1。样品的固定和树脂包埋<ol><li>新鲜的组织和细胞的样品至少2个小时是固定的，在2％多聚甲醛和2.5％，0.1 M磷酸缓冲液pH值在7.4戊二醛。样本大小应不超过0.150毫米的厚度，以便有足够的渗透或固定液，不应该超过12小时修复左。 </li><li>放置在20毫升玻璃闪烁瓶样品和冲洗三次，每次5分钟，在0.1M cacodylate缓冲区。 </li><li> 1.5％（W / V）在0.1 M的cacodylate缓冲区为30分钟（0.1M，pH值7.4）亚铁氰化钾和1％（W / V）的四氧化锇染色。 </li><li>在0.1M cacodylate缓冲区的1.0％（W / V）30分钟的四氧化锇染色。 </li><li> 3分钟，用双蒸水冲洗一次。 </li><li> 1％（W / V）30分钟，双蒸水醋酸铀染色。 </li><li> 5分钟双蒸水冲洗的部分，然后脱水梯度酒精系列，每个变化（1 × 50％× 70％，1 × 90％× 95，1％，2 × 100％）2分钟。 </li><li>嵌入在用乙醇Durcupan树脂混合30分钟，每一个变化，50％的乙醇Durcupan开始浓度的增加，然后依次为：70％，90％，95％和100％。 </li><li>更换新鲜Durcupan和搅拌4小时慢慢。 </li><li>放置节，使用鸡尾酒​​枝木，与模具分离剂涂在玻璃显微镜幻灯片，并在65个地点° C的烘箱24小时。 </li></ol> 2。 FIB / SEM样品准备<ol start="11"><li>单独的树脂层，包含的样本，从两者之间的玻璃显微镜幻灯片，并彻底清洗，以消除任何模具分离剂。 </li><li>使用一个透射光显微镜，具有低功耗的目标或传输照明显微镜解剖，找出感兴趣的区域内的树脂片。 </li><li>切一小（3毫米x 3毫米）周围地区使用刀片的利益方，并坚持这一空白树脂与丙烯酸酯胶块（图1）。胶变硬，等待5分钟，然后进行下一步。 </li><li>块和钳入ulramicrotome持有人，使用附加的体视显微镜，修剪周围地区用刀片的兴趣，直到材料仍然只是一个小金字塔。 </li><li>进一步削减块，使用玻璃刀固定到ultramicrotome，使感兴趣的区域，可以精确地尊重位于块的表面（图1）的尺寸。剪切块的一个边缘，邻近地区的利益，使垂直第一步是通过削减样品（图1和2）。这一步形成的成像面（图2）。 </li><li>删除从ultramicrotome和照片的块内的利息区域块，使用透射光显微镜。 </li><li>切修剪街区之遥，从剩余的树脂存根。这是通过使用一个珠宝商的锯和保证，只有一小块放在里面的FIB / SEM。这个区块的总高度不应超过5毫米。 </li><li>胶块，​​碳糊铝存根，确保成像面的最外边缘（图1）。 </li><li>在已经干涸的胶水，外套用薄金层（&gt; 20纳米; Cressington真空蒸发系统）的块。 </li></ol> 3。 FIB / SEM成像<ol start="20"><li>在低倍率，使用二次电子成像（5千伏，0.5 NAMP），定位块，使选择的地区的利益和侧块进行成像和正面临着经营者（图2 C，D）。 </li><li>东方在于使面部成像块铣削束平行。这将意味着在54 °面对这种情况（图2 B），电子束导向。 </li><li>使用离子束电流13 - 30千伏27 NAMPS从前面的成像区域中删除的树脂的窄带。 </li><li>切换到反向散射成像模式查看碾磨面覆盖在感兴趣的区域。 </li><li>使用光学显微镜的参考图像（第16步）和碾磨面对的形象被碾碎和成像（图2），定位块的确切地区。 </li><li>存款的碳（或金属）的保护层（约1微米厚），感兴趣的区域（图2），使用显微镜的气体喷射系统，到块的表面，上面。 </li><li>使用目前的700 picoAmps，精细磨块区域内的最终图像将采取。 </li><li>离开铣削束完全磨，直到没有铣削工件可以在脸上看到的形象面对。面对的一个完整的轧机检查通过观察每一个横跨整个视野的后续形象变化。不足铣也可以看作是白色条纹或出现Vertica的“窗帘”LLY中的形象。 </li><li>保留任何消散的温度变化至少2小时的显微镜。这降低了风险，块面漂移在成像过程中，造成对齐图像。 </li><li>面对连续成像显微镜参数选择。确保电子束的电压足够低，唯一的形象在块面的材料的深度很浅。这也应该比要删除的面厚度较浅。成像的典型参数电压介于1.2 - 2.0千伏之间的像素大小的4 - 20纳米。像素停留时间的需求将保持约10个这样的总时间磨脸，并获得一个图像保持低于2分钟（图3）μsecs。 </li></ol> 4。成功的秘诀 步骤1）确保样本不超过150微米的厚度。这将使足够渗透到不同的污渍和树脂材料 。 这样就可以实现由一个vibratome切片后立即固定样本。这个厚度是适合于脑组织 。 其他组织需要进行测试，以确保有足够的固定和染色。 步骤3），样品必须自由地分布在整个解决方案，而不是一起成群在一个小瓶的一部分，而这一次要固定液介绍。 这将确保最大穿透样品，并减少样品，成为这个固定过程中扭曲。 这是通过轻轻摇动小瓶样品后，立即解决的办法是添加。 第15步）确保感兴趣的区域，位于下方（&lt;30微米）的块面。这将减少离子束铣削量，在图像采集阶段 。 第22步）的离子束扫描时间需要adequte，以确保整个成像面对的是完全碾磨。不足铣床将显示为白色条纹，或垂直向下成像面出现“窗帘”。 步骤26）最后碾磨面对必须大于成像面。这是因为在附近的块面块和redeposits弹出铣削树脂 。 这再沉积可以侵犯到成像领域的观点，并避免铣得多大面积比成像。对于成像的窗口，是宽20微米，精块面是大于30微米，宽 。 步骤29），这是关键，从出现的第一个块的表面几纳米的电子所产生的图像是由电子束参数等。 这acheved最小的电压低于2千伏和保证，没有电子穿透太远。这也是帮助探测器上使用的电网电压，因此，只有具有最高能量的电子有助于最终图像。 通常情况下，1.3千伏电网的张力是用于一个1.5千伏的成像电压。 5。代表性的成果： <img src="/files/ftp_upload/2588/2588fig1.jpg" alt="图1"/> 图1）树脂制备树脂块。嵌入steromicroscope查看通过一个成年大鼠脑冠状切面（80微米厚）。图像清楚地显示不同的大脑部分使用手术刀刀片（二）可以从削减地区。在这里，从1平方毫米的皮层被删除，（三）坚持一个空白树脂块D）树脂块，然后与装在一个ultramicrotome玻璃刀修剪，一旦区块已减少到只有离开感兴趣的区域（E），一步步被切断嵌入式材料的前表面垂直。F）修剪这整个地区，然后从其余的树脂削减，并安装到一的铝金属涂层的存根，在成像扫描电子显微镜。 <img src="/files/ftp_upload/2588/2588fig2.jpg" alt="图2"/> 图2。准备FIB / SEM成像的块面。a）和b）显示块和边缘研磨创建的成像面（与黑色的双箭头表示）中的插图。离子束（白）的位置是平行的成像面，并显示54 °打脸的离子束，电子束（灰色）。C）与电子束块二次电子成像。沿着这一侧块较暗的区域（虚线白盒表示）显示部分已大致碾磨面对（双头显示ED黑色箭头），以揭示底层样品。D）当本地区只用背散射电子成像包埋组织中可以看出。在这里，全宽包埋组织部分，是一个双头白色箭头指示。规模在 C栏）为100微米。 <img src="/files/ftp_upload/2588/2588fig3.jpg" alt="图3"/> 图3。成像与各向同性体素脑组织量A）反背散射图像块的对比显示大鼠脑区域内面对的超微结构。所有的膜是可见的，以及大型的大分子结构。B）共1600图像收集在5 nm的图像堆栈 C）本协议栈可在任何平面成像同一决议的各向同性体素的间距。此图像显示了在xy平面和yz平面成像的单突触连接。

<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>

FIB / SEM技术工程优化感兴趣的区域时不会太大。影像量上限取决于离子束和地区，它可以始终磨多小时反复。然而，到目前为止，已与约60 × 60微米的地区进行，成像这整个地区是不可能与一个合适的图像分辨率，主要是由于在收集这样一个大的图像的时间因素。作为一个例子，一个4 KX 4 K像素图像停留时间10微秒将采取2分40秒获得，并为60微米的领域，这将只给出了15纳米的像素大小。对于成像的细胞和组织的超微结构，一个更小的像素尺寸更适合;通常在4和10 nm /像素之间。对于一个面积为60 × 60微米的图像将在10小时内被收购，即使在显微镜有这个能力。基于这些原因，在显微镜图像卷，在10 × 10 × 10微米，或整个细胞显着的地区秩序的理想工具。这可以很容易地在48小时内完成，在此过程对比样品。 到目前为止，该协议主要用于脑组织，以及坚持以盖玻片培养的哺乳动物细胞的各类。然而，固定和染色过程，可用于许多其他类型的生物材料，包括植物材料，生产同样的对比的图像堆栈。 因此，这项技术的主要优势是它的各向同性体素的能力形象卷。这种类型的图像堆栈允许使用任何飞机在堆栈拍摄的图像的3D量的分析。这样做的好处是让来自任何方向的图像系列，并提供一个更大的图像细节（图3）的图像特征的观察员。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>氰基丙烯酸酯胶</td><td></td><td></td></tr><tr><td>解剖显微镜：莱卡MZ8 </td><td>徕卡显微系统，德国</td><td></td></tr><tr><td> Durcupan树脂</td><td> Sigma - Aldrich公司Chemie GmbH德国，瑞士</td><td></td></tr><tr><td> FIB / SEM显微镜（蔡司Nvision公司40） </td><td></td><td></td></tr><tr><td>玻璃组织学幻灯片</td><td>德国门泽尔格拉泽， </td><td> AA00008032E </td></tr><tr><td>玻璃刀制造商：徕卡EM KMR2 </td><td>徕卡显微系统，德国</td><td></td></tr><tr><td>玻璃闪烁瓶（20毫升） </td><td> （EMS），美国</td><td> 72634 </td></tr><tr><td>戊二醛</td><td> （EMS），美国</td><td> 16222 </td></tr><tr><td>珠宝匠的锯</td><td> （EMS），美国</td><td> 72010 </td></tr><tr><td>模具分离剂</td><td>瑞士Glorex， </td><td> 62407445 </td></tr><tr><td>四氧化锇</td><td> （EMS），美国</td><td> 19110 </td></tr><tr><td>多聚甲醛</td><td> （EMS），美国</td><td> RT 19208 </td></tr><tr><td>磷酸盐缓冲液中的磷酸盐</td><td> Sigma - Aldrich公司Chemie GmbH德国，瑞士</td><td> 71642和71496 </td></tr><tr><td>钠cacodylate </td><td> Sigma - Aldrich公司Chemie GmbH德国，瑞士</td><td> 20840 </td></tr><tr><td>与黄金的目标溅射涂布机</td><td>美国Cressington， </td><td></td></tr><tr><td> Tris碱公司（TBS） </td><td> Sigma - Aldrich公司Chemie GmbH德国，瑞士</td><td> T1378 </td></tr><tr><td> Ultramicrotome：莱卡UCT </td><td>徕卡显微系统，德国</td><td></td></tr><tr><td>醋酸铀</td><td> （EMS），美国</td><td> RT 22451 </td></tr></tbody></table>

focussed ion beam milling and scanning electron microscopy of brain tissue

这个协议描述的生物样本，如脑组织，可以使用聚焦离子束/扫描电子显微镜（FIB / SEM）在三个层面成像。该样本是固定的醛，重金属，使用四氧化锇和醋酸铀染色。然后，他们用酒精脱水和树脂，然后硬化渗透。利用光镜和玻璃刀ultramicrotome，小块状，含有接近地表的地区的利益。块，然后放在FIB / SEM和离子束大约一个垂直的面磨沿块的一侧，接近这一地区。采用背散射电子图像的基本结构，然后碾磨面较小的一个更好的离子束表面审议更密切合作，以确定脸的确切面积成像和碾磨。在显微镜的参数，然后使面部反复碾磨，使成像序列图像块的体积通过收集。图像堆栈通常会包含各向同性体素尺寸小的每个方向4海里。这在任何成像平面的图像质量，使用户能够分析在任何视角图像的堆栈内的细胞超微结构。

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

这个协议描述树脂嵌入脑组织如何准备和聚焦离子束在三个层面成像，扫描电子显微镜。

聚焦离子束铣削和脑组织的扫描电子显微镜

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

focussed-ion-beam-milling-scanning-electron-microscopy-brain

Research

JoVE Journal

Neuroscience

27.8K 次观看.  École Polytechnique Fédérale de Lausanne. 这个协议描述树脂嵌入脑组织如何准备和聚焦离子束在三个层面成像，扫描电子显微镜。

视频: 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.

制备小鼠脑组织中的免疫电镜

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

科研

神经科学

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.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

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.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

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.

脑组织中的双管和同心微电极用于测量细胞外离子的信号

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.

表征使用原子力显微镜，冲击压痕和流变测定脑组织的多尺度力学性能

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.

使用串行块面部扫描电子显微镜脑线粒体分析

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.

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

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.

成人脑组织切片中钾离子选择性电极的制备、检测及应用

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

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.

鼠脑水平海马片

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 ...

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In addition, some features of dendritic spines, such as volume of the spine and post-synaptic density (PSD) (representing post-synaptic part of the synapse), presence of presynaptic terminal, and smooth endoplasmic reticulum or atypical form of PSD (e.g., multi-innervated spines), can be observed only with 3D EM. By employing serial block-face scanning electron microscopy (SBEM) it is possible to obtain 3D EM data easier and in a more reproducible manner than when performing traditional serial sectioning. Here we show how to prepare mouse hippocampal samples for SBEM analysis and how this protocol can be combined with immunofluorescence study of dendritic spines. Mild fixation perfusion allows us to perform immunofluorescence studies with light microscopy on one half of the brain, while the other half was prepared for SBEM. This approach reduces the number of animals to be used for the study.

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

Serial Block-Face Scanning Electron Microscopy (SBEM) is applied to image and analyze dendritic spines in the murine hippocampus.

用于登德里脊柱研究的串行块面扫描电子显微镜 （SBEM）

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...