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Techniques to Investigate Mitochondrial Function in Neurons
Author:
Strack Stefan / Usachev Yuriy M
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Springer;Humana Press
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2017
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Springer Science+Business Media LLC 2017

Stefan Strack and Yuriy M. Usachev (eds.) Techniques to Investigate Mitochondrial Function in Neurons Neuromethods 10.1007/978-1-4939-6890-9_1

1. Three-Dimensional Reconstruction of Neuronal Mitochondria by Electron Tomography

Guy Perkins 1

(1)

National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, School of Medicine, University of California - San Diego, Biomedical Sciences Building room 1000, 9500 Gilman Dr, La Jolla, CA 92093, USA

Guy Perkins

Email:

Abstract

Electron tomography (ET) is a method that uses higher voltage electron microscopes and computer image processing to generate reconstructed volumes of cells, organelles and macromolecules. A renaissance in the study of neuronal mitochondrial structure and function is being fueled by improved techniques for ET with the ability now to generate volumes with nanometer resolution at relatively high throughput. High-quality ET requires knowledge and skill and even though generally performed by well-trained scientists, its use can be extended to those with no training in electron microscopy. This premise is the goal of this chapter. Detailed instructions for performing ET on neuronal mitochondria in situ are presented here. Modern microscopes and the software used to collect tilt series and perform the various image processing tasks have become sufficiently user friendly that scientists trained in the biological sciences can learn them relatively quickly, especially with guidance from personnel at ET facilities.

Key words

Electron tomography Image processing Mitochondria Movie Reconstruction Segmentation Specimen preparation Tilt series

Introduction

A goal of neurobiology is to generate a theoretical framework that merges structural, physiological, and molecular knowledge of neuronal mitochondrial function. These realms do not advance in synchrony ; advances in one realm define new experiments in other realms. For example, in the last few decades, our understanding of mitochondrial function has been driven by physiological and molecular techniques. These advances demand higher resolution structural images of mitochondria in situ. During the 1990s and 2000s, a renaissance in mitochondrial structurefunction research was fueled by improved techniques for electron tomography (ET) with the ability now to generate volumes with nanometer resolution. Over the last 18 years, ET has been applied to neuronal mitochondria with special attention to the crista and crista junction architectures.

What is ET? ET is a method that uses higher voltage electron microscopes and computer image processing to generate 3D images from multiple 2D projection images of an object, obtained over a wide range of viewing directions, i.e., tilt angles. The 3D reconstruction is generated by using a computer algorithm to back-project each 2D image with appropriate weighting []. As such, ET is comparable to the medical imaging method of X-ray computerized axial tomography (CAT) in that it generates projection images to provide a 3D view of an object, yet with nanometer resolution. The 3D perspective it provides has revised our understanding of cellular and organellar organizations, their dynamics in normal development, and their perturbations due to disease.

The quality of the tomographic reconstruction depends on each step of the process and care must be exercised to perform each step in the best way to preserve the original structure. The steps that require the most attention to detail are, in order,

Specimen preparation, including the application of fiducial markers.
Tilt series collection, paying attention to optimal imaging parameters.
Image processing, with consideration of the strengths and limitations of common methods.

Other steps that follow are for presenting the 3D data in ways that highlight the rich structural information and focus on interrelationships between structural components and include,

Segmentation of mitochondrial compartments for display and measurements.
Figure and movie making for analysis and presentation.

Methods

2.1 Specimen Preparation for ET

Excellent ultrastructural preservation lays the foundation for a high-quality ET reconstruction and starts with attention to detail. The process is relatively long and the accurate execution of nearly every step is important. Although neurons may be grown in culture, either with or without supporting astrocytes, the focus here is tissues and applies to both tissues in the central nervous system and peripheral nerves. The preparation will address mice and rats only (Note ).

2.1.1 Preparing Primary Fixative

For 50 ml of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer:

Heat 20 ml of double-distilled (dd)water in a glass beaker to above 60 C, but less then 100 C (i.e., not boiling).
In a fume hood, add 1 g paraformaldehyde (Prills EM quality). Stir with a stir bar.
Add 2 drops 1 N NaOH solution into the stirring solution.
When the solution clears, take the beaker off the hotplate (and after cooling close to RT) filter through a #1 Whatman filter, using a funnel into a clean beaker.
Add 5 ml of 25% glutaraldehyde (EM grade).
Add 16 ml of a 0.3 M cacodylate stock solution pH 7.4.
Bring total volume to 50 ml with dd-water.
Warm to 37 C in a water bath before perfusion fixation.

2.1.2 Preparing Ringers Solution

250 ml is needed to perfuse 46 mice.

Add the following components together from stock solutions:
- 2.5 ml Na2HPO4 (18 g/L).
- 24.8 ml NaCl (80 g/L).
- 2.5 ml KCl (38 g/L).
- 2.5 ml MgCl2. 6 H2O (20 g/L).
- 6.3 ml NaHCO3 (50 g/L).
- 0.5 g dextrose.
Bring the total volume to 250 ml with dd-water.
Bubble 95% air/5% CO2 through the solution at 37 C for 5 min.
Add 2.5 ml CaCl22 H2O (30.0 g/l).
Continue to bubble the gas until switching the line to the fixative.
Because high quality ultrastructure is desired, add 2.5 ml xylocaine (anesthetizes smooth muscle) and 5 ml heparin (prevents blood clotting).

2.1.3 Perfusing the Animal (Example Given Is a Mouse)

For mice, use a 25-G needle.

For medium rats, use a 22-G needle.

For large rats, use an 18-G needle

Set out the necessary surgical instruments: large and small scissors, hemostat, tweezers, and iris scissors.
Anesthetize the mouse by giving an intraperitoneal injection of nembutol (0.1 cc/100 gm body weight). Ensure the needle is in the peritoneal cavity by drawing back needle and checking for air (no blood).
Allow the anesthetic to take effect and test the consciousness of the mouse by using a tail pinch and a paw prick. Ensure that breathing does not stop because mitochondrial structure often changes after even only 1 min of ischemia.
Pin the anesthetized mouse to a board.
Using small scissors and tweezers, lift up the skin below the rib cage and cut until the liver is visible. Cut upward along the sides of the body cavity until the diaphragm is visible. Cut the diaphragm and then quickly cut up along the sides of the body, being very careful not to damage the heart.

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