4.4 Article

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy

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JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/63496

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  1. MRC [MC_U12266B, MC_UU_00012/6]
  2. European Research Council [ERC-2013StG-337057]

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Transmission electron microscopy (TEM) has been the standard technique for visualizing cellular ultrastructure, but it is limited to two-dimensional analysis. Volume electron microscopy (vEM) techniques provide a way to study cellular ultrastructure in three dimensions at different resolutions. This article presents a straightforward workflow for acquiring vEM data using serial section TEM and covers the entire process from sample processing to 3D reconstruction. The usefulness of this technique is demonstrated by studying the 3D ultrastructural relationship between the endoplasmic reticulum and mitochondria in liver hepatocytes.
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 similar to 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.

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