TY - JOUR
T1 - Direct comparison of optical and electron microscopy methods for structural characterization of extracellular vesicles
AU - Noble, Jade M.
AU - Roberts, La Deidra Monét
AU - Vidavsky, Netta
AU - Chiou, Aaron E.
AU - Fischbach, Claudia
AU - Paszek, Matthew J.
AU - Estroff, Lara A.
AU - Kourkoutis, Lena F.
N1 - Publisher Copyright:
© 2020 The Authors
PY - 2020/4/1
Y1 - 2020/4/1
N2 - As interest in the role of extracellular vesicles in cell-to-cell communication has increased, so has the use of microscopy and analytical techniques to assess their formation, release, and morphology. In this study, we evaluate scanning electron microscopy (SEM) and cryo-SEM for characterizing the formation and shedding of vesicles from human breast cell lines, parental and hyaluronan synthase 3-(HAS3)-overexpressing MCF10A cells, grown directly on transmission electron microscopy (TEM) grids. While cells imaged with conventional and cryo-SEM exhibit distinct morphologies due to the sample preparation process for each technique, tubular structures protruding from the cell surfaces were observed with both approaches. For HAS3-MCF10A cells, vesicles were present along the length of membrane protrusions. Once completely shed from the cells, extracellular vesicles were characterized using nanoparticle tracking analysis (NTA) and cryo-TEM. The size distributions obtained by each technique were different not only in the range of vesicles analyzed, but also in the relative proportion of smaller-to-larger vesicles. These differences are attributed to the presence of biological debris in the media, which is difficult to differentiate from vesicles in NTA. Furthermore, we demonstrate that cryo-TEM can be used to distinguish between vesicles based on their respective surface structures, thereby providing a path to differentiating vesicle subpopulations and identifying their size distributions. Our study emphasizes the necessity of pairing several techniques to characterize extracellular vesicles.
AB - As interest in the role of extracellular vesicles in cell-to-cell communication has increased, so has the use of microscopy and analytical techniques to assess their formation, release, and morphology. In this study, we evaluate scanning electron microscopy (SEM) and cryo-SEM for characterizing the formation and shedding of vesicles from human breast cell lines, parental and hyaluronan synthase 3-(HAS3)-overexpressing MCF10A cells, grown directly on transmission electron microscopy (TEM) grids. While cells imaged with conventional and cryo-SEM exhibit distinct morphologies due to the sample preparation process for each technique, tubular structures protruding from the cell surfaces were observed with both approaches. For HAS3-MCF10A cells, vesicles were present along the length of membrane protrusions. Once completely shed from the cells, extracellular vesicles were characterized using nanoparticle tracking analysis (NTA) and cryo-TEM. The size distributions obtained by each technique were different not only in the range of vesicles analyzed, but also in the relative proportion of smaller-to-larger vesicles. These differences are attributed to the presence of biological debris in the media, which is difficult to differentiate from vesicles in NTA. Furthermore, we demonstrate that cryo-TEM can be used to distinguish between vesicles based on their respective surface structures, thereby providing a path to differentiating vesicle subpopulations and identifying their size distributions. Our study emphasizes the necessity of pairing several techniques to characterize extracellular vesicles.
KW - Breast cancer
KW - Cellular communication
KW - Cryo-scanning electron microscopy
KW - Cryo-transmission electron microscopy
KW - Exosomes
KW - Extracellular vesicles
KW - Glycocalyx
KW - Microvesicles
KW - Nanoparticle tracking analysis
UR - http://www.scopus.com/inward/record.url?scp=85079167019&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2020.107474
DO - 10.1016/j.jsb.2020.107474
M3 - Article
C2 - 32032755
AN - SCOPUS:85079167019
SN - 1047-8477
VL - 210
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 1
M1 - 107474
ER -