Chun Chung, Gary Hong;
Gissen, Paul;
Stefan, Christopher J;
Burden, Jemima J;
(2022)
Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy.
Journal of Visualized Experiments
, 184
, Article e63496. 10.3791/63496.
Preview |
Text
Burden_jove-protocol-63496-three-dimensional-characterization-interorganelle-contact-sites.pdf Download (1MB) | Preview |
Abstract
Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is often limited to two dimensions, hampering the ability to fully describe the three-dimensional (3D) ultrastructure and functional relationship between organelles. Volume electron microscopy (vEM) describes a collection of techniques that enable the interrogation of cellular ultrastructure in 3D at mesoscale, microscale, and nanoscale resolutions. This protocol provides an accessible and robust method to acquire vEM data using serial section transmission EM (TEM) and covers the technical aspects of sample processing through to digital 3D reconstruction in a single, straightforward workflow. To demonstrate the usefulness of this technique, the 3D ultrastructural relationship between the endoplasmic reticulum and mitochondria and their contact sites in liver hepatocytes is presented. Interorganelle contacts serve vital roles in the transfer of ions, lipids, nutrients, and other small molecules between organelles. However, despite their initial discovery in hepatocytes, there is still much to learn about their physical features, dynamics, and functions. Interorganelle contacts can display a range of morphologies, varying in the proximity of the two organelles to one another (typically ~10-30 nm) and the extent of the contact site (from punctate contacts to larger 3D cisternal-like contacts). The examination of close contacts requires high-resolution imaging, and serial section TEM is well suited to visualize the 3D ultrastructural of interorganelle contacts during hepatocyte differentiation, as well as alterations in hepatocyte architecture associated with metabolic diseases.
Type: | Article |
---|---|
Title: | Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy |
Open access status: | An open access version is available from UCL Discovery |
DOI: | 10.3791/63496 |
Publisher version: | https://doi.org/10.3791/63496 |
Language: | English |
Additional information: | © 2022 JoVE Creative Commons Attribution-NonCommercial 3.0 License |
UCL classification: | UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Lab for Molecular Cell Bio MRC-UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL |
URI: | https://discovery.ucl.ac.uk/id/eprint/10150048 |
Archive Staff Only
View Item |