1. Sample fixation and resin embedding
<ol>
<li>Samples of fresh tissue and cells are fixed for at least 2 hours in 2% paraformaldehyde, and 2.5% glutaraldehyde in 0.1 M phosphate buffer at a pH of 7.4. The sample size should not exceed 0.150 mm in thickness to allow adequate penetration or fixative, and should not be left in the fix for longer than 12 hours.</li>
<li>Place the samples in 20ml glass scintillation vials and rinse them three times, 5 minutes each, in 0.1M cacodylate buffer.</li>
<li>Stain with 1.5 % (w/v) potassium ferrocyanide and 1% (w/v) osmium tetroxide in 0.1 M cacodylate buffer (0.1 M, pH 7.4) for 30 minutes.</li>
<li>Stain with 1.0 % (w/v) osmium tetroxide in 0.1M cacodylate buffer for 30 minutes.</li>
<li>Rinse once with double distilled water for 3 minutes.</li>
<li>Stain with 1 % (w/v) uranyl acetate in double distilled water for 30 minutes.</li>
<li>Rinse sections for 5 minutes in double distilled water, and then dehydrate in graded alcohol series, 2 mins each change (1 x 50 %, 1 x 70 %, 1 x 90 %, 1 x 95 %, 2 x 100 %).</li>
<li>Embed in increasing concentrations of Durcupan resin mixed with ethanol, 30 minutes each change, starting at 50% Durcupan in ethanol, then followed by: 70%, 90%, 95%, and then 100%.</li>
<li>Replace with fresh Durcupan and agitate slowly for 4 hours.</li>
<li>Place sections, using wooden cocktail sticks, on glass microscopes slides coated with mould separating agent, and place in 65&deg; C oven for 24 hours.</li>
</ol>
2. Preparing the sample for the FIB/SEM
<ol start="11">
<li>Separate the resin layer, containing the samples, from between the two glass microscope slides and wash thoroughly to remove any mould separating agent.</li>
<li>Using a transmitted light microscope with low power objectives, or dissecting microscope with transmitted illumination, to identify the region of interest within the slice of resin.</li> 
<li>Cut a small (3 mm x 3 mm) square around the region of interest using a razor blade and stick this to the top of blank resin block with acrylic glue (Figure 1). Wait 5 minutes for the glue to harden, and then proceed with the next step.</li>
<li>Clamp the block into the holder of the ulramicrotome and, using the attached stereomicroscope, trim around the region of interest with a razor blade until only a small pyramid of material remains.</li>
<li>Further trim the block, using a glass knife fixed into the ultramicrotome, so that the region of interest can be precisely located with respect the dimensions of the block&#x2019;s surface (Figure 1). Cut one edge of the block, close to the region of interest, so that a perpendicular step is cut through the sample (Figure 1 and 2). This step forms the imaging face (Figure 2).</li> 
<li>Remove the block from the ultramicrotome and photograph the region of interest within the block, using a transmitted light microscope.</li>
<li>Cut the trimmed block away from the remaining resin stub. This is done by using a jeweller&#x2019;s saw and ensures that only a small block is placed inside the FIB/SEM. The total height of this block should not be more than 5 mm.</li>
<li>Glue the block to an aluminium stub with carbon paste ensuring that the side to be imaged is on the outermost edge (Figure 1).</li>
<li>After the glue has dried, coat the block with a thin layer of gold (&gt; 20 nm; Cressington vacuum evaporation system).</li>
</ol>
3. Imaging in the FIB/SEM
<ol start="20">
<li>At a low magnification, and using secondary electron imaging ( 5 kV, 0.5 nAmp), orient the block so that the chosen region of interest and the side of the block to be imaged is facing the operator (Figure 2 c, d).</li>
<li>Orient the block so that the face to be imaged lies parallel to the milling beam. This will mean that electron beam is oriented at 54 &deg; to this face (Figure 2 b).</li>
<li>Using an ion beam current of 13 - 27 nAmps at 30 kV remove a narrow band of resin from the front of the region to be imaged.</li>
<li>Switch to backscattered imaging mode to view the milled face that overlies the region of interest. </li>
<li>Using the light microscopy reference image (taken at step 16) and the image of the milled face locate the exact region on the block to be milled and imaged (Figure 2).</li>
<li>Deposit a protective layer (approximately 1 micron thick) of carbon (or metal), using the gas injection system of the microscope, onto the surface of the block, above the region of interest (Figure 2).</li>
<li>Using a current of 700 picoAmps, finely mill the region of the block within which the final images will be taken.</li>
<li>Leave the milling beam to completely mill this image face until no milling artifacts can be seen on the face. A complete mill of the face is checked by observing changes in each subsequent image across the entire field of view. Inadequate milling can also seen as white streaks or &#x2018;curtains&#x2019; appearing vertically in the image.</li>
<li>Leave the microscope for a least 2 hours for any thermal changes to dissipate. This reduces the risk that the block face will drift during imaging, resulting in misaligned images.</li>
<li>Select the microscope parameters for imaging the face serially. Ensure that the electron beam has a voltage that is low enough to only image a very shallow depth of material in the block face. This should also be much shallower than the thickness of the face to be removed. Typical parameters for imaging are voltages of between 1.2 - 2.0 kV with pixel sizes of between 4 - 20 nm. The pixel dwell time needs to be kept around 10 &mu;secs so that the total time to mill the face and acquire one image is maintained below 2 minutes (Figure 3).</li>
</ol>
4. Secrets to success
Step 1) Ensure the samples are not thicker than 150 microns. This will enable adequate penetration of the different stains and resin into the material. This can be achieved by sectioning the samples with a vibratome immediately after the fixation. This thickness is appropriate for brain tissue. Other tissues need to be tested to ensure adequate fixation and staining.
Step 3) The samples must be freely distributed throughout the solution, and not clumped together in one part of the vial while this secondary fixative is introduced. This will ensure maximum penetration of the sample and reduce the sample becoming distorted during this fixation process. This is achieved by gently swirling the samples within the vial immediately after the solution is added.
Step 15) Ensure that the region of interest is located immediately below (&lt; 30 microns) the surface of the block. This will reduce the amount of milling by the ion beam during the image acquisition phase.
Step 22) The scanning time of the ion beam needs to be adequte to ensure that the entire imaging face is completely milled. Inadequate milling will be shown as white streaks, or &#x2018;curtains&#x2019; appearing vertically down the imaging face.
Step 26) The final milled face must be larger than the face that is imaged. This is because the milled resin is ejected from the block and redeposits on the block surface in the immediate vicinity. This redeposition can encroach onto the imaging field of view, and is avoided by milling a much large area than is imaged. For an imaging window that is 20 microns wide, the milled block face is greater than 30 microns wide.
Step 29) It is critical that the electron beam parameters are such that the image is generated by electrons that emerge from only the first few nanometres of the block&#x2019;s surface. This a acheved by minimising the voltage to below 2 kV and ensuring that no electrons penetrate too far. This is also helped by using a grid voltage on the detector so that only the electron with the highest energies contribute to the final image. Typically, a grid tension of 1.3 kV is used for an imaging voltage of 1.5 kV.
5. Representative Results:
<img src="/files/ftp_upload/2588/2588fig1.jpg" alt="Figure 1"/> Figure 1. Preparation of resin block. a) Resin embedded coronal section (80 microns thick) through an adult rat brain viewed in a steromicroscope. The image clearly shows the different brain regions that can be cut from the section (b) using a scalpel blade. Here, a 1 mm square from the cortex is removed and (c) stuck to a blank resin block. d) The resin block is then trimmed with a glass knife mounted in an ultramicrotome, and once the block has been reduced to only leave the region of interest (e), a step is cut perpendicular to the front surface of the embedded material. f) This entire trimmed region is then cut from the rest of the resin and mounted onto a aluminium stub ready for metal coating and imaging in the scanning electron microscope.
<img src="/files/ftp_upload/2588/2588fig2.jpg" alt="Figure 2"/> Figure 2. Preparing the block face for FIB/SEM imaging. a) and b) show illustrations of the block and the edge that is milled to create the imaging face (indicated with the black double-headed arrow). The position of the ion beam (white) is shown parallel to the imaging face, and the electron beam (grey) is shown hitting the face at 54&deg; to the ion beam. c) shows a view of the block taken with the electron beam and imaged with the secondary electrons. Along this side of the block a darker region (indicated with dotted white box) shows a portion of the face that has been roughly milled (shown with double-headed black arrow) to reveal the underlying sample. d) When this region is imaged using only the backscattered electrons the embedded tissue can be seen. Here, the full width of the embedded tissue section is indicated with a double headed white arrow. Scale bar in c) is 100 microns.
<img src="/files/ftp_upload/2588/2588fig3.jpg" alt="Figure 3"/> Figure 3. Imaging the a volume of brain tissue with isotropic voxels. a) Reversed contrast backscattered image of the block face showing the ultrastructure within a region of the rat brain. All membranes are visible as well as large macromolecular structures. b) A total of 1600 images were collected at 5 nm spacing resulting in an image stack with isotropic voxels. c) This stack can be imaged in any plane with the same resolution. This image shows single synaptic connection that is imaged in the x y plane and the y z plane.

<ol>
	<li>Knott, G., Marchman, H., Wall, D., Lich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+section+scanning+electron+microscopy+of+adult+brain+tissue+using+focused+ion+beam+milling.">Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling.</a> J Neurosci. 28, 2959-2964 (2008).</li><li>Hekking, L. H., Lebbink, M. N., De Winter, D. A., Schneijdenberg, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+ion+beam-scanning+electron+microscope:+exploring+large+volumes+of+atherosclerotic+tissue.">Focused ion beam-scanning electron microscope: exploring large volumes of atherosclerotic tissue.</a> J Microsc. 235, 336-347 (2009).</li><li>Armer, H. E., Mariggi, G., Png, K. M., Genoud, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+transient+blood+vessel+fusion+events+in+zebrafish+by+correlative+volume+electron+microscopy.">Imaging transient blood vessel fusion events in zebrafish by correlative volume electron microscopy.</a> PLoS One. 4, e7716-e7716 (2009).</li></ol>

No conflicts of interest declared.

The technique of FIB/SEM works optimally when the region of interest is not too large. The upper limit for an imaged volume depends on the ion beam and the area that it can consistently mill repeatedly over many hours. So far this has been done with areas of around 60 x 60 microns, however, imaging this entire area is not possible with a suitable image resolution, mainly due to the time factor in collecting such a large image. As an example, a 4 k x 4 k image with a pixel dwell time to 10 microseconds would take 2 minutes and 40 seconds to acquire, and for a field of view of 60 microns this would only give a pixel size of 15 nm. For imaging the ultrastructure of cells and tissue, a smaller pixel size is more suitable; usually between 4 and 10 nm / pixel. For an area of 60 x 60 microns the image would be acquired in 10 hours, even if the microscope had this capability. For these reasons, the microscope is an ideal tool for image volumes in the order of 10 x 10 x 10 microns, or significant regions of whole cells. This can easily be done within a 48 hour period on samples that are well-contrasted with this procedure.
So far the protocol has been used mainly with brain tissue, as well as various types of cultured mammalian cells adhering to coverslips. However, the fixation and staining procedure can be used with many other types of biological material, including plant material to produce equally well-contrasted image stacks.
Therefore the main advantage of this technique is its capacity to image volumes with isotropic voxels. Image stacks of this type allow for analyses of the 3d volume using images taken at any plane in the stack. The benefit of this is to allow the observer to image features in the image series from any direction and providing a greater image detail (Figure 3).

<table border="1">
 <tbody>
 <tr>
 <th>Name of Reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 <td>Cyanoacrylate glue</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dissecting microscope: Leica MZ8</td>
 <td>Leica Microsystems, Germany</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Durcupan resin</td>
 <td>Sigma-Aldrich Chemie GmbH, Switzerland</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FIB/SEM microscope (Zeiss, NVision 40)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass histology slides</td>
 <td>Menzel-Gl&auml;ser, Germany</td>
 <td>AA00008032E </td>
 </tr>
 <tr>
 <td>Glass knife maker: Leica EM KMR2</td>
 <td>Leica Microsystems, Germany</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass scintillation vials (20ml)</td>
 <td>EMS, USA</td>
 <td>72634</td>
 </tr>
 <tr>
 <td>Glutaraldehyde</td>
 <td>EMS, USA</td>
 <td>16222</td>
 </tr>
 <tr>
 <td>Jeweler&#x2019;s saw</td>
 <td>EMS, USA</td>
 <td>72010</td>
 </tr>
 <tr>
 <td>Mould separating agent</td>
 <td>Glorex, Switzerland</td>
 <td>62407445</td>
 </tr>
 <tr>
 <td>Osmium tetroxide</td>
 <td>EMS, USA</td>
 <td>19110</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>EMS, USA</td>
 <td>RT 19208</td>
 </tr>
 <tr>
 <td>Phosphate salts for phosphate buffer</td>
 <td>Sigma-Aldrich Chemie GmbH, Switzerland</td>
 <td>71642 and 71496</td>
 </tr>
 <tr>
 <td>Sodium cacodylate</td>
 <td>Sigma-Aldrich Chemie GmbH, Switzerland</td>
 <td>20840</td>
 </tr>
 <tr>
 <td>Sputter Coater with gold target</td>
 <td>Cressington, USA</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tris base (TBS)</td>
 <td>Sigma-Aldrich Chemie GmbH, Switzerland</td>
 <td>T1378</td>
 </tr>
 <tr>
 <td>Ultramicrotome: Leica UCT</td>
 <td>Leica Microsystems, Germany</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Uranyl acetate</td>
 <td>EMS, USA</td>
 <td>RT 22451</td>
 </tr>
 </tbody>
</table>

focussed ion beam milling and scanning electron microscopy of brain tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

Watch this Scientific Journal Video about Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue at JoVE.com

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

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Research

JoVE Journal

Neuroscience

27.9K Views.  École Polytechnique Fédérale de Lausanne. Questo protocollo descrive come il tessuto cerebrale resina incorporati possono essere preparati e ripreso nelle tre dimensioni del fascio di ioni focalizzato, microscopio elettronico a scansione.

Video: Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

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.

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, ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K+ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K+ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K+-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K+ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K+ concentrations in the nervous system.

Potassium ions contribute&#160;to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices.

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase uptake of antibodies and engagement through transient opening of the blood-brain barrier (BBB). In our laboratory, we are developing therapeutic approaches for neurodegenerative diseases in which an antibody in various formats is delivered across the BBB using microbubbles, concomitant with focused ultrasound application through the skull targeting multiple spots, an approach we refer to as scanning ultrasound (SUS). The mechanical effects of microbubbles and ultrasound on blood vessels increases paracellular transport across the BBB by transiently separating tight junctions and enhances vesicle- mediated transcytosis, allowing antibodies and therapeutic agents to effectively cross. Moreover, ultrasound also facilitates the uptake of antibodies from the interstitial brain into brain cells such as neurons where the antibody distributes throughout the cell body and even into neuritic processes. In our studies, fluorescently labeled antibodies are prepared, mixed with in-house prepared lipid-based microbubbles and injected into mice immediately before SUS is applied to the brain. The increased antibody concentration in the brain is then quantified. To account for alterations in normal brain homeostasis, microglial phagocytosis can be used as a cellular marker. The generated data suggest that ultrasound delivery of antibodies is an attractive approach to treat neurodegenerative diseases.

Presented here is a protocol to transiently open the blood-brain barrier (BBB) either focally or throughout a mouse brain to deliver fluorescently-labeled antibodies and activate microglia. Also presented is a method to detect the delivery of antibodies and microglia activation by histology.

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...