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).

伦理学声明:涉及动物主题程序已经批准的机构动物护理和使用委员会（IACUC）在弗吉尼亚理工大学。 注意:极端的预防措施必须处理和处理在本协议中使用的几个组件时服用。使用前，地方机构的指导方针，健康和安全的做法，必须建立并遵守，特别是对四氧化锇，这是易失性和剧毒，醋酸铀，这既是一种重金属和放射性源，和硝酸铅，这是重金属毒物。二氨基硫脲（TCH）能分解产生易爆，有毒气体，如果处理不当。许多机构将在这些试剂通常利用并能提供援助EM核心设施。 1.脑组织及SBFSEM成像的制备<ol><li>麻醉年轻（〜2 - 4月龄）C57黑色的鼠标（C57BL / 6J株）使用4 - 5％isofluraNE以下的机构准则。通过监控肌张力，缺乏随意运动和厌恶刺激像一条尾巴捏响应损失确认麻醉。 </li><li>引脚上的解剖盘鼠标，并沿腹中线皮肤切口。使在皮肤进一步切口以暴露小鼠的左肋。切开隔膜，然后小心地解剖出来的胸腔沿周边露出跳动的心脏，然后使在右心房中的切口。 </li><li>使用蝴蝶插管，导管插入左心室。灌注transcardially用10鼠标 - 20毫升磷酸盐缓冲盐水（PBS）pH值7.2，直到放血通过在肝脏的颜色变化确认。 注意:在肝脏的颜色的变化被用作指导确定放血的程度。确认从红棕色肝脏颜色变为淡粉色。 </li><li>灌注transcardially使用10鼠标 - 20毫升2％戊二醛的醛和4％多聚甲醛在0.10M的二甲砷酸缓冲液（pH 7.2）制成，从内部迅速修复脑组织。固定是通过观察尾加强监视。 注意:在80ml水中溶解2.14克二甲胂酸钠的制备0.10M的二甲胂缓冲液（pH 7.2），添加盐酸以调节pH，并且使体积至100毫升水。 </li><li>以下灌注，斩首鼠标，在4℃解剖大脑，随后固定在含有0.10M的二甲胂酸钠缓冲液（pH 7.2）2％戊二醛和4％多聚甲醛48小时</li><li> 48小时后，使使用振动400μm的冠状切片，然后小心地解剖感兴趣的区域中的脑（例如，海马）的解剖显微镜42下。精心修剪的组织，并采取保护方向的画面。 注意:在SBFSEM组织染色的后处理方法结合了各种重金属染色方法为了提高分辨率，并且基于由Deerinck 等人 40以前开发的方法。 </li><li>洗戊二醛固定组织3次，每次在0.1M二甲胂酸盐缓冲液（pH 7.2）5分钟。 </li><li>通过在0.1M二甲胂酸钠缓冲液（pH 7.2）单宁酸中溶解制备0.1％的单宁酸溶液。漩涡直至溶解，如有必要，通过0.45微米的滤网过滤。 </li><li>后缀用二甲胂酸盐组织缓冲的0.1％单宁酸通过在1ml试剂30孵育 - 在RT 60分钟。 注意:温育时间取决于组织的大小，但30分钟最适合大多数组织中。 </li><li>洗组织3次，每次在二甲砷酸缓冲液5分钟（pH值7.2）。 </li><li>溶解0.3克亚铁氰化钾和0.86克次硫酸钠二甲胂在蒸馏水中H 2 O 10毫升（卫生署2 O）。保持冰的亚铁氰化钾溶液。使用前，加入10 mL 4％四氧化锇（OSO 4）。 </li><li>染色，用2％的os组织90分钟溢价 - 亚铁氰化物溶液，在冰上。洗3次，每次5分钟用蒸馏水2 O </li><li>通过在10毫升的dh 2 O溶解0.1克TCH制备1％氨基硫脲（TCH）溶液由漩每隔10分钟，直到完全溶解溶解在60℃。遵守安全注意事项，同时处理TCH，特别注意避免使用金属，加热至高温，或允许解决干燥。 </li><li>对待新鲜配制的1％TCH样品在室温下20分钟。洗3次，每次5分钟用蒸馏水2 O </li><li>稀释4％OSO 4比2％与卫生署2 O，染色组织由1小时孵育2％水溶液四氧化锇。洗组织3次，每次5分钟用卫生署2 O. </li><li>孵育样品O / N在1％醋酸铀用蒸馏水2 O 4℃。 </li><li>准备沃尔顿的领先解决方案，天门冬氨酸（如Deerinck 等 40条所描述）。 <ol><li>溶解0.998克L-天门冬氨酸250毫升卫生署2 O然后以逐滴的方式添加10 N氢氧化钾（KOH），直到pH达到5.5。 pH调节后，在10ml天冬氨酸库存和热加0.066克硝酸铅至60℃30分钟。 </li></ol></li><li>冲洗组织用蒸馏水2 O和沃尔顿的天门冬氨酸铅染色在60℃烘箱30分钟孵化。洗3次，每次5分钟用蒸馏水2 O </li><li>通过一个分级系列使用的20％，50％，75％，85％和95％的乙醇冷却溶液，每次5分钟，醇的脱水的样品，然后100％乙醇3次，每次10分钟。 注意:从刚刚打开的瓶用100％的乙醇，作为开乙醇吸收空气中的水，并导致嵌入失败。较长的孵育将需要较大的组织样本。 </li><li>洗样品2次，每次在氧化丙烯15分钟。 </li><li>使使用25毫升树脂，10.5毫升DDSA15.5毫升NMA（纳迪克甲基酸酐）和1ml塑料包埋树脂（十二碳烯基琥珀酸酐），DMP-30（2,4,6-三二甲基氨基甲基苯酚）。通过摇动混合树脂。旋转并允许树脂直立，直到泡沫的决心。 注:这是标准的中等硬度的配方。其他类型的电子显微镜（EM）的塑料树脂均可使用，但应不是所有树脂SBFSEM工作预先用非必要的对照样品进行测试。 </li><li>孵育组织O / N在50:嵌入树脂和环氧丙烷的50混合，在使氧化丙烯蒸发在大约8的期间即最初上限，则未加帽后2小时的小瓶 - 10小时。 </li><li>转移组织至100％的新鲜嵌入树脂在干净小瓶中2小时。 </li><li>在新鲜嵌入树脂中嵌入的样品，在平面模具含有印刷纸的标签，并在60℃下固化它们的烘箱中48小时。经过约1小时后，再次检查的组织安排和调整，如果需要进行调整。 </li><li>修剪样品的感兴趣的区域，并安装在使用gellin铝销克氰基丙烯酸酯强力胶或导电环氧树脂，然后涂上胶态银膏周围块的两侧，以提供到铝销的导电路径。 </li><li>检查使用配备在室超微级和低千伏背散射电子检测器32的扫描电子显微镜系统的组织标本。 注:获取在扫描电子显微镜（SEM）使用仪器 - 和现场培训和成为授权用户。另外，与小项目的研究人员或核心设施的合作是可能的。作为中小企业产生X射线辐射的训练也可能是必需的。 </li><li>图像样本，请使用以下设置:2.25千伏，在5 - 10纳米/像素的分辨率，拥有80间场大小 - 在×250微米，Y（其他字段大小可能），50层厚 - 100纳米，总共有250 - 在一个16 600片 - 20小时的时间段。 注意:设置不同的显微镜之间有很大差异，INDI维杜阿尔样品，和所需的分辨率。这些设置应该产生对于许多样品容易解释的图像。 </li></ol> 2.分析成像数据集注:图片焦耳/斐济软件用于分析数据集和依赖于TrakEM2插件。可以使用各种的软件来执行的预处理步骤，可以是广泛的或次要根据经验水平并获得栈。使用开源软件的主要转换（ImageJ的版本1.50b，斐济下载2015年10月1日）的说明。 <ol><li>通过在软件中打开和选择菜单项的图像→类型→8位从原来的专有的16位图像将图像转换为8位TIFF格式。 <ol><li>如果在这一步自动对比度/亮度转换是不理想的图像，重新打开16位图像，然后按图像→调整→亮度/对比度。选择所有图像工作的范围，并点击应用。然后执行Çonversion。注意:在一些中小型企业，这一步可能需要显微镜制造商的软件。 </li><li>如果需要的话，由于切片之间不可接受的图像运动（ 如漂流，由于充电），注册/对齐图像堆栈（菜单项→插件→登记线性StackAlignmentWithSIFT）。在大多数的注册软件，"翻译 - 只有"模式而不是"刚性体"进行设置。 注意:许多方法和软件可能工作:SIFT注册插件作品对于许多应用，有一个虚拟堆栈版本。 <ol><li>如果需要的话，放大画布大小登记前（图像→调整→CanvasSize）或将其降低到感兴趣的区域（图像→作物）。 注:可能会出现一些漂移或张开，并尝试其他插件或软件可能会产生更好的效果。手动选择也可以（ 如 ImageJ的/斐济，插件→注册→ManualLandmarkSelection）。 </li></ol></li><li>如果渴望DesirED，使用ImageJ（图像→缩放）规模图像更小，更易于管理的大小（ 如 25％）。 </li></ol></li><li>启动软件，选择文件→导入→图像序列，然后选择TIFF文件。 </li><li>通过选择文件→新建→TrakEM2（空白）启动插件。两个窗口将打开;另一个负责管理项目和area_lists和其他管理跟踪，并作为"画布"被提及。 </li><li>通过在画布进口右键点击加载图像堆栈到插件。选择导入并单击导入堆栈。在弹出的选项，请检查堆栈的虚拟框。 注意:虽然在图像栈中的软件已经打开，它们也必须在插件打开。如果虚拟堆栈复选框被选中的计算机可能运行更平稳。 </li><li>创建后的贴图和栈被加载后，右键单击命名插件窗口下的项目。在模板山坳右键单击"新项目"UMN，并选择"添加新的孩子该项目。右击再次选择&#39;area_list"。 </li><li>设置项目与在画布上右击原电子显微镜照片设置同意的Z轴的比例，然后单击显示并选择校准。此外，通过在插件窗口中选择所有图层设置Z-规模，右击选择规模Z和厚度。 </li><li>单击并拖动"项目"和所有的孩子"到项目对象部分在画布窗口中的"Z空间"选项卡下创建"区域列表"。 </li><li>选择"区域列表"，然后右键单击，选择并设置一个颜色。现在，使用画笔工具跟踪对象，如线粒体，在电子显微镜图像。使用"Shift +点击"填写一个封闭的圈子，"按Ctrl +滚动"来放大和缩小，以及"Alt +单击"打开画笔光标变为一个橡皮擦。 </li><li>选择10〜两个领域 - 15微米10 - 15微米的在图像的左上角和右下角，并确定这些区域内的所有线粒体，以允许每个数据集线粒体的偏采样。 </li><li>在"画布"，观察和跟踪线粒体整个章节。注:线粒体是很暗/致密的细胞器出现，类似规模的直径和松散的圆柱形。由内膜形成了独特的嵴此细胞器内很容易分辨。 </li><li>当跟踪完成后，右键单击在Z空间选项卡下的area_list，选择"显示在3D"。这将启动3D查看器插件来查看跟踪图像的三维重建。 <ol><li>为了进行线粒体体积测量，在3D查看器中选择对象，单击"编辑"标签，选择"对象属性"。线粒体体积估计与其他几个测量列在一起。 </li></ol></li></ol>

The authors declare that they have no competing financial interests. Emily K Benson is a paid employee of Renovo Neural Inc., and Grahame J Kidd serves as Scientific Director for 3DEM at Renovo Neural Inc., which is a commercial provider of serial block-face SEM services and analysis.

在神经系统的复杂性造成在重建大组织体积和分析的形态和细胞器的分布的显著挑战例如用足够的分辨率线粒体。多细胞，包括神经元，少突胶质细胞和扩展在三个维度众多流程的星形胶质细胞的脑组织43的内部交互。由于线粒体无论是在细胞和遥远的过程的SOMA所在，线粒体形态是神经系统（ 图1）非常多形性。具有足够分辨率足够三维结构信息，因此无法通过常规的光学显?...

线粒体是改变它们的形状和根据蜂窝线索和需要的位置，在与细胞骨架紧密相互作用动态的细胞器，并响应于细胞事件例如在神经元1钙电流。线粒体也相互作用与其他细胞器，例如内质网，从而调节其动力学和代谢2。线粒体形态示出了在不同的细胞类型异质性即细胞器的形状从管状变化到该组成的片材，袋和椭圆3。它已经表明，线粒体融合和分裂周期蛋白可调节的位置，大小，形状和线粒体4的分布。此外，在线粒体的形状变化与神经变性，神经元可塑性，肌肉萎缩，钙信号传导，活性氧产生以及寿命和细胞死亡牵连塔相关联T细胞特异性线粒体形态是维持正常细胞功能5-11的关键。 线粒体的主要生物能量的功能是通过执行一系列涉及通过 TCA循环的营养物质完全崩溃（ 即葡萄糖，脂肪，酸或氨基酸）和氧化磷酸化途径12代谢反应，产生三磷酸腺苷（ATP）。人脑只占2％体重但是它消耗〜生产使它极其能量苛刻器官13的总能量的20％。因此，并不奇怪，在人类线粒体功能障碍导致的大量神经系统表现14-17中。在OXPHOS部件遗传变异的损害ATP生成导致线粒体紊乱17,18，它们是疾病的临床异质组〜1的患病率:5000的个人，和m的最常见的原因之一etabolic障碍的儿童和成人。线粒体来源的ATP的赤字影响多器官系统具有高能量要求的器官，如脑，心脏和骨骼肌被在这些患者14,17,18主要影响。近年来，多研究已经在这两个神经发育和神经变性疾病15-17,19,20线粒体功能障碍提供了证据。因为线粒体是必要的，对大脑发育和功能是至关重要的，必须开发出能分析健康和患病状态下的脑线粒体形态，结构，尺寸，数量和分布的变化的协议。小鼠模型与线粒体针对性的绿色荧光蛋白（GFP）已经生产可视化线粒体运动和定位大脑中的21,22。虽然这是检查线粒体运动和一般分布一个非常有用的工具，也有一些缺点，其中大型的源码Ë有限的分辨率和荧光显微镜的灵敏度。这些属性使得它难以跟踪的相对小的尺寸的线粒体。同样地，串行传输电子显微镜已成功地用于查看突触线粒体23，但这种方法是非常耗时。线粒体形态是已知的，因为它们经受连续裂变和聚变周期是高度动态的，并且在大多数细胞线粒体维持一个高度连接的网络24-26。神经元是高度极化与多个树突和延长轴突，并形成在所述电池主体连接的网状网络线粒体细胞可能具有分开，因为它们使自己通过这些突起（ 图1）的方式。这使得脑线粒体的大小和形状极其多样。例如，使用串行块面扫描电子显微镜（SBFSEM）技术，我们先前观察到，在突触外mitochondr的体积或尺寸的差十六个褶皱27 IA存在于神经末梢线粒体可以是一样多。 有用于执行体积几种方法分析28，它包括连续切片的TEM 29，自动化磁带收集超微SEM 30，聚焦离子束扫描电镜31以及SBFSEM 32。该SBFSEM分析具有在于其具有的分辨率，以提供对形态学形状，尺寸，分布和细胞器的数量的定量数据的优点，如在区域线粒体到大脑的1毫米。该技术操作也最苛刻的，与以往缺乏经验EM许多生物实验室的能力范围之内的数据采集和分析。商业仪器用于产生串行像部分的图像的出现使得组织的三维超微结构分析常规技术，其中，还允许以快速和可重复的方式28的无偏体积分析</SUP&gt;。所述SBFSEM在2004年首次32描述和使用在神经生物学的领域中，基于由顿于1981年33。多项研究引入从那时起已经建立这种技术，因为在神经回路34的重建分析的主要工具的想法。此外，对于许多较小规模的项目，它提供的重建分析，以确定细胞器27,35-39。一直以来，获得的图像由低电压背散射电子衍生，结合不同的已知重金属染色技术的新染色方法被开发来提高分辨率40。 在本文中，我们提供了利用三维电子显微镜成像和基于已使用过被我们和其他人38,39,41方法脑线粒体的体积分析的协议。所使用的组织的后处理方法进行了先前由Deerinck 等人描述40。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson laboratory&#160;</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VETone, tradename Fluriso</td>
			<td>501017</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection tray</td>
			<td>Fisher scientific&#160;</td>
			<td>S65105&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Ted Pella Inc.</td>
			<td>1316</td>
			<td></td>
		</tr>
		<tr>
			<td>Butterfly canula</td>
			<td>Exel International</td>
			<td>26704</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter (0.45 micron)</td>
			<td>EMD Millipore</td>
			<td>NC0813356</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome sectioning system</td>
			<td>Ted Pella Inc.</td>
			<td>Vibratome 3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacodylate</td>
			<td>EMS</td>
			<td>12300</td>
			<td></td>
		</tr>
		<tr>
			<td>Tannic Acid</td>
			<td>EMS</td>
			<td>21700</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Ferrocyanide</td>
			<td>J.T. Baker</td>
			<td>14459-95-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide 4% Solution</td>
			<td>EMS</td>
			<td>19150</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiocarbohydrazide</td>
			<td>EMS</td>
			<td>21900</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A93100</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Acros Organics</td>
			<td>43731000</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead Nitrate</td>
			<td>EMS</td>
			<td>17900</td>
			<td></td>
		</tr>
		<tr>
			<td>EMbed-812 EMBEDDING KIT</td>
			<td>EMS</td>
			<td>14120</td>
			<td>Contains Embed 812&#160; resin, DDSA, NMA, and DMP-30.</td>
		</tr>
		<tr>
			<td>Glutaraldehyde 25% EM Grade</td>
			<td>Polysciences Inc.</td>
			<td>1909</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>EMS</td>
			<td>19202</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>EMS</td>
			<td>22400</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>EMS</td>
			<td>15055</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene Oxide</td>
			<td>EMS</td>
			<td>20400</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding Mold</td>
			<td>EMS</td>
			<td>70907</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum specimen pin</td>
			<td>EMS</td>
			<td>70446</td>
			<td></td>
		</tr>
		<tr>
			<td>Colloidal Silver Liquid</td>
			<td>EMS</td>
			<td>12630</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor</td>
			<td>EMS</td>
			<td>72000</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Glue (Loctite Gel Control)</td>
			<td>Loctite</td>
			<td>234790</td>
			<td>Hardware/craft stores carry this item</td>
		</tr>
		<tr>
			<td>Conductive epoxy</td>
			<td>Ted Pella Inc.</td>
			<td>16043</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Zeiss</td>
			<td>Sigma VP</td>
			<td></td>
		</tr>
		<tr>
			<td>In chamber ultramicrotome for SEM</td>
			<td>Gatan Inc.</td>
			<td>3View2</td>
			<td>Can be designed for other SEMs</td>
		</tr>
		<tr>
			<td>Trimming microscope for pin preparation</td>
			<td>Gatan Inc.</td>
			<td colspan="2">supplied as part of 3View system</td>
		</tr>
		<tr>
			<td>Low kV backscattered electron detector</td>
			<td>Gatan Inc.</td>
			<td>3V-BSED</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
	</tbody>
</table>

analysis of brain mitochondria using serial block-face scanning electron microscopy

人的大脑是一个高耗能的器官，主要依靠葡萄糖作为燃料来源。葡萄糖通过脑线粒体经由糖酵解，三羧酸（TCA）循环和氧化磷酸化（OXPHOS）途径分解代谢以产生三磷酸腺苷（ATP）形式的细胞能量。线粒体ATP生产的减值导致线粒体病，这与著名的神经和肌病临床症状出现。线粒体缺陷也存在于神经发育障碍（ 例如自闭症谱系障碍）和神经变性疾病（ 如肌萎缩性侧索硬化症，阿尔茨海默氏症和帕金森氏病）。因此，存在在该领域的兴趣增加为下都健康和疾病状态进行线粒体形态，结构和分布的三维分析。大脑线粒体形态极其多样，一些线粒体尤其是突触区域在&lt;200纳米的直径，这低于传统的光学显微镜的分辨率极限的范围内。表达在大脑中线粒体靶向的绿色荧光蛋白（GFP）显著增强了共焦显微镜的细胞器检测。然而，它不克服上检测相对小尺寸的线粒体的灵敏度的限制，而不过饱和的大尺寸的线粒体中的图像。而串行传输电子显微镜已成功地用于在神经元突触表征线粒体，这种技术是非常费时比较多个样品时，更是如此。串行块面扫描电子显微镜（SBFSEM）技术涉及切片，组织和数据采集的成像块的自动化过程。在这里，我们提供从啮齿动物的大脑进行限定区域的SBFSEM迅速重建和可视化线粒体形态的协议。这种高科技NIQUE还可以用于提供一个限定大脑区域对线粒体数目，体积，尺寸和分布的准确信息。由于所获得的图像分辨率高（一般低于10纳米）的任何毛线粒体形态的缺陷也可以被检测。

我们表明，脑线粒体形态和大小是不同的神经元的子隔间异类。与慢病毒表达线粒体针对性的绿色荧光蛋白转低密度神经细胞共聚焦显微镜显示，居住在神经元胞体线粒体形成网状网，而那些居住在轴突远端表现出离散细长的形态（ 图1 AB）。使用SBFSEM技术，线粒体形态，大小，数量和分布在鼠脑进行分析。线粒体的三维（3D）图像是从在小鼠大脑海马既轴突?...

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

使用串行块面部扫描电子显微镜脑线粒体分析

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, ...

analysis-brain-mitochondria-using-serial-block-face-scanning-electron

Research

JoVE Journal

Neuroscience

14.0K 次观看.  Virginia Tech Carilion Research Institute. 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. 

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

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.

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.

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 ...

科研

神经科学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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 ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

在脑核糖凝视与原子力显微镜

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

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 ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

常见马莫塞的全皮质电皮质阵列的慢性植入

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

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.

使用聚焦扫描超声将抗体输送到大脑中

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

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 ...

使用串行块面部扫描电子显微镜脑线粒体分析

摘要

探索更多视频

此视频中的章节

聚焦离子束铣削和脑组织的扫描电子显微镜

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

小脑颗粒神经元的遗传操作

嗅觉系统作为模型来研究轴突生长的模式和形态

在脑核糖凝视与原子力显微镜

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

常见马莫塞的全皮质电皮质阵列的慢性植入

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

使用聚焦扫描超声将抗体输送到大脑中

用于登德里脊柱研究的串行块面扫描电子显微镜（SBEM）

使用串行块面部扫描电子显微镜脑线粒体分析

摘要

探索更多视频

此视频中的章节

聚焦离子束铣削和脑组织的扫描电子显微镜

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

小脑颗粒神经元的遗传操作

嗅觉系统作为模型来研究轴突生长的模式和形态

在脑核糖凝视与原子力显微镜

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

常见马莫塞的全皮质电皮质阵列的慢性植入

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

使用聚焦扫描超声将抗体输送到大脑中

用于登德里脊柱研究的串行块面扫描电子显微镜 （SBEM）

用于登德里脊柱研究的串行块面扫描电子显微镜（SBEM）