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 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.

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 institutions will have an EM core facility in which these reagents are routinely utilized and can provide assistance.

1. Preparation of Brain Tissue and SBFSEM Imaging

1. Anesthetize a young (~2 - 4 months old) C57 black mouse (C57BL/6J strain) using 4 - 5% isoflurane following the institutional guidelines. Confirm anesthetization by monitoring the loss of muscle tone, lack of voluntary movements and responses to aversive stimuli like a tail pinch.
2. Pin the mouse on a dissection tray and make an incision on the skin along ventral midline. Make further incisions in the skin to expose the rib cage of the mouse. Incise the diaphragm, and carefully dissect out the chest cavity along the periphery to expose the beating heart, and then make an incision on the right atrium.
3. Using a butterfly cannula, cannulate the left ventricle. Perfuse the mouse transcardially using 10 - 20 ml of phosphate buffer saline (PBS) pH 7.2, until exsanguination is confirmed by a change in the color of the liver.
   Note: A change in the color of liver is used as a guide to determine the extent of exsanguination. Confirm that the liver color changes from reddish brown to pale pink.
4. Perfuse the mouse transcardially using 10 - 20 ml of 2% glutaraldehyde and 4% paraformaldehyde made in 0.10 M cacodylate buffer (pH 7.2), to fix the brain tissue rapidly from within. Fixation is monitored by observing the tail stiffening.
   Note: Prepare 0.10 M cacodylate buffer (pH 7.2) by dissolving 2.14 g of sodium cacodylate in 80 ml water, add hydrochloric acid to adjust the pH, and make the volume to 100 ml with water.
5. Following perfusion, decapitate the mouse, dissect the brain, followed by fixation in 0.10 M sodium cacodylate buffer (pH 7.2) containing 2% glutaraldehyde and 4% paraformaldehyde for 48 hr at 4 ^oC.
6. After 48 hr make 400 µm coronal sections using a vibratome and then carefully dissect the region of interest in the brain (for example, the hippocampus) under the dissection microscope ⁴². Carefully trim the tissue and take a picture for preserving orientation.
   Note: Post processing methods of tissue staining in SBFSEM combines a variety of heavy metal staining methods in order to improve resolution and are based on the method developed previously by Deerinck et al⁴⁰.
7. Wash the glutaraldehyde-fixed tissues 3 times, 5 min each in 0.1 M cacodylate buffer (pH 7.2).
8. Prepare 0.1% tannic acid solution by dissolving tannic acid in 0.1 M sodium cacodylate buffer (pH 7.2). Swirl until dissolved and filter through 0.45 µm filter, if necessary.
9. Postfix the tissues with cacodylate buffered 0.1% tannic acid by incubating in 1 ml reagent for 30 - 60 min at RT.
   Note: The incubation time is dependent on tissue size, but 30 min works best for most tissues.
10. Wash tissues 3 times, 5 min each in cacodylate buffer (pH 7.2).
11. Dissolve 0.3 g potassium ferrocyanide and 0.86 g sodium cacodylate in 10 ml distilled H₂O (dH₂O). Keep the potassium ferrocyanide solution on ice. Just before use, add 10 ml of 4% osmium tetroxide (OsO₄).
12. Stain the tissues with 2% osmium-ferrocyanide solution for 90 min, on ice. Wash 3 times, 5 min each in dH₂O.
13. Prepare 1% thiocarbohydrazide (TCH) solution by dissolving 0.1 g TCH in 10 ml dH₂O. Dissolve at 60 °C by swirling every 10 min until fully dissolved. Observe safety precautions while handling TCH, particularly take care to avoid use of metals, heating to high temperature, or allowing solution to dry out.
14. Treat the samples with freshly prepared 1% TCH for 20 min at RT. Wash 3 times, 5 min each in dH₂O.
15. Dilute 4% OsO₄ to 2% with dH₂O, stain tissues with 2% aqueous osmium tetroxide by incubating for 1 hr. Wash tissues 3 times, 5 min each with dH₂O.
16. Incubate the samples O/N in 1% uranyl acetate in dH₂O at 4 °C.
17. Prepare Walton's lead aspartate solution (as described by Deerinck et al⁴⁰).
    1. Dissolve 0.998 g L-Aspartate in 250 ml dH₂O and then add 10 N potassium hydroxide (KOH) in a dropwise fashion until the pH reaches 5.5. After pH adjustment, add 0.066 g of lead nitrate in 10 ml aspartic acid stock and heat to 60 °C for 30 min.
18. Rinse tissues in dH₂O and incubate with Walton's lead aspartate stain for 30 min in a 60 °C oven. Wash 3 times, 5 min each in dH₂O.
19. Dehydrate samples through a graded series of alcohol using chilled solutions of 20%, 50%, 75%, 85% and 95% ethanol for 5 min each, followed by 100% ethanol 3 times, 10 min each.
    Note: Use 100% ethanol from freshly opened bottle, as opened ethanol absorbs water from the air and cause embedding to fail. Longer incubations will be necessary with larger tissue samples.
20. Wash samples 2 times, 15 min each in propylene oxide.
21. Make plastic embedding resin using 25 ml resin, 10.5 ml DDSA (dodecenyl succinic anhydride), 15.5 ml NMA (nadic methyl anhydride) and 1 ml DMP-30 (2,4,6-Tris dimethylaminomethyl phenol). Mix the resin by shaking. Spin and allow the resin to stand until bubbles resolve.
    Note: This is the standard medium hardness recipe. Other types of electron microscopy (EM) plastic resin may be used, but should be tested with a non-essential control sample in advance as not all resins work for SBFSEM.
22. Incubate the tissues O/N in a 50 : 50 mix of embedding resin and propylene oxide, in a vial that is capped initially, then uncapped after 2 hr so that the propylene oxide evaporates over the period of about 8 - 10 hr.
23. Transfer the tissues to 100% fresh embedding resin in clean vials for 2 hr.
24. Embed samples in fresh embedding resin, in flat molds containing printed paper labels, and cure them in an oven at 60 °C for 48 hr. After about 1 hr, check the tissue placement and alignment again, and adjust if necessary.
25. Trim samples to the area of interest and mount on an aluminum pin using gelling cyanoacrylate superglue or a conductive epoxy resin, then coat with colloidal silver paste around the sides of the block to provide a conductive path to the aluminum pin.
26. Examine tissue specimens using a scanning electron microscope system equipped with an in-chamber ultramicrotome stage and low kV backscattered electron detector ³².
    Note: Obtain instrument- and site-training in scanning electron microscopy (SEM) use and become an authorized user. Alternatively, collaboration with a researcher or core facility for small projects may be possible. Radiation training may also be required as SEMs generate x-rays.
27. To image the samples, use the following settings: 2.25 kV, at 5 - 10 nm/pixel resolution, with field sizes between 80 - 250 µm in x,y (other field sizes possible), and slice thickness of 50 - 100 nm, with a total of 250 - 600 slices in a 16 - 20 hr time period.
    Note: Settings vary substantially between different microscopes, individual samples, and desired resolution. These settings should produce images that are readily interpretable for many samples.

2. Analyzing the Imaging Dataset

Note: The Image J/Fiji software is used to analyze the dataset and relies upon the TrakEM2 plugin. Preprocessing steps may be performed using a variety of software, and may be extensive or minor depending on experience level and the stacks obtained. The main transformations using the open-source software (ImageJ ver 1.50b, FIJI download Oct 1, 2015) are described here.

1. Convert images to 8 bit tiff format from the original proprietary 16-bit images by opening in the software and selecting menu items Image→Type→8 bit.
   1. If automatic contrast/brightness conversion during this step is not ideal for images, reopen the 16-bit images, and press Image→Adjust→Brightness/Contrast. Select a range that works for all images, and press Apply. Then perform conversion. Note: On some SEMs, this step may require microscope manufacturer software.
   2. If required, due to unacceptable image movement between slices (e.g. drift due to charging), register/align the image stacks (menu items Plugins→Registration→Linear StackAlignmentWithSIFT). In most registration software, set for "translation-only" mode rather than "rigid body".
      Note: Many approaches and software may work: the SIFT registration plug-in works for many applications and there is a virtual stack version.
      1. If required, enlarge the canvas size prior to registration (Image→Adjust→CanvasSize) or reduce it to an area of interest (Image→Crop).
         Note: Some drift or splaying may occur, and trying other plugins or software may produce better results. Manual options are also available (e.g. ImageJ/FIJI, Plugins→Registration→ManualLandmarkSelection).
   3. If desired, scale images to smaller, more manageable size (e.g. 25%) using ImageJ (Image→Scale).
2. Launch the software, select File→import→image sequence and then select the tiff files.
3. Launch the plugin by selecting File→new→TrakEM2 (Blank). Two windows will open; one manages the project and area_lists and the other manages tracing, and is referred to as the 'canvas'.
4. Load the image stack into the plugin by making a right-click on the canvas import. Select import and click on import stack. In the pop-up options, check the box for virtual stacks.
   Note: Although the image stacks are already opened in the software, they must also be opened in the plugin. The computer may run more smoothly if the virtual stacks box is checked.
5. After the mipmaps are created and the stack is loaded, right click to name the project under the plugin window. Right click on 'new project' in the template column, and select 'add new child' for that project. Right click again to select 'area_list'.
6. Set the Z-axis scale of the project to agree with the original electron micrograph settings by right clicking on the canvas, then click display and select calibration. Also, set the Z-scale by selecting all layers in the plugin window, right click to select scale Z and thickness.
7. Click and drag the 'project' and all 'children' into the project objects section to create the 'area lists' under the 'Z space' tab in the canvas window.
8. Select the 'area list' and right click to select and set a color. Now, use the paint brush tool to trace objects such as mitochondria, in the electron micrograph images. Use 'Shift + click' to fill in an enclosed circle, 'Ctrl + scroll' to zoom in and out, and 'Alt + click' to turn paint brush cursor into an eraser.
9. Select two areas of ~10 - 15 µm by 10 - 15 µm at the top left corner and bottom right corner of the image and identify all mitochondria within these areas to allow unbiased sampling of mitochondria in each dataset.
10. On the 'canvas', observe and trace mitochondria throughout the sections. Note: Mitochondria are quite dark/dense appearing organelles, of similar size in diameter and loosely cylindrical. The unique cristae formed by the inner membrane are easily distinguishable inside this organelle.
11. When finished with tracing, right click on the area_list under the Z space tab, select 'Show in 3D'. This will launch the 3D viewer plugin to view a 3D reconstruction of the traced image.
    1. To perform mitochondrial volume measurements, select object in 3D viewer, click on 'Edit' tab and select 'Object Properties'. The mitochondrial volume estimate is listed along with several other measurements.

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