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

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 tissues^1,2. 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 Styrofoam^3,4. Almost 70 years later, there have been considerable advances in numerous aspects of the process, from microscope performance, serial section col....

Protocol

All animals were housed in accordance with the UK Home Office guidelines, and the tissue harvesting was carried out in accordance with the UK Animal (Scientific Procedures) Act 1986.

1. Specimen fixation and preparation

1. Dissect the liver tissue into appropriate size pieces, approximately 8 mm x 8 mm x 3 mm, and place the pieces in warm phosphate-buffered saline (PBS, 37 °C).
2. Inject room temperature (20-25 °C) fixative (1.5% glutaraldehyde in 1% sucrose, 0.1 M sodium cacodylate) into the liver pieces and transfer them from PBS to fixative for up to 20 min at room temperature. Always keep the tissue subm....

Results

For this technique, regions of interest are selected based on the biological research aim and identified prior to the trimming and sectioning of embedded tissue. Similarly, the size of the block face may be dictated by the research question; in this case, the sample was trimmed to leave a block face of approximately 0.3 mm x 0.15 mm (Figure 4A). This allowed for two grids of 9 serial sections per grid, providing 18 serial sections and incorporating a volume of liver tissue of a volume of app.......

Discussion

An accessible vEM technique for visualizing organelle structure and interactions in 3D is described in this protocol. The morphology of interorganelle contacts in hepatocytes is presented as a case study here. However, this approach has also been applied to investigate a variety of other samples and research areas, including Schwann cell-endothelial interactions in peripheral nerves⁴⁵, Weibel Palade Body biogenesis in endothelial cells⁴⁶, cargo secretion in kidney cells

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 µm syringe filter	Sarstedt	83.1826.001
Aluminum trays	Agar Scientific	AGG3912
Amira v6	ThermoFisher		https://www.thermofisher.com
Chloroform	Fisher	C/4960/PB08
DDSA/Dodecenyl Succinic Anhydride	TAAB	T027	Epon ingredient
Diamond knife	DiaTOME	ultra 45°
DMP-30/2,4,6-tri (Dimethylaminomethyl) phenol	TAAB	D032	Epon ingredient
Dumont Tweezers N5	Agar Scientific	AGT5293
Fiji			https://imagej.net/
Fiji TrakEM2 plugin			https://imagej.net/
Formaldehyde 36% solution	TAAB	F003
Formvar coated slot grid	Homemade		Alternative: EMS diasum (FF2010-Cu)
Glass bottle with applicator rod	Medisca	6258
Glass vials	Fisher Scientific	15364769
Gluteraldehyde 25% solution	TAAB	G011
MNA/Methyl Nadic Anhydride	TAAB	M011	Epon ingredient
Osmium Tetroxide 2% solution	TAAB	O005
Potassium Ferricyanide	Sigma-Aldrich	P-8131
Propylene oxide	Fisher Scientific	E/0050/PB08
Reuseable adhesive	Blue Tack
Reynolds Lead Citrate	TAAB	L037	Section stain
Sodium Cacodylate	Sigma-Aldrich	C-0250	to make 0.1 M Caco buffer
Super Glue	RS Components	918-6872	Cyanoacrylate glue, Step 1.3
TAAB 812 Resin	TAAB	T023	Epon ingredient
Tannic acid	TAAB	T046
Triton X-100	Sigma-Aldrich	T9284
Two part Epoxy Resin	RS Components	132-605	Alternative: Step 2.13
Ultramicrotome	Leica	UC7
Vibrating microtome	Leica		100 µm thick slices, 0.16 mm/s cutting at 1 mm amplitude .
Weldwood Original Contact cement	DAP	107	Contact adhesive: Step 3.1.4