使用串行块面部扫描电子显微镜脑线粒体分析
Summary
Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.
Abstract
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<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。
Introduction
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>。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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| C57BL/6J mice | Jackson laboratory | 664 | |
| Isoflurane | VETone, tradename Fluriso | 501017 | |
| Dissection tray | Fisher scientific | S65105 | |
| Dissection scissors | Ted Pella Inc. | 1316 | |
| Butterfly canula | Exel International | 26704 | |
| Phosphate buffer saline | Sigma-Aldrich | P4417-100TAB | |
| Filter (0.45 micron) | EMD Millipore | NC0813356 | |
| Dissection microscope | Olympus | SZ61 | |
| Vibratome sectioning system | Ted Pella Inc. | Vibratome 3000 | |
| Sodium Cacodylate | EMS | 12300 | |
| Tannic Acid | EMS | 21700 | |
| Potassium Ferrocyanide | J.T. Baker | 14459-95-1 | |
| Osmium Tetroxide 4% Solution | EMS | 19150 | |
| Thiocarbohydrazide | EMS | 21900 | |
| L-Aspartic Acid | Sigma-Aldrich | A93100 | |
| Potassium Hydroxide | Acros Organics | 43731000 | |
| Lead Nitrate | EMS | 17900 | |
| EMbed-812 EMBEDDING KIT | EMS | 14120 | Contains Embed 812 resin, DDSA, NMA, and DMP-30. |
| Glutaraldehyde 25% EM Grade | Polysciences Inc. | 1909 | |
| Paraformaldehyde | EMS | 19202 | |
| Uranyl Acetate | EMS | 22400 | |
| Ethanol | EMS | 15055 | |
| Propylene Oxide | EMS | 20400 | |
| Embedding Mold | EMS | 70907 | |
| Aluminum specimen pin | EMS | 70446 | |
| Colloidal Silver Liquid | EMS | 12630 | |
| Razor | EMS | 72000 | |
| Super Glue (Loctite Gel Control) | Loctite | 234790 | Hardware/craft stores carry this item |
| Conductive epoxy | Ted Pella Inc. | 16043 | |
| Scanning electron microscope | Zeiss | Sigma VP | |
| In chamber ultramicrotome for SEM | Gatan Inc. | 3View2 | Can be designed for other SEMs |
| Trimming microscope for pin preparation | Gatan Inc. | supplied as part of 3View system | |
| Low kV backscattered electron detector | Gatan Inc. | 3V-BSED | |
| ImageJ/ Fiji processing package | ImageJ ver 1.50b, FIJI download Oct 1, 2015 | http://zoi.utia.cas.cz/files/imagej_api.pdf | |
| http://rsb.info.nih.gov/ij/ | |||
| http://www.icmr.ucsb.edu/programs/3DWorkshop/Uchic-2015_FIJI_Tutorial.pdf | |||
| http://fiji.sc/TrakEM2 | |||
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