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, Eulji University researchers 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.
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 3Dreconstruction 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.