesenchymal stem/stromal cells derived from a reproductive tissue niche under oxidative stress have high aldehyde dehydrogenase activity. Stem Cell Rev. Rep. 12, 285–297 (2016).

    CAS  Article  Google Scholar 

  32. Borges, F. T., Reis, L. A. & Schor, N. Extracellular vesicles: structure, function, and potential clinical uses in renal diseases. Braz. J. Med. Biol. Res. 46, 824–830 (2013).

    CAS  Article  Google Scholar 

  33. Thakur, B. K. et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res. 24, 766–769 (2014).

    CAS  Article  Google Scholar 

  34. Chen, I. H. et al. Phosphoproteins in extracellular vesicles as candidate markers for breast cancer. Proc. Natl. Acad. Sci. USA 114, 3175–3180 (2017).

    CAS  Article  Google Scholar 

  35. Baker, M. J. et al. Using Fourier transform IR spectroscopy to analyze biological materials. Nat. Protoc. 9, 1771–1791 (2014).

    CAS  Article  Google Scholar 

  36. McCarthy, S. A., Davies, G.-L. & Gun’ko, Y. K. Preparation of multifunctional nanoparticles and their assemblies. Nat. Protoc. 7, 1677–1693 (2012).

    CAS  Article  Google Scholar 

  37. Martin, F. L. et al. Distinguishing cell types or populations based on the computational analysis of their infrared spectra. Nat. Protoc. 5, 1748–1760 (2010).

    CAS  Article  Google Scholar 

  38. Dazzi, A. et al. AFM–IR: combining atomic force microscopy and infrared spectroscopy for nanoscale chemical characterization. Appl. Spectrosc. 66, 1365–1384 (2012).

    CAS  Article  Google Scholar 

  39. Butler, H. J. et al. Using Raman spectroscopy to characterize biological materials. Nat. Protoc. 11, 664–687 (2016).

    CAS  Article  Google Scholar 

  40. Zhang, Y., Zhao, S., Zheng, J. & He, L. Surface-enhanced Raman spectroscopy (SERS) combined techniques for high-performance detection and characterization. Trends Anal. Chem. 90, 1–13 (2017).

    Article  Google Scholar 

  41. Tatischeff, I., Larquet, E., Falcón-Pérez, J. M., Turpin, P.-Y. & Kruglik, S. G. Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy. J. Extracell. Vesicles 1, 19179 (2012).

  42. Park, J. et al. Exosome classification by pattern analysis of surface-enhanced Raman spectroscopy data for lung cancer diagnosis. Anal. Chem. 89, 6695–6701 (2017).

    CAS  Article  Google Scholar 

  43. Grasso, L., Wyss, R. & Vogel, H. Use of fourier transform infrared spectroscopy analysis of extracellular vesicles isolated from body fluids for diagnosing, prognosing and monitoring pathophysiological states and method therfor. EPFL patent WO2016097996 (A1) (2016).

  44. Ramer, G., Aksyuk, V. A. & Centrone, A. Quantitative chemical analysis at the nanoscale using the photothermal induced resonance technique. Anal. Chem. 89, 13524–13531 (2017).

    CAS  Article  Google Scholar 

  45. Jaffar, J. et al. Greater cellular stiffness in fibroblasts from patients with idiopathic pulmonary fibrosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 315, L59–L65 (2018).

    CAS  Article  Google Scholar 

  46. Mihály, J. et al. Characterization of extracellular vesicles by IRspectroscopy: fast and simple classification based on amide and CH stretching vibrations. Biochim. Biophys. Acta Biomembr. 1859, 459–466 (2017).

    Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

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.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-018-0109-3

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing