Name: analysis of brain mitochondria using serial block-face scanning electron microscopy
Uploaded: 2016-07-09T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy at JoVE.com

We thank Sidney Walker for providing technical help. This work was supported in part by a grant from the National Institute of Health (1R01EY024712-01A1).

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 nit...

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...

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 regulate the location, size, shape and distribution of mitochondria 4. Moreover, changes in mitochondrial shape are associated with neurodegeneration, neuronal plasticity, muscle atrophy, calcium signaling, reactive oxygen species generation as well as lifespan and cell death implicating that cell-specific mitochondrial morphology is critical for the maintenance of normal cellular function 5-11.
A major bioenergetic function of mitochondria is to generate adenosine triphosphate (ATP) by executing a series of metabolic reactions that involve complete breakdown of nutrients (i.e. glucose, fatty-acids or amino-acids) via the TCA cycle and OXPHOS pathways 12. The human brain constitutes only 2% of body weight however it consumes ~20% of total energy produced making it an extremely energy demanding organ 13. It is therefore not surprising that mitochondrial dysfunction in humans leads to a large number of neurological manifestations 14-17. Genetic mutations in OXPHOS components that impair ATP generation leads to mitochondrial disorders 17,18, which are clinically heterogeneous group of disorders with a prevalence of ~1 : 5,000 individuals, and one of the most common cause of metabolic disorders in children and adults. Deficit of mitochondria-derived ATP affects multiple organ systems with high energy demanding organs such as brain, heart and skeletal muscles being predominantly affected in these patients 14,17,18. In recent years, multiple studies have provided evidence for mitochondrial dysfunction in both neurodevelopmental and neurodegenerative disorders 15-17,19,20. Since mitochondria are essential and critical for brain development and function, it is imperative to develop protocols that can analyze changes in brain mitochondrial morphology, structure, size, number and distribution under both healthy and diseased states. Mouse models with mitochondrially-targeted green fluorescent protein (GFP) have been produced to visualize mitochondrial movements and localization in the brain 21,22. While this is an extremely useful tool to examine mitochondrial motility and general distribution, there are some drawbacks which include limited resolution and sensitivity of fluorescence microscopy. These attributes make it difficult to track the relatively small sized mitochondria. Similarly, serial transmission electron microscopy has been successfully used to view synaptic mitochondria 23, but this method is very time consuming. Mitochondrial morphology is known to be highly dynamic as they undergo continuous fission and fusion cycles, and in most cells mitochondria maintain a highly connected network 24-26. Neurons are highly polarized cells with multiple dendrites and extended axons, and mitochondria that form a connected reticular network in the cell body may have to separate as they make their way through these neurites (Figure 1). This makes brain mitochondria extremely varied in size and shape. For example, using serial block-face scanning electron microscopy (SBFSEM) technique, we previously observed that the difference in the volume or size of extrasynaptic mitochondria to mitochondria present in the nerve terminals may be as much as sixteen fold 27.
There are several approaches for performing volume analyses 28, which includes serial section TEM 29, automated tape collecting ultramicrotome SEM 30, focused ion beam SEM 31, and SBFSEM 32. The SBFSEM analysis has advantages in that it has the resolution to provide quantitative data on the morphological shape, size, distribution and number of organelles such as mitochondria in areas up to 1 mm of the brain. The technical operation is also the least demanding, with data acquisition and analysis within capabilities of many biological labs that lack previous EM experience. The advent of commercial instruments for generating serial section-like images has made 3D ultrastructural analysis of tissues a routine technique, which further permits an unbiased volumetric analysis in a rapid and repeatable manner28. The SBFSEM was first described and used in the field of neurobiology in 2004 32, based on an idea introduced by Leighton in 1981 33. Multiple studies since then have established this technique as a major tool in reconstruction analysis of neuronal circuitry 34. Furthermore, for many smaller scale projects, it provides reconstruction analysis to identify cellular organelles 27,35-39. Since, the acquired images are derived from low voltage back scatter electrons, new staining protocols which combine different known heavy metal staining techniques were developed to increase the resolution 40.
In this paper, we provide a protocol for utilizing 3D electron microscopy imaging and volumetric analysis of brain mitochondria based on methods that have previously been used by us and others 38,39,41. The tissue post-processing methods used were as previously described by Deerinck et al40.

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			<td>C57BL/6J mice</td>
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			<td>664</td>
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			<td>Isoflurane</td>
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			<td>Fisher scientific&#160;</td>
			<td>S65105&#160;</td>
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			<td>Ted Pella Inc.</td>
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			<td>Butterfly canula</td>
			<td>Exel International</td>
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			<td>Phosphate buffer saline</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-100TAB</td>
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			<td>Filter (0.45 micron)</td>
			<td>EMD Millipore</td>
			<td>NC0813356</td>
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			<td>Ted Pella Inc.</td>
			<td>Vibratome 3000</td>
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			<td>EMS</td>
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			<td>21700</td>
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			<td>J.T. Baker</td>
			<td>14459-95-1</td>
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			<td>Sigma-Aldrich</td>
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			<td>Acros Organics</td>
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			<td>Lead Nitrate</td>
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			<td>EMbed-812 EMBEDDING KIT</td>
			<td>EMS</td>
			<td>14120</td>
			<td>Contains Embed 812&#160; resin, DDSA, NMA, and DMP-30.</td>
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			<td>Glutaraldehyde 25% EM Grade</td>
			<td>Polysciences Inc.</td>
			<td>1909</td>
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			<td>EMS</td>
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			<td>EMS</td>
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			<td>EMS</td>
			<td>70907</td>
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			<td>EMS</td>
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			<td>EMS</td>
			<td>12630</td>
			<td></td>
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			<td>Razor</td>
			<td>EMS</td>
			<td>72000</td>
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			<td>Super Glue (Loctite Gel Control)</td>
			<td>Loctite</td>
			<td>234790</td>
			<td>Hardware/craft stores carry this item</td>
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			<td>Conductive epoxy</td>
			<td>Ted Pella Inc.</td>
			<td>16043</td>
			<td></td>
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			<td>Scanning electron microscope</td>
			<td>Zeiss</td>
			<td>Sigma VP</td>
			<td></td>
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		<tr>
			<td>In chamber ultramicrotome for SEM</td>
			<td>Gatan Inc.</td>
			<td>3View2</td>
			<td>Can be designed for other SEMs</td>
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		<tr>
			<td>Trimming microscope for pin preparation</td>
			<td>Gatan Inc.</td>
			<td colspan="2">supplied as part of 3View system</td>
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			<td>Low kV backscattered electron detector</td>
			<td>Gatan Inc.</td>
			<td>3V-BSED</td>
			<td></td>
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			<td>ImageJ/ Fiji processing package&#160;</td>
			<td colspan="2">ImageJ ver 1.50b, FIJI download Oct 1, 2015</td>
			<td><a href="http://zoi.utia.cas.cz/files/imagej_api.pdf">http://zoi.utia.cas.cz/files/imagej_api.pdf</a></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>http://rsb.info.nih.gov/ij/</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td><a href="http://www.icmr.ucsb.edu/programs/3DWorkshop/Uchic-2015_FIJI_Tutorial.pdf">http://www.icmr.ucsb.edu/programs/3DWorkshop/Uchic-2015_FIJI_Tutorial.pdf</a></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td><a href="http://fiji.sc/TrakEM2">http://fiji.sc/TrakEM2</a></td>
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analysis of brain mitochondria using serial block-face scanning electron microscopy

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&#39;s and Parkinson&#39;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 &#60;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.

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...

Watch this Scientific Journal Video about Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy at JoVE.com

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

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.

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, ...

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