Skip to main content
SpringerLink
Log in

Atomic force microscopy analysis of extracellular vesicles

  • Original Article
  • Published:
European Biophysics Journal Aims and scope Submit manuscript

Abstract

Extracellular vesicles (EVs) are small vesicles ensuring transport of molecules between cells and throughout the body. EVs contain cell type-specific signatures and have been proposed as biomarkers in a variety of diseases. Their small size (<1 μm) and biological and physical functions make them obvious candidates for therapeutic agents in immune therapy, vaccination, regenerative medicine and drug delivery. However, due to the complexity and heterogeneity of their origin and composition, the actual mechanism through which these vesicles exert their functions is still unknown and represents a great biomedical challenge. Moreover, because of their small dimensions, the quantification, size distribution and biophysical characterization of these particles are challenging and still subject to controversy. Here, we address the advantage of atomic force microscopy (AFM), for the characterization of isolated EVs. We review AFM imaging of EVs immobilized on different substrates (mica, glass) to identify the influence of isolation and deposition methods on the size distribution, morphology and mechanical properties of EVs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Akers JC, Ramakrishnan V, Kim R, Skog J, Nakano I, Pingle S, Kalinina J, Hua W, Hesari S, Mao Y, Breakefield XO, Hochberg FH, Van Meir EG, Carter BS, Chen CC (2013) MiR-21 in the extracellular vesicles (EVs) of cerebrospinal fluid (CSF): a platform for glioblastoma biomarker development. PLoS ONE 8:e78115. doi:10.1371/journal.pone.0078115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Anderson W, Lane R, Korbie D, Trau M (2015) Observations of tunable resistive pulse sensing for exosome analysis: improving system sensitivity and stability. Langmuir 31:6577–6587. doi:10.1021/acs.langmuir.5b01402

    Article  CAS  PubMed  Google Scholar 

  • Ashcroft BA, de Sonneville J, Yuana Y, Osanto S, Bertina R, Kuil ME, Oosterkamp TH (2012) Determination of the size distribution of blood microparticles directly in plasma using atomic force microscopy and microfluidics. Biomed Microdevice 14:641. doi:10.1007/s10544-012-9642-y

    Article  CAS  Google Scholar 

  • Bourkoula E, Mangoni D, Ius T, Pucer A, Isola M, Musiello D, Marzinotto S, Toffoletto B, Sorrentino M, Palma A, Caponnetto F, Gregoraci G, Vindigni M, Pizzolitto S, Falconieri G, De Maglio G, Pecile V, Gruaro M, Gri G, Parisse P, Casalis L, Scoles G, Skrap M, Beltrami CA, Beltrami AP, Cesselli D (2014) Glioma-associated stem cells: a novel class of tumor-supporting cells able to predict prognisis of human low-grade gliomas. Stem Cells 32:1239–1253. doi:10.1002/stem.1605

    Article  CAS  PubMed  Google Scholar 

  • Calò A, Reguera D, Oncincs G, Persuy MA, Sanz G, Lobasso S, Corcelli A, Pajot-Augy E, Gomila G (2014) Force measurements on natural membrane nanovesicles reveal a composition-independent, high Young’s modulus. Nanoscale 21:2275–2285. doi:10.1039/c3nr05107b

    Article  Google Scholar 

  • Canet-Ferrer J, Coronado E, Forment-Aliaga A, Pinilla-Cienfuegos E (2014) Correction of the tip convolution effects in the imaging of nanostructures studied through scanning force microscopy. Nanotechnology 25:395703. doi:10.1088/0957-4484/25/39/395703

    Article  PubMed  Google Scholar 

  • Caponnetto F, Manini I, Skrap M, Palmai-Pallaga T, Loreto C, Beltrami AP, Cesselli D, Ferrari E (2017) Size-dependent cellular uptake of exosomes. Nanomed NMB 13:1011. doi:10.1016/j.nano.2016.12.009

    Article  CAS  Google Scholar 

  • Chao Y, Zhang T (2011) Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy. Appl Microbiol Biotechnol 92:381. doi:10.1007/s00253-011-3551-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chernyshev VS, Rachamadugu R, Tseng YH, Belnap DM, Jia Y, Branch KJ, Butterfield AE, Pease LF III, Bernard PS, Skliar M (2015) Size and shape characterization of hydrated and desiccated exosomes. Anal Bioanal Chem 407:3285–3301. doi:10.1007/s00216-015-8535-3

    Article  CAS  PubMed  Google Scholar 

  • Danielson KM, Estanislau J, Tigges J, Toxavidis V, Camacho V, Felton EJ, Khoory J, Kreimer S, Ivanov AR, Mantel P-Y, Jones J, Akuthota P, Das S, Ghiran I (2016) Diurnal variations of circulating extracellular vesicles measured by nano flow cytometry. PLoS ONE 11:e0144678. doi:10.1371/journal.pone

    Article  PubMed  PubMed Central  Google Scholar 

  • DiNoto G, Bugatti A, Zendrini A, Mazzoldi EL, Montanelli A, Caimi L, Rusnati M, Ricotta D, Bergese P (2016) Merging colloidal nanoplasmonics and surface plasmon resonance spectroscopy for enhanced profiling of multiple myeloma-derived exosomes. Biosens Bioelectron 77:518. doi:10.1016/j.bios.2015.09.061

    Article  CAS  Google Scholar 

  • Dragovic RA, Gardiner C, Brooks AS, Ds Tannetta, Ferguson DJ, Hole P, Carr B, Redman CW, Harris AL, Dobson PJ, Harrison P, Sargent IL (2011) Sizing and phenotyping of cellular vesicles using nanoparticle tracking analysis. Nanomedicine 7:780. doi:10.1016/j.nano.2011.04.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fais S, O’Driscoll L, Borras FE, Buzas E, Camussi G, Cappello F, Carvalho J, Cordeiro da Silva A, Del Portillo H, El Andaloussi S, Ficko Trcek T, Furlan R, Hendrix A, Gursel I, Kralj-Iglic V, Kaeffer B, Kosanovic M, Lekka ME, Lipps G, Logozzi M, Marcilla A, Sammar M, Llorente A, Nazarenko I, Oliveira C, Pocsfalvi G, Rajendran L, Raposo G, Rohde E, Siljander P, van Niel G, Vasconcelos MH, Yanez-Mo M, Yliperttula ML, Zarovni N, Zavec AB, Giebel B (2016) Evidence-based clinical use of nanoscale extracellular vesicles in nanomedicine. ACS Nano 10:3886. doi:10.1021/acsnano.5b08015

    Article  CAS  PubMed  Google Scholar 

  • Gajos K, Kamińska A, Awsiuk K, Bajor A, Gruszczyński K, Pawlak A, Żądło A, Kowalik A, Budkowski A, Stępień E (2017) Immobilization and detection of platelet-derived extracellular vesicles on functionalized silicon substrate: cytometric and spectrometric approach. Anal Bioanal Chem 409:1109. doi:10.1007/s00216-016-0036-5

    Article  CAS  PubMed  Google Scholar 

  • Gardiner C, Di Vizio D, Sahoo S, Thery C, Witwer KW, Wauben M, Hill AF (2016) Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey. J Extracell Ves 5:32945. doi:10.3402/jev.v5.32945

    Article  Google Scholar 

  • Gholizadeh S, Draz M, Zarghooni M, Nezhad AS, Ghavami S, Shafiee H, Akbari M (2017) Microfluidic approaches for isolation, detection, and characterization of extracellular vesicles: current status and future directions. Biosens Bioelectron 91:588. doi:10.1016/j.bios.2016.12.062

    Article  CAS  PubMed  Google Scholar 

  • Hardij J, Cecchet F, Berquand A, Gheldof D, Chatelain C, Mullier F, Chatelain B, Dogné JM (2013) Characterisation of tissue factor-bearing extracellular vesicles with AFM: comparison of air-tapping-mode AFM and liquid peak force AFM. J Extracell Vesicles 2:21045. doi:10.3402/jev.v2i0.21045

    Article  Google Scholar 

  • Hernando-Perez M, Miranda R, Aznar M, Carrascosa JL, Schaap IAT, Reguera D, de Pablo PJ (2012) Direct measurement of phage phi29 stiffness provides evidence of internal pressure. Small 8:2366. doi:10.1002/smll.201200664

    Article  CAS  PubMed  Google Scholar 

  • Iwai K, Minamisawa T, Suga K, Yajima Y, Shiba K (2016) Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations. J Extracell Vesicles. doi:10.3402/jev.v5.30829

    PubMed  PubMed Central  Google Scholar 

  • Jorgensen M, Baek R, Pedersen S, Sondergaarf EKL, Kristensen SR, Varming K (2013) Extracellular vesicle (EV) array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping. J Extracell Vesicles 2:20920. doi:10.3402/jev.v2i0.20920

    Article  Google Scholar 

  • Khatun Z, Bhat A, Sharma S, Sharma A (2016) Elucidating diversity of exosomes: biophysical and molecular characterization methods. Nanomedicine 11:2359–2377. doi:10.2217/nnm-2016-0192

    Article  CAS  PubMed  Google Scholar 

  • Li S, Eghiaian F, Sieben C, Herrmann A, Schaap IAT (2011) Bending and puncturing the influenza lipid envelope. Biophys J 100:637. doi:10.1016/j.bpj.2010.12.3701

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liang X, Mao G, Ng KYS (2004) Mechanical properties and stability measurement of cholesterol-containing liposome on mica by atomic force microscopy. Colloids Surf B 34:41. doi:10.1016/j.jcis.2004.05.042

    Article  CAS  Google Scholar 

  • Minciacchi V, Freeman MR, Di Vizio D (2015) Extracellular vesicles in cancer: exosomes, microvesicles and the emerging role of large oncosomes semin. Cell Develop Biol 40:41–51. doi:10.1016/j.semcdb.2015.02.010

    Article  CAS  Google Scholar 

  • Nguyen DB, Ly TBT, Wesseling MC, Hittinger M, Torge A, Devitt A, Perrie Y, Bernhardt I (2016) Characterization of microvesicles released from human red blood cells. Cell Physiol Biochem 38:1085. doi:10.1159/000443059

    Article  CAS  PubMed  Google Scholar 

  • Palanisamy V, Sharma S, Deshpande A, Zhou H, Gimzewski J, Wong DT (2010) Nanostructural and transcriptomic analyses of human saliva derived exosomes. PLoS ONE 5:e8577. doi:10.1371/journal.pone.0008577

    Article  PubMed  PubMed Central  Google Scholar 

  • Paolini L, Zendrini A, Di Noto G, Busatto S, Lottini E, Radeghieri A, Dossi A, Caneschi A, Ricotta D, Bergese P (2016) Residual matrix from different separation techniques impacts exosome biological activity. Sci Rep 6:23550. doi:10.1038/srep23550

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Park JW (2010) Sulfatide incorporation effect on mechanical properties of vesicles. Colloids Surf B 80:59. doi:10.1016/j.colsurfb.2010.05.031

    Article  CAS  Google Scholar 

  • Rauti R, Lozano N, Leon V, Scaini D, Musto M, Rago I, Ulloa Severino FP, Fabbro A, Casalis L, Vazquez E, Kostarelos K, Prato M, Ballerini L (2016) Graphene oxide nanosheets reshape synaptic function in cultured brain networks. ACS Nano 10:4459. doi:10.1021/acsnano.6b00130

    Article  CAS  PubMed  Google Scholar 

  • Regev-Rudzki N, Wilson DW, Carvalho TG, Sisquella X, Coleman BM, Rug M, Bursac D, Angrisano F, Gee M, Hill AF, Baum J, Cowman AF (2013) Cell-cell communication between malaria-infected red blood cells via exosome-like vesicles. Cell 153:1120. doi:10.1016/j.cell.2013.04.029

    Article  CAS  PubMed  Google Scholar 

  • Roma-Rodrigues C, Fernandes AR, Baptista PV (2014) Exosome in tumour microenvironment: overview of the crosstalk between normal and cancer cells. Biomed Res Int 2014:179486. doi:10.1155/2014/179486

    Article  PubMed  PubMed Central  Google Scholar 

  • Roos WH, Bruinsma R, Wuite GJL (2010) Physical virology. Nat Phys 6:733. doi:10.1038/nphys1797

    Article  CAS  Google Scholar 

  • Sebaihi N, De Boeck B, Yuana Y, Nieuwland R, Pétry J (2017) Dimensional characterization of extracellular vesicles using atomic force microscopy. Meas Sci Technol 28:034006. doi:10.1088/1361-6501/28/3/034006

    Article  Google Scholar 

  • Sharma S, Gimzewski JK (2012) The quest for characterizing exosomes: circulating nano-sized vesicles. J Nanomed Nanotechol 3:e115. doi:10.4172/2157-7439.1000e115

    Article  Google Scholar 

  • Sharma S, Rasooi HI, Palanisamy V, Mathisen C, Schmidt M, Wong DT, Gimzewski (2010) Structural-mechanical characterization of nanoparticle exosomes in human saliva, using correlative AFM, FESEM, and force spectroscopy. ACS Nano 4:1921. doi:10.1021/nn901824n

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sharma S, Gillespie BM, Palanisamy V, Gimzewski J (2011) Quantitative nanostructural and single-molecule force spectroscopy biomolecular analysis of human-saliva-derived exosomes. Langmuir 27:14394. doi:10.1021/la2038763

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sharma S, Das K, Woo J-R, Gimzewski JK (2014) Nanofilaments on glioblastoma exosomes revealed by peak force microscopy. J R Soc Interface 11:20131150. doi:10.1098/rsif.2013.1150

    Article  PubMed  PubMed Central  Google Scholar 

  • Siedlecki CA, Wang IW, Higashi JM, Kottke-Marchant K, Marchant RE (1999) Platelet-derived microparticles on synthetic surfaces observed by atomic force microscopy and fluorescence microscopy. Biomaterials 20:1521. doi:10.1016/S0142-9612(99)00065-4

    Article  CAS  PubMed  Google Scholar 

  • Skog J, Wurdinger T, van Rijn S, Meijer DH, Gainche L, Sena-Esteves M, Curry WT, Carter BS, Krichevsky AM, Breakefield XO (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 10:1470. doi:10.1038/ncb1800 (Epub 2008 Nov 16)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Svensson KJ, Kucharzewska P, Christianson HC, Skold S, Lofstedt T, Johansson MC, Morgelin M, Bengzon J, Ruf Belting M (2011) Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells. PNAS 198:13147. doi:10.1073/pnas.1104261108

    Article  Google Scholar 

  • Tian T, Wang Y, Wang H, Zhu Z, Xiao Z (2010) Visualizing of the cellular uptake and intracellular trafficking of exosomes by live-cell microscopy. J Cell Biochem 111:488. doi:10.1002/jcb.22733

    Article  CAS  PubMed  Google Scholar 

  • Tickner JA, Urquhart AJ, Stephenson S, Richard DJ, O’Byrne KJ (2014) Functions and therapeutic roles of exosomes in cancer. Front Oncol 4:127. doi:10.3389/fonc.2014.00127.eCollection

    Article  PubMed  PubMed Central  Google Scholar 

  • Van der Pol E, Hoekstra AG, Sturk A, Otto C, van Leeuwen TG, Nieuwland R (2010) Optical and non-optical methods for detection and characterization of microparticles and exosomes. J Thromb Haemost 8:2596. doi:10.1111/j.1538-7836.2010.04074.x

    Article  PubMed  Google Scholar 

  • Van der Pol E, van Gemert MJ, Sturk A, Nieuwland R, van Leeuwen TG (2012) Single vs. swarm detection of microparticles and exosomes by flow cytometry. J Thromb Haemost 10:919 doi: 10.1111/j.1538-7836.2012.04683.x

  • Vella LJ (2014) The emerging role of exosomes in epithelial-mesenchymal-transition in cancer. Front Oncol 4:361. doi:10.3389/fonc.2014.00361

    Article  PubMed  PubMed Central  Google Scholar 

  • Vogel R, Coumans FAW, Maltesen RG, Boing AN, Bonnington KE, Broekman ML, Broom MF, Buzas EI, Christiansen G, Hajji N, Kristensen SR, Kuehn MJ, Lund SM, Maas SLN, Nieuwland R, Osteikoetxea X, Schnoor R, Scicluna BJ, Shambrook M, de Vrij J, Mann SI, Hill AF, Pedersen S (2016) A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing. J Extracell Vesicles 5:31242. doi:10.3402/jev.v5.31242

    Article  PubMed  Google Scholar 

  • Vorselen D, MacKintosh FC, Roos WH, Wuite GJL (2017) Competition between bending and internal pressure governs the mechanics of fluid nanovesicles. ACS Nano 11:2628. doi:10.1021/acsnano.6b07302

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Whitehead B, Wu L, Hvam ML, Aslan H, Dong M, Dyrskjot L, Ostenfeld MS, Moghimi SM, Howard KA (2015) Tumour exosomes display differential mechanical and complement activation properties dependent on malignant state: implications in endothelial leakiness. J Extracell Ves 4:29685. doi:10.3402/jev.v4.29685

    Article  Google Scholar 

  • Woo JR, Sharma S, Gimzewski J (2016) The role of isolation methods on a nanoscale surface structure and its effect on the size of exosomes. J Circ Biomark 5:11. doi:10.5772/64148

    Article  PubMed  PubMed Central  Google Scholar 

  • Yacoot A, Koenders L (2008) Aspects of scanning force microscope probes and their effects on dimensional measurement. J Phys D Appl Phys 41:103001. doi:10.1088/0022-3727/41/10/103001

    Article  Google Scholar 

  • Yuana Y, Oosterkamp TH, Bahatyrova S, Ashcroft B, Garcia Rodriguez P, Bertina RM, Osanto S (2010) Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles. J Thromb Haemost 8:315. doi:10.1111/j.1538-7836.2009.03654.x

    Article  CAS  PubMed  Google Scholar 

  • Zhang S, Aslan H, Besenbacher F, Dong M (2014) Quantitative biomolecular imaging by dynamic nanomechanical mapping. Chem Soc Rev 43:7412. doi:10.1039/c4cs00176a

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors acknowledge funding from the Italian Ministry of Education (FIRB grant RBAP11ETKA-005) and European Regional Development Fund Interreg V-A Italia–Austria 2014–2020 (EXOTHERA ITAT1036).

Author information

Authors and Affiliations

  1. INSTM-ST Unit, Trieste, Italy

    P. Parisse, E. Ambrosetti & L. Casalis

  2. Elettra, Sincrotrone Trieste S.C.p.A., Trieste, Italy

    P. Parisse, I. Rago, L. Ulloa Severino, F. Perissinotto, E. Ambrosetti, P. Paoletti & L. Casalis

  3. University of Trieste, Trieste, Italy

    I. Rago, L. Ulloa Severino, F. Perissinotto & E. Ambrosetti

  4. SISSA, Scuola Internazionale Superiore di Studi Avanzati, Trieste, Italy

    P. Paoletti

  5. Biological and Soft Systems, Cavendish Laboratory, Cambridge University, Cambridge, UK

    M. Ricci

  6. Department of Medical and Biological Sciences, University of Udine, Udine, Italy

    A. P. Beltrami & D. Cesselli

Authors
  1. P. Parisse
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. I. Rago
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. L. Ulloa Severino
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F. Perissinotto
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. E. Ambrosetti
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. P. Paoletti
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M. Ricci
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A. P. Beltrami
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. D. Cesselli
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. L. Casalis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to P. Parisse.

Additional information

Special issue: Regional Biophysics Conference 2016.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Parisse, P., Rago, I., Ulloa Severino, L. et al. Atomic force microscopy analysis of extracellular vesicles. Eur Biophys J 46, 813–820 (2017). https://doi.org/10.1007/s00249-017-1252-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00249-017-1252-4

Keywords

Search

Navigation