Methods to Analyze EVs

  • Bernd GiebelEmail author
  • Clemens Helmbrecht
Part of the Methods in Molecular Biology book series (MIMB, volume 1545)


Research in the field of extracellular vesicles (EVs) is challenged by the small size of the nano-sized particles. Apart from the use of transmission and scanning electron microscopy, established technical platforms to visualize, quantify, and characterize nano-sized EVs were lacking. Recently, methodologies to characterize nano-sized EVs have been developed. This chapter aims to summarize physical principles of novel and conventional technologies to be used in the EV field and to discuss advantages and limitations.

Key words

Nanoparticle tracking analysis Electron microscopy Dynamic light scattering Flow cytometry Extracellular vesicles Resistive pulse sensing 


  1. 1.
    Witwer KW, Buzas EI, Bemis LT, Bora A, Lasser C, Lotvall J, Nolte-'t Hoen EN, Piper MG, Sivaraman S, Skog J, Thery C, Wauben MH, Hochberg F (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2:PMID: 24009894. doi: 10.3402/jev.v2i0.20360 CrossRefGoogle Scholar
  2. 2.
    Ludwig AK, Giebel B (2012) Exosomes: small vesicles participating in intercellular communication. Int J Biochem Cell Biol 44(1):11–15. doi: 10.1016/j.biocel.2011.10.005 CrossRefPubMedGoogle Scholar
  3. 3.
    Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200(4):373–383. doi: 10.1083/jcb.201211138 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Harding C, Heuser J, Stahl P (1984) Endocytosis and intracellular processing of transferrin and colloidal gold-transferrin in rat reticulocytes: demonstration of a pathway for receptor shedding. Eur J Cell Biol 35(2):256–263PubMedGoogle Scholar
  5. 5.
    Harding C, Heuser J, Stahl P (1983) Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J Cell Biol 97(2):329–339CrossRefPubMedGoogle Scholar
  6. 6.
    Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9(6):654–659. doi: 10.1038/ncb1596, ncb1596 [pii]CrossRefPubMedGoogle Scholar
  7. 7.
    Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, van Eijndhoven MA, Hopmans ES, Lindenberg JL, de Gruijl TD, Wurdinger T, Middeldorp JM (2010) Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci U S A 107(14):6328–6333. doi: 10.1073/pnas.0914843107, 0914843107 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    El Andaloussi S, Mager I, Breakefield XO, Wood MJ (2013) Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 12(5):347–357. doi: 10.1038/nrd3978, nrd3978 [pii]CrossRefGoogle Scholar
  9. 9.
    Lener T, Gimona M, Aigner L, Borger V, Buzas E, Camussi G, Chaput N, Chatterjee D, Court FA, Del Portillo HA, O'Driscoll L, Fais S, Falcon-Perez JM, Felderhoff-Mueser U, Fraile L, Gho YS, Gorgens A, Gupta RC, Hendrix A, Hermann DM, Hill AF, Hochberg F, Horn PA, de Kleijn D, Kordelas L, Kramer BW, Kramer-Albers EM, Laner-Plamberger S, Laitinen S, Leonardi T, Lorenowicz MJ, Lim SK, Lotvall J, Maguire CA, Marcilla A, Nazarenko I, Ochiya T, Patel T, Pedersen S, Pocsfalvi G, Pluchino S, Quesenberry P, Reischl IG, Rivera FJ, Sanzenbacher R, Schallmoser K, Slaper-Cortenbach I, Strunk D, Tonn T, Vader P, van Balkom BW, Wauben M, Andaloussi SE, Thery C, Rohde E, Giebel B (2015) Applying extracellular vesicles based therapeutics in clinical trials – an ISEV position paper. J Extracell Vesicles 4:30087. doi: 10.3402/jev.v4.30087 CrossRefPubMedGoogle Scholar
  10. 10.
    Sokolova V, Ludwig AK, Hornung S, Rotan O, Horn PA, Epple M, Giebel B (2011) Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf B Biointerfaces 87(1):146–150. doi: 10.1016/j.colsurfb.2011.05.013, S0927-7765(11)00272-4 [pii]CrossRefPubMedGoogle Scholar
  11. 11.
    Dragovic RA, Gardiner C, Brooks AS, Tannetta DS, 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(6):780–788. doi: 10.1016/j.nano.2011.04.003 PubMedPubMedCentralGoogle Scholar
  12. 12.
    Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, Geuze HJ (1996) B Lymphocytes secrete antigen-presenting vesicles. J Exp Med 183(3):1161–1172CrossRefPubMedGoogle Scholar
  13. 13.
    Klar TA, Hell SW (1999) Subdiffraction resolution in far-field fluorescence microscopy. Opt Lett 24(14):954–956CrossRefPubMedGoogle Scholar
  14. 14.
    Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793):1642–1645. doi: 10.1126/science.1127344 CrossRefPubMedGoogle Scholar
  15. 15.
    Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91(11):4258–4272. doi: 10.1529/biophysj.106.091116 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10):793–795. doi: 10.1038/nmeth929 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Grigorieff N, Harrison SC (2011) Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy. Curr Opin Struct Biol 21(2):265–273. doi: 10.1016/ CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Yuana Y, Koning RI, Kuil ME, Rensen PC, Koster AJ, Bertina RM, Osanto S (2013) Cryo-electron microscopy of extracellular vesicles in fresh plasma. J Extracell Vesicles 2:PMID:24455109. doi: 10.3402/jev.v2i0.21494 CrossRefGoogle Scholar
  19. 19.
    Afework A, Beynon MD, Bustamante F, Cho S, Demarzo A, Ferreira R, Miller R, Silberman M, Saltz J, Sussman A, Tsang H (1998) Digital dynamic telepathology—the virtual microscope. Proceedings/AMIA annual symposium, pp 912–916Google Scholar
  20. 20.
    Grasselli JG, Bulkin BJ (1991) Analytical Raman spectroscopy. Wiley, New York, NYGoogle Scholar
  21. 21.
    Seinfeld JH, Pandis SN (2006) Atmospheric chemistry and physics: from air pollution to climate change. Wiley, New York, NYGoogle Scholar
  22. 22.
    Bohren C, Huffman DR (1998) Absorption and scattering of light by small particles. Wiley Science Paperback Series, New York, NYCrossRefGoogle Scholar
  23. 23.
    Van De Hulst HC (1982) Light scattering by small particles. Peter Smith Publisher Incorporated, Gloucester, MAGoogle Scholar
  24. 24.
    Affolabi D, Torrea G, Odoun M, Senou N, Ali Ligali M, Anagonou S, Van Deun A (2010) Comparison of two LED fluorescence microscopy build-on modules for acid-fast smear microscopy. Int J Tuberc Lung Dis 14(2):160–164PubMedGoogle Scholar
  25. 25.
    Affatato S, Spinelli M, Squarzoni S, Traina F, Toni A (2009) Mixing and matching in ceramic-on-metal hip arthroplasty: an in-vitro hip simulator study. J Biomech 42(15):2439–2446. doi: 10.1016/j.jbiomech.2009.07.031 CrossRefPubMedGoogle Scholar
  26. 26.
    Appidi JR, Grierson DS, Afolayan AJ (2008) Foliar micromorphology of Hermannia icana Cav. Pak J Biol Sci 11(16):2023–2027CrossRefPubMedGoogle Scholar
  27. 27.
    Ashafa AO, Grierson DS, Afolayan AJ (2008) Foliar micromorphology of Felicia muricata Thunb., a South African medicinal plant. Pak J Biol Sci 11(13):1713–1717CrossRefPubMedGoogle Scholar
  28. 28.
    Rigon A, Soda P, Zennaro D, Iannello G, Afeltra A (2007) Indirect immunofluorescence in autoimmune diseases: assessment of digital images for diagnostic purpose. Cytometry B Clin Cytom 72(6):472–477. doi: 10.1002/cyto.b.20356 CrossRefPubMedGoogle Scholar
  29. 29.
    Brown RG (1987) Dynamic light scattering using monomode optical fibers. Appl Optics 26(22):4846–4851. doi: 10.1364/AO.26.004846 CrossRefGoogle Scholar
  30. 30.
    Afaj AH, Sultan MA (2005) Mineralogical composition of the urinary stones from different provinces in Iraq. ScientificWorldJournal 5:24–38. doi: 10.1100/tsw.2005.2 CrossRefPubMedGoogle Scholar
  31. 31.
    Filipe V, Hawe A, Jiskoot W (2010) Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm Res 27(5):796–810. doi: 10.1007/s11095-010-0073-2 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Phillips JB, Smit X, De Zoysa N, Afoke A, Brown RA (2004) Peripheral nerves in the rat exhibit localized heterogeneity of tensile properties during limb movement. J Physiol 557(Pt 3):879–887. doi: 10.1113/jphysiol.2004.061804 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Afonso C, Fenselau C (2003) Use of bioactive glass slides for matrix-assisted laser desorption/ionization analysis: application to microorganisms. Anal Chem 75(3):694–697CrossRefPubMedGoogle Scholar
  34. 34.
    Carmanchahi PD, Aldana Marcos HJ, Ferrari CC, Affanni JM (1998) A simple method for taking photographs of histological sections without using neither photographic camera nor microscope. BioCell 22(3):207–210PubMedGoogle Scholar
  35. 35.
    Konokhova AI, Yurkin MA, Moskalensky AE, Chernyshev AV, Tsvetovskaya GA, Chikova ED, Maltsev VP (2012) Light-scattering flow cytometry for identification and characterization of blood microparticles. J Biomed Opt 17(5):057006. doi: 10.1117/1.JBO.17.5.057006 CrossRefPubMedGoogle Scholar
  36. 36.
    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(12):2596–2607. doi: 10.1111/j.1538-7836.2010.04074.x CrossRefPubMedGoogle Scholar
  37. 37.
    Lacroix R, Robert S, Poncelet P, Kasthuri RS, Key NS, Dignat-George F, Workshop IS (2010) Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. J Thromb Haemost 8(11):2571–2574. doi: 10.1111/j.1538-7836.2010.04047.x CrossRefPubMedGoogle Scholar
  38. 38.
    Orozco AF, Lewis DE (2010) Flow cytometric analysis of circulating microparticles in plasma. Cytometry A 77(6):502–514. doi: 10.1002/cyto.a.20886 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Freyssinet JM, Toti F (2010) Membrane microparticle determination: at least seeing what's being sized! J Thromb Haemost 8(2):311–314. doi: 10.1111/j.1538-7836.2009.03679.x CrossRefPubMedGoogle Scholar
  40. 40.
    Clayton A, Court J, Navabi H, Adams M, Mason MD, Hobot JA, Newman GR, Jasani B (2001) Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods 247(1-2):163–174, doi:S0022-1759(00)00321-5 [pii]CrossRefPubMedGoogle Scholar
  41. 41.
    Thery C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3:Unit 22. doi: 10.1002/0471143030.cb0322s30 PubMedGoogle Scholar
  42. 42.
    Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C (2005) Exosomal-like vesicles are present in human blood plasma. Int Immunol 17(7):879–887. doi: 10.1093/intimm/dxh267, dxh267 [pii]CrossRefPubMedGoogle Scholar
  43. 43.
    Rabesandratana H, Toutant JP, Reggio H, Vidal M (1998) Decay-accelerating factor (CD55) and membrane inhibitor of reactive lysis (CD59) are released within exosomes during In vitro maturation of reticulocytes. Blood 91(7):2573–2580PubMedGoogle Scholar
  44. 44.
    Steen HB (2004) Flow cytometer for measurement of the light scattering of viral and other submicroscopic particles. Cytometry A 57(2):94–99. doi: 10.1002/cyto.a.10115 CrossRefPubMedGoogle Scholar
  45. 45.
    Hercher M, Mueller W, Shapiro HM (1979) Detection and discrimination of individual viruses by flow cytometry. J Histochem Cytochem 27(1):350–352CrossRefPubMedGoogle Scholar
  46. 46.
    Nolte-'t Hoen EN, van der Vlist EJ, Aalberts M, Mertens HC, Bosch BJ, Bartelink W, Mastrobattista E, van Gaal EV, Stoorvogel W, Arkesteijn GJ, Wauben MH (2012) Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles. Nanomedicine 8(5):712–720. doi: 10.1016/j.nano.2011.09.006 PubMedGoogle Scholar
  47. 47.
    van der Vlist EJ, Nolte-'t Hoen EN, Stoorvogel W, Arkesteijn GJ, Wauben MH (2012) Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat Protoc 7(7):1311–1326. doi: 10.1038/nprot.2012.065 CrossRefPubMedGoogle Scholar
  48. 48.
    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(5):919–930. doi: 10.1111/j.1538-7836.2012.04683.x CrossRefPubMedGoogle Scholar
  49. 49.
    Tatischeff I, Larquet E, Falcon-Perez JM, Turpin PY, Kruglik SG (2012) Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy. J Extracell Vesicles 1:PMCID:PMC3760651. doi: 10.3402/jev.v1i0.19179 CrossRefGoogle Scholar
  50. 50.
    Deckert-Gaudig T, Deckert V (2011) Nanoscale structural analysis using tip-enhanced Raman spectroscopy. Curr Opin Chem Biol 15(5):719–724. doi: 10.1016/j.cbpa.2011.06.020 CrossRefPubMedGoogle Scholar
  51. 51.
    Binnig G, Quate CF, Gerber C (1986) Atomic force microscope. Phys Rev Lett 56(9):930–933CrossRefPubMedGoogle Scholar
  52. 52.
    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(2):315–323. doi: 10.1111/j.1538-7836.2009.03654.x CrossRefPubMedGoogle Scholar
  53. 53.
    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 Microdevices 14(4):641–649. doi: 10.1007/s10544-012-9642-y CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Sharma S, Rasool HI, Palanisamy V, Mathisen C, Schmidt M, Wong DT, Gimzewski JK (2010) Structural-mechanical characterization of nanoparticle exosomes in human saliva, using correlative AFM, FESEM, and force spectroscopy. ACS Nano 4(4):1921–1926. doi: 10.1021/nn901824n CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    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(1), e8577. doi: 10.1371/journal.pone.0008577 CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Syvitski JPM (ed) (1991) Principles, methods and application of particle size analysis. Cambridge University Press, CambridgeGoogle Scholar
  57. 57.
    Vogel R, Willmott G, Kozak D, Roberts GS, Anderson W, Groenewegen L, Glossop B, Barnett A, Turner A, Trau M (2011) Quantitative sizing of nano/microparticles with a tunable elastomeric pore sensor. Anal Chem 83(9):3499–3506. doi: 10.1021/ac200195n CrossRefPubMedGoogle Scholar
  58. 58.
    Afanassiev V, Hanemann V, Wolfl S (2000) Preparation of DNA and protein micro arrays on glass slides coated with an agarose film. Nucleic Acids Res 28(12), E66CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Ito T, Sun L, Henriquez RR, Crooks RM (2004) A carbon nanotube-based coulter nanoparticle counter. Acc Chem Res 37(12):937–945. doi: 10.1021/ar040108+ CrossRefPubMedGoogle Scholar
  60. 60.
    Citkowicz A, Petry H, Harkins RN, Ast O, Cashion L, Goldmann C, Bringmann P, Plummer K, Larsen BR (2008) Characterization of virus-like particle assembly for DNA delivery using asymmetrical flow field-flow fractionation and light scattering. Anal Biochem 376(2):163–172. doi: 10.1016/j.ab.2008.02.011 CrossRefPubMedGoogle Scholar
  61. 61.
    Kang D, Oh S, Ahn SM, Lee BH, Moon MH (2008) Proteomic analysis of exosomes from human neural stem cells by flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry. J Proteome Res 7(8):3475–3480. doi: 10.1021/pr800225z CrossRefPubMedGoogle Scholar
  62. 62.
    Zak KP, Filatova RS, Afanasyeva VV (1983) Cytochemical and ultracytochemical studies of peroxidase activity in rabbit blood granulocytes under hydrocortisone effect. Folia Haematol 110(4):490–502Google Scholar
  63. 63.
    Afifi F, Peignoux M, Auclair C (1980) Modifications of bile secretion and liver microsomal enzymes by aldosterone and spironolactone. Hepatogastroenterology 27(1):9–16PubMedGoogle Scholar
  64. 64.
    Baalousha M, Stolpe B, Lead JR (2011) Flow field-flow fractionation for the analysis and characterization of natural colloids and manufactured nanoparticles in environmental systems: a critical review. J Chromatogr A 1218(27):4078–4103. doi: 10.1016/j.chroma.2011.04.063 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Institute for Transfusion Medicine, University Hospital EssenUniversity Duisburg-EssenEssenGermany
  2. 2.Particle Metrix GmbHMeerbuschGermany

Personalised recommendations