Abstract
Transmission electron microscopy (TEM) and transmission scanning electron Microscopy (TSEM), which denotes application of a scanning electron microscope (SEM) in the transmission mode, have been used to detect and characterize particles down to an imaging resolution of ~1 nm. In the field of EVs, TEM also has been valued for its capability to detect and characterize single EV. Furthermore, employing immunogold labeling in TEM could give information regarding biochemical properties of EV surface proteins. Significant shortcomings in TEM such as dehydration, chemical fixation, and/or staining of the biological specimens are eluded by the use of cryo-TEM. In cryo-TEM imaging, samples are directly applied onto an EM grid, vitrified and visualized, thus allowing for characterization of EVs near its native state. In this chapter, we describe a step-by-step guide for preparing EVs on the grid before TEM and cryo-TEM imaging. Finally, we provide a guide to an automated image-processing analysis to provide the size distribution of EVs.
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References
van der Pol E, Boing AN, Gool EL, Nieuwland R (2016) Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles. J Thromb Haemost 14(1):48–56
Yuana Y, Sturk A, Nieuwland R (2013) Extracellular vesicles in physiological and pathological conditions. Blood Rev 27(1):31–39
van Der Pol E, Boing AN, Harrison P, Sturk A, Nieuwland R (2012) Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 64(3):676–705
Yanez-Mo M, Siljander PR, Andreu Z et al (2015) Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4:27066
Lener T, Gimona M, Aigner L et al (2015) Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper. J Extracell Vesicles 4:30087
van der Pol E, Coumans FA, Grootemaat AE, Gardiner C, Sargent IL, Harrison P, Sturk A, van Leeuwen TG, Nieuwland R (2014) Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost 12(7):1182–1192
Lotvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C, Gho YS, Kurochkin IV, Mathivanan S, Quesenberry P, Sahoo S, Tahara H, Wauben MH, Witwer KW, Thery C (2014) Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 3:26913
Crescitelli R, Lasser C, Szabo TG, Kittel A, Eldh M, Dianzani I, Buzas EI, Lotvall J (2013) Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes. J Extracell Vesicles 2:20677
Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200(4):373–383
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 2:21494
Hoog JL, Lotvall J (2015) Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy. J Extracell Vesicles 4:28680
Arraud N, Linares R, Tan S, Gounou C, Pasquet JM, Mornet S, Brisson AR (2014) Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration. J Thromb Haemost 12(5):614–627
Yuana Y, Boing AN, Grootemaat AE, van der Pol E, Hau CM, Cizmar P, Buhr E, Sturk A, Nieuwland R (2015) Handling and storage of human body fluids for analysis of extracellular vesicles. J Extracell Vesicles 4:29260
Linares R, Tan S, Gounou C, Arraud N, Brisson AR (2015) High-speed centrifugation induces aggregation of extracellular vesicles. J Extracell Vesicles 4:29509
Boing AN, van der Pol E, Grootemaat AE, Coumans FA, Sturk A, Nieuwland R (2014) Single-step isolation of extracellular vesicles by sizeexclusion chromatography. J Extracell Vesicles 3:23430
Nordin JZ, Lee Y, Vader P, Mager I, Johansson HJ, Heusermann W, Wiklander OP, Hallbrink M, Seow Y, Bultema JJ, Gilthorpe J, Davies T, Fairchild PJ, Gabrielsson S, Meisner-Kober NC, Lehtio J, Smith CI, Wood MJ, Andaloussi SE (2015) Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomedicine 11(4):879–883
Lozano-Ramos I, Bancu I, Oliveira-Tercero A, Armengol MP, Menezes-Neto A, Del Portillo HA, Lauzurica-Valdemoros R, Borras FE (2015) Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples. J Extracell Vesicles 4:27369
Acknowledgment
Both authors thank the technical supports given by A.E. Grootemaat from Department of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, Dr. R.I. Koning from the Molecular Cell Biology Department at the Leiden University Medical Center, and Dr. G. Posthuma from Cell Microscopy Center, Department of Cell Biology of University Medical Center Utrecht, The Netherlands.
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Cizmar, P., Yuana, Y. (2017). Detection and Characterization of Extracellular Vesicles by Transmission and Cryo-Transmission Electron Microscopy. In: Kuo, W., Jia, S. (eds) Extracellular Vesicles. Methods in Molecular Biology, vol 1660. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7253-1_18
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DOI: https://doi.org/10.1007/978-1-4939-7253-1_18
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