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. 2020 Jul 23;10:12372. doi: 10.1038/s41598-020-69256-9

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Workflow for 3D reconstruction of axonal varicosities in the myenteric plexus. (a) A diamond knife (blue) was used cut 50- or 70-nm ultrathin slices from the top of the block-face in SEM (SBF-SEM). In this method, an image is captured after every retraction of the knife, and the cutting and imaging cycle is automatically repeated to acquire serial images. (b) SBF-SEM can produce a series of hundreds or even thousands of orthoslices where segmentation of various ultrastructures can be performed with different software. (c) Following acquisitions and semi-automatic or manual segmentation, orthoslice series and structures were rendered into 3D ultrastructure and quantified. (d) Top: representative (1st, 5th, 10th and 15th) images from an actual stack at 50-nm thickness. Middle: axon (light blue), synaptic vesicles (yellow), and mitochondria (red) were marked in semi-automatic and manual segmentation, and traced using TrakEM2 (https://imagej.net/TrakEM2), Amira version 5.6 (https://www.fei.com/software/amira/), and Microscopy Image Browser. Bottom: three-dimensionally (3D) rendered axon (blue), synaptic vesicles (yellow), mitochondria (red), and merged image.