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Structural Analysis of Exosomes Using Different Types of Electron Microscopy
Applied Microscopy 2017;47:171-5
Published online September 30, 2017
© 2017 Korean Society of Microscopy.

Hyosun Choi1, and Ji Young Mun1,2,*

1BK21 Plus Program, Department of Senior Healthcare, Graduate School, Eulji University, Daejeon 34824, Korea, 2Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam 13135, Korea
Correspondence to: Mun JY, Tel: +82-31-740-7380, Fax: +82-31-740-7354, E-mail: mjy1026@gmail.com
Received June 12, 2017; Revised July 24, 2017; Accepted July 24, 2017.
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Negative staining has been traditionally used for exosome imaging; however, the technique is limited to surface topology only and can cause staining artifacts. Therefore, to analyze the internal structure of exosomes, we employed a method of block preparation, thin sectioning, and electron tomography. In addition, an automatic serial sectioning technique with 15-nm thickness through focused ion beam was employed to observe the three-dimensional structure of exosomes of various sizes. Cryo-transmission electron microscopy revealed the near-to-native structure of exosomes.

Keywords : Exosome, Electron tomography, Serial block face section through focused ion beam, Cryo-transmission electron microscopy
Figures
Fig. 1. Sectioned images of exosomes after block preparation. (A) Cryo-transmission electron microscopy (TEM) image from a chemically fixed block. (B) TEM image from an HPF-FS block of exosome-treated cells. Arrow indicates exosome.
Fig. 2. Three-dimensional (3D) reconstruction of exosome serial sections by focused ion beam (serial block face)-SEM (FIB-SEM). The FIB-SEM setup is a combination of ion milling (by gallium ions) and SEM imaging (by backscattered electrons) in the same instrument. The sections are cut automatically using the gallium ion beam and then images of the surface block face are prepared using the backscattered electron beam. Serial sections are easy to align for 3D reconstruction because of automatic sectioning and imaging without loss of samples. The 3D structures are reconstructed using the IMOD software. Each exosome can be represented as a different color.
Fig. 3. Electron tomography. The 61 tilt images (2° intervals at ±60°) of sectioned exosomes with 60-nm thickness were used for electron tomography. The tomogram was obtained using the through ETOMO software and showed improved resolution. Tilted images (left panel) do not show a clear detailed inner structure of exosomes, unlike the electron tomogram. Arrows show exosomes.
Fig. 4. Cryo-fixed exosomes. (A, B) Cryo-transmission electron microscopy images. The bar represents 100 nm.
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