Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Summary

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.

Abstract

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer's and Parkinson's diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of <200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Introduction

Mitochondria are dynamic organelles which change their shape and location depending on the cellular cues and needs, in tight interaction with cell cytoskeleton, and in response to cellular events such as calcium currents in neurons ¹. Mitochondria also interact with other cellular organelles e.g. endoplasmic reticulum, which in turn regulates their dynamics and metabolism². Mitochondrial morphology shows heterogeneity in different cell types i.e. the shape of the organelle varies from tubular to that consisting of sheets, sacks and ovals ³. It has been shown that mitochondrial fusion and fission cycle proteins can reg....

Protocol

Ethics Statement: Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Tech.

Caution: Extreme precautions must be taken when handling and disposing several components used in this protocol. Before use, the local institutional guidelines and health and safety practices must be established and followed, particularly for osmium tetroxide, which is volatile and extremely poisonous, uranyl acetate, which is both a heavy metal and source of radioactivity, and lead nitrate, which is a heavy metal poison. Thiocarbohydrazide (TCH) can decompose to produce explosive and poisonous gases, if incorrectly handled. Many ins....

Results

We demonstrate that the brain mitochondrial morphology and size is heterogeneous in different neuronal sub-compartments. Confocal microscopy on low density neuronal cultures transduced with lentivirus expressing mitochondrially-targeted green fluorescent protein showed that mitochondria residing in neuronal soma form a reticular network, whereas those residing in distal neurites exhibit a discrete elongated morphology (Figure 1 A-B). Using the SBFSEM technique, the mitoch.......

Discussion

The complexity of the nervous system poses a significant challenge in reconstructing large tissue volumes and analyzing the morphology and distribution of organelles such as mitochondria with adequate resolution. Multiple cells including neurons, oligodendrocytes and astrocytes with numerous processes extended in three dimensions interact within the brain tissue ⁴³. Since mitochondria resides both in the soma of cells and distant processes, mitochondrial morphology is extremely pleomorphic in the nervous syste.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6J mice	Jackson laboratory	664
Isoflurane	VETone, tradename Fluriso	501017
Dissection tray	Fisher scientific	S65105
Dissection scissors	Ted Pella Inc.	1316
Butterfly canula	Exel International	26704
Phosphate buffer saline	Sigma-Aldrich	P4417-100TAB
Filter (0.45 micron)	EMD Millipore	NC0813356
Dissection microscope	Olympus	SZ61
Vibratome sectioning system	Ted Pella Inc.	Vibratome 3000
Sodium Cacodylate	EMS	12300
Tannic Acid	EMS	21700
Potassium Ferrocyanide	J.T. Baker	14459-95-1
Osmium Tetroxide 4% Solution	EMS	19150
Thiocarbohydrazide	EMS	21900
L-Aspartic Acid	Sigma-Aldrich	A93100
Potassium Hydroxide	Acros Organics	43731000
Lead Nitrate	EMS	17900
EMbed-812 EMBEDDING KIT	EMS	14120	Contains Embed 812  resin, DDSA, NMA, and DMP-30.
Glutaraldehyde 25% EM Grade	Polysciences Inc.	1909
Paraformaldehyde	EMS	19202
Uranyl Acetate	EMS	22400
Ethanol	EMS	15055
Propylene Oxide	EMS	20400
Embedding Mold	EMS	70907
Aluminum specimen pin	EMS	70446
Colloidal Silver Liquid	EMS	12630
Razor	EMS	72000
Super Glue (Loctite Gel Control)	Loctite	234790	Hardware/craft stores carry this item
Conductive epoxy	Ted Pella Inc.	16043
Scanning electron microscope	Zeiss	Sigma VP
In chamber ultramicrotome for SEM	Gatan Inc.	3View2	Can be designed for other SEMs
Trimming microscope for pin preparation	Gatan Inc.	supplied as part of 3View system
Low kV backscattered electron detector	Gatan Inc.	3V-BSED
ImageJ/ Fiji processing package	ImageJ ver 1.50b, FIJI download Oct 1, 2015	http://zoi.utia.cas.cz/files/imagej_api.pdf
http://rsb.info.nih.gov/ij/
http://www.icmr.ucsb.edu/programs/3DWorkshop/Uchic-2015_FIJI_Tutorial.pdf
http://fiji.sc/TrakEM2