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

Gary Hong Chun Chung; Paul Gissen; Christopher J. Stefan; Jemima J. Burden

doi:10.3791/63496

Method Article

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

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DOI:

10.3791/63496

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June 9th, 2022

Gary Hong Chun Chung¹ , Paul Gissen¹^,² , Christopher J. Stefan¹ , Jemima J. Burden¹

¹MRC Laboratory for Molecular Cell Biology, University College London, ²NIHR Great Ormond Street Hospital Biomedical Research Centre, University College London

Summary

A simple and comprehensive protocol to acquire three-dimensional details of membrane contact sites between organelles in hepatocytes from the liver or cells in other tissues.

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.

Introduction

Since their invention in the 1930s, electron microscopes have allowed researchers to visualize the structural components of cells and tissues¹^,². Most investigations have provided 2D information, as building 3D models requires painstaking serial section collection, manual photography, negative processing, manual tracing, and the creation and assembly of 3D models from sheets of glass, plastic, or Styrofoam³^,⁴. Almost 70 years later, there have been considerable advances in numerous aspects of the process, from microscope performance, serial section col....