这项工作是由马克斯·普朗克学会（KMD，BD，AWM，结核病，台湾企银，粉喷桩，和星期），法兰克福卓越“大分子复合物”的集群由德意志研究联合会（星期）和一个博士后EMBO长期资金支持奖学金（VAMG）。

从差速离心细胞或组织1。制备线粒体本节介绍了完整的线粒体来自不同真核生物的隔离一般程序。精确的缓冲部件和离心速度需要为每种组织进行优化/物种的影响。 <ol><li>破细胞在等渗缓冲液（ 例如 250mM的蔗糖，10mM的HEPES pH 7.4）中，用玻璃珠磨机（真菌菌丝体）28，细胞壁（ 酿酒酵母 ）29，滚珠轴承匀浆的酶消化（单胰岛β细胞真核生物/培养细胞/线虫）30或搅拌机（动物或植物组织）31。 </li><li>通过细布接着通过低速离心（2000 XG，4℃，10分钟），通过过滤除去细胞碎片。 </li><li>收集上清和沉淀线粒体由高速离心（9,000 XG，4℃，10分钟）。 </li><li>必要时，使用的等渗浓度梯度的步骤为线粒体部分32的进一步纯化。 </li></ol> 2，准备线粒体的冷冻电子断层扫描以下部分介绍如何获取冷冻水合样品低温ET。注：该方法包括使用极冷液态氮和乙烷，这可能会导致严重的皮肤灼伤。护目镜和低温保护必须戴手套。液态乙烷，这也是易燃的，必须在通风橱中进行处理。 <ol><li>重悬沉淀线粒体中的250mM海藻糖，10mM的HEPES缓冲液在pH 7.4至大约5毫克/毫升的总蛋白质的浓度。 </li><li>辉光放电多孔碳EM电网，碳面朝上，在根据制造商的说明真空装置。 </li><li>通过将乙烷气体流至液氮酷内侧液化乙烷几毫升编辑铝制容器。 </li><li>混合蛋白A结合的黄金基准停牌1：1与线粒体悬液，并立即申请3微升至镊子举行的辉光放电电磁网格。 </li><li>把镊子的玻璃化装置， 例如 ，一个自制断头台。吸去多余的液体用滤纸楔（〜5秒或直至液体停止扩频），并立即通过释放扳机沉浸网格成液态乙烷。 </li><li>传送从液态乙烷网格到液氮中。在转移过程中，从电网用滤纸除去过量的乙烷。放置楔形片滤纸的尖端到液体乙烷。由于液态乙烷上升，轻轻拖动网格了滤​​纸，但保持低于液体的前沿。液态乙烷从电网通过毛细作用移除并一次全部液体乙烷已被删除，网格立即转移至液氮中。 </li><li>将玻璃化电网成格收纳盒，第二液氮下保存以备后用适当的。 </li></ol> 3，录音断层倾角系列以下部分介绍了如何设置和收集断层倾角系列配备了后能量过滤器和CCD相机Polara电子显微镜线粒体。类似的协议适用于所有电子低温显微镜配备了CCD或直接电子探测器相机。 <ol><li>对准显微镜<ol><li>将试样如石墨和金群岛上的多孔碳薄膜。 </li><li>选择搜索模式，在显微镜的低剂​​量的系统。 </li><li>把样品eucentric高度。这是最小的xy移动的点倾斜的试样夹持器时。居中的关注点在0°倾斜，倾斜的阶段到20°并重新中心的点，通过改变Z轴高度。返回到0°，并重复直到横向偏移被最小化。 </li><li>选择曝光模式。选择所需的放大倍率收集的断层图像（ 例如 ，25,000X对探测器≈0.6 nm的试样像素大小）。 </li><li>选择一个小的冷凝器孔径（50-70毫米），并选择一个点的大小和束强度，以使光束正好大于成像装置更宽的，并给出了60 E A像素读取- /像素（CCD）或13 E - /像素/秒（直接电子探测器，计数模式）。 </li><li>中心冷凝器光圈。 </li><li>查找高斯焦点，例如，点的最小对比度。复位显微镜离焦读数和正确的枢转点，并根据制造商的说明旋转中心。 </li><li>拨打所需的散焦录制断层扫描。注：高离焦（8微米）增加对比度，而且降低了分辨率，而小散焦（2-4微米），在对比度为代价提高分辨率。 </li><li>在一个空孔，生成新的增益参考，并调整能量过滤器根据制造商的说明。 </li><li>调整搜索和曝光模式。在曝光模式，围绕一个兴趣点，并切换到搜索模式。 1500倍的倍率选择（对检测器样品的0.033微米/像素）和散焦为100μm（用于增加对比度）。把兴趣点回用图像移位线圈中心。 </li><li>在搜索模式中，调整光斑大小和光束的强度，以使光束正好大于成像装置更宽的，并给出了〜20个电子的像素读- /像素（CCD）或〜7 E - /像素/秒（直接电子检测器，计数模式）。 </li></ol></li><li>找到一个好的标本区<ol><li>插入电网与冷冻水合线粒体成在液氮温度下的电子显微镜（参照EM制造商的说明）。 </li><li>在搜索模式中，搜索网格适当的冰层厚度和标本的质量方面。采取有前途的领域有6秒的图像搜索，以确定是否适合断层图像采集。博特小时内和外线粒体膜应该在这个放大率可见。 </li></ol></li><li>录制断层倾角系列<ol><li>一旦一个好的试样区域被发现，倾斜阶段±60°，以确定最大倾斜范围是可用的，没有任何阻碍的曝光或由尘棒或冰块集中区。 </li><li>在相似的外观附近的冰填充孔，改变曝光模式，调整光束强度或图像采集时间，让每一个记录的图像具有30-50Ë电子剂量- /像素的CCD芯片或6-8ë - /像素/ s的直接电子探测器，计数模式。 </li><li>计算出的剂量分布比（I 0 / I 60）除以平均电子数为0°与60°的图像的获取的1秒的图像。这个比例说明了增加曝光时间，以保持每幅图像的恒定电子计数随倾斜角度（曝光时间= 1 / C要求OS（α）n其中（I 0 / I 60）= 2 n）的 。该比例也可以作为冰厚的一个很好的迹象。线粒体良好断层通常是记录与I 0 / I 60 = 2.3-2.6。 </li><li>在一个空孔，收购曝光模式下1秒的图像，并注意每2电子计数。考虑到剂量分布比例，计算出可以记录特定电子总剂量图像的总数（ 例如 ，&lt;40ë - / 2的结构测定和〜160ë - / 2的形态）。 </li><li>通过将总的倾斜范围确定为断层图像采集相应的倾斜的时间间隔（ 例如 ，120°±60°），通过在3.3.4计算图像的总数目。 </li></ol></li><li>建立并记录上面使用适当的自动数据采集软件3确定的参数的断层3，34。倾斜系列通常开始在±20°和为了最大限度地低倾斜的图像，它是通过增大电子的剂量破坏了信息内容到达高倾斜之前经过0°。 </li></ol> 4，建立和层析卷分割本节介绍如何线粒体断层卷从倾斜系列和卷是如何存在于一般观赏生成。 <ol><li>保存断层倾角系列到适当的目录。生成图像的堆栈，并转换成与开放源代码软件，如dm2mrc或tif2mrc（IMOD包）一个适当的文件格式，转换.dm3，.dm4或tif档案，以MRC栈。 MRC栈所需的断层重建IMOD 35。其他包需要不同的格式。 </li><li>对齐图像，并按照在IMOD教程详细步骤生成的断层（ <ahref=&quot;http://bio3d.colorado.edu/imod/doc/etomoTutorial.html&quot;&gt;http://bio3d.colorado.edu/imod/doc/etomoTutorial.html ). </li><li>提高使用分布式与IMOD非线性的各向异性扩散滤波器的断层图像的对比度。此过滤器可以很好地用于膜和膜结合粒子如ATP合酶。 </li><li>对于可视化，手动分段的断层图像使用可商购的方案，例如，阿米拉。分配对应于内或外的膜的体素，并生成一个表面。使用的EM-包插件AMIRA 36唱首歌选项标记ATP合酶粒子的位置。 </li></ol> ATP合酶二聚体5 Subtomogram平均和配件X射线结构的以下部分介绍了如何对ATP合成酶二聚体subtomogram平均可以得到。 <ol><li>使用该标记的颗粒作为输入和适当的软件程序包，如&#39;部分ICLE估计电子断层扫描“程序，计算subtomogram平均水平。 </li><li>对于分辨率的估计，比较两个独立决定subtomogram平均傅立叶壳的相关性37。 </li><li>如果有的话，船坞已知X射线结构进subtomogram平均通过刚性体，无论是手动装配或使用自动连续对接例程，如那些在程序中嵌合体38。 </li></ol>

作者宣称，他们没有竞争的经济利益。

这里介绍的协议进行了介绍冷冻ET和线粒体subtomogram平均，但基本上相同的方法可应用于任何其他细胞区室或膜。为了获得最佳的数据，在手术过程中的关键步骤是样品制备，暴跌冻结过程和数据采集策略。样品的质量，这是至关重要的成功，取决于一个优化的冻结协议，以确保合适的冰的厚度，这对于良好的图像对比度非常重要的。最佳的数据采集策略取决于仪器和样品。要优化的参数包括每幅图像的电子剂量，倾斜机制和散焦。取得了良好的样本的良好断层扫描使所有进一步的处理步骤更容易，并确保一个满意的最终结果。 冷冻的ET结合subtomogram平均和原子模型拟合提供了如何蛋白质复合物被布置在第细节EIR天然细胞环境。该技术同样适合用于研究的大的膜蛋白复合物，诸如呼吸链supercomplexes（1.7 MDA），ATP合酶的二聚体（2×500 kDa的），或核孔复合物（〜120 MDA）8，9，17的结构。的ATP合酶的二聚体的组织进行不能由如X射线晶体学，核磁共振或单粒子冷冻电镜高分辨率技术可以观察到，因为该二聚体行由洗涤剂提取，这是在隔离的必要步骤打乱和的膜蛋白复合物的纯化。 在沿行嵴隆起二聚体的ATP合成酶的安排，是所有物种的线粒体的普遍组织原则。电子传递链的质子泵配合物，特别是复合物I（NADH脱氢酶），分别位于所述膜的区域中的行8的任一侧9，该组织在呼吸链中的具有线粒体生物能量产生深远的影响。如果该二聚体行不能由于缺乏二聚体特异性蛋白亚基组成，该细胞表现出更长的生成时间和减少细胞的健身，如在图3中41所示的酵母突变体观察到，随着在线粒体疾病的越来越大的兴趣，有详细治线粒体超微结构和功能的分子基础的认识是非常重要的。电子冷冻断层提供在所述膜的纳米尺度由高分辨率的方法测定蛋白质的结构和分布与这些蛋白质的结构之间的联系。这使得冷冻ET在健康和疾病的理解线粒体结构和功能的基本工具。 在低温ET技术上的进一步发展和改进包括蛋白质标记策略，以确定protei的位置Ñ​​亚基的大分子复合物或更小或更独特的蛋白（&lt;0.5 MDA）在细胞中的位置。此外，混合动力EM的加工方法，其中结合subtomogram平均单粒子分析或螺旋重建最近确定的蛋白结构，以〜8 A 42，43。这些处理方法目前仅限于纯化的蛋白质，这是很好的分离在冰面或形成螺旋集会，目前并不适用于拥挤的细胞环境，如线粒体嵴膜。数据收集，正在开发新的计算和硬件工具，使自动化断层图像采集，增加图像的对比度和降低的总要求电子剂量。在低温ET的唯一基本限制是辐射损坏样品的电子束。这就是说，只有非常低的电子剂量可用于记录的断层倾斜系列的每个图像，从而产生信号叔差Ø信噪比，最终限制了可实现的分辨率。新的直接电子探测器，发布不到一年前，正在彻底改变单粒子冷冻电镜44,45领域。这些新的检测器提供更高的对比度和更好的分辨率更低的电子剂量。对于低温电子断层扫描，这意味着更小的步长，甚至双轴断层的倾角系列无需过多有关辐射损伤（1 CCD图像是在电子剂量相当于5直接电子探测器图像）的关注被收集。海量通过这些检测器产生的数据中创建数据的处理，处理和存储他们自己的挑战，这将必须被克服。 此外，相板，类似的原则与在光学显微镜经常使用的工作，以提高相衬，目前正在透射电子显微镜46，4开发7，这应该允许断层图像被收集更接近焦点，并因此以较高的分辨率，而同时保持低的分辨率提供必要的对准和断层体积的解释。总之，这些技术进步将极大地扩大了可以通过冷冻ET来解决生物学问题的范围。

线粒体是电房小区。通过将电化学质子梯度跨线粒体膜成化学键的能量，线粒体ATP合酶产生最驱动的细胞过程中的ATP。为了理解后面线粒体能量转换机制，我们需要确定在原位 ATP合酶的结构，并发现它是如何布置和分布在线粒体内膜。虽然可用的大多数的整个复杂4线粒体ATP合成酶组分1-3和低分辨率图的高分辨率结构，它建立在工作酶的结构和构象中的膜是很重要的。的ATP合成酶的线粒体内膜的分布已被广泛地认为是随机的，但早期发现5和我们自己的初步结果6所示，这是不吨他的情况下。从我们的基团随后的研究和其他7已经证实，ATP合酶被布置在长行沿线粒体内膜嵴8的紧密弯曲的脊部的二聚体，而电子传递链的质子泵可能在位于两侧的行9。这种结构具有用于线粒体能量转换机制具有重要意义。 我们已经使用，以确定该布置的技术是电子冷冻断层摄影（冷冻ET）。冷冻ET是目前，在分子的分辨率提供了细胞，细胞区室或细胞器中的精确的三维（3D）的卷的唯一方法。冷冻ET特别适合用于研究的大复合体中的生物薄膜，因为该膜出现了具有良好对比度和很容易就可以跟踪在3D断层扫描体积。 其他的方法来研究细胞或细胞器的三维结构ES不提供分子细节。超分辨率光镜10，11是极好的露出位置或附接至具有几十nm的精度兴趣蛋白质的发光标签之间的距离，但它并没有揭示该蛋白本身的结构，即使在低的分辨率。连续切片12或块面的成像扫描塑料包埋的生物样品的电镜13透射电子显微镜提供蜂窝体积的低分辨率的意见，但同样没有揭示分子细节。原子力显微镜14在原理上可以实现分子甚至原子级的分辨率，但是仅在物体上的原子级平坦，固体支持物的表面上。最后，X射线断层摄影术15或强烈的X射线脉冲从自由电子激光散射16不太可能揭示大的，复杂的，非周期的对象的结构，例如在米全细胞或细胞器olecular分辨率在可预见的未来。因此，在目前，有没有替代冷冻ET的细胞或细胞器的三维结构的纳米级分辨率的研究。 冷冻ET是选择的方法用于检查的膜结合蛋白组合件包括核孔复合体17中，流感尖峰配合物18的结构和构象，和鞭毛马达蛋白22，23，而且整个细菌细胞19的机构和进入致病病毒如艾滋病病毒在细胞20-23。冷冻ET是无价的可视化丝状蛋白及其在细胞间的相互作用，包括肌动蛋白丝24或轴突25。分辨率可以通过subtomogram平均26，由此反复，常规功能子卷是由单粒子图像的亲切出一个断层量，平均提高到2纳米或更高cessing技术。 冷冻ET涉及收购了一系列的薄样品（&lt;250纳米），在不同的倾斜角度在透射电子显微镜（TEM）拍摄的投影图像。试样必须是薄的，这样的电子，这与物质的强烈的相互作用，分散不超过一次。多次散射，使所得到的图像难以解释，并降低对比度。所选择的样本区域的图像的对齐相对于每个其它与投影到3D空间中通过合适的计算机程序，产生的样品的三维体积。的图像的排列是由金的基准标记，它与冷冻前的样品混合资助。理想的情况是10或更均匀分布的基准标记应该是存在于每一个图像，以实现良好的取向。 观察分子的细节，样品急跌冷冻在液态乙烷，它保留了其天然的水合状态。冷冻在液态乙烷是如此之快（〜10 5℃/秒）27使得水不会结晶而是保持在玻璃化，玻璃样状态。冰晶形成的损害敏感的生物结构。作为生物样品从辐射损伤患，是有一定限度的散射事件的标本可以容忍的总数。图像中的低剂量模式这样获取：感兴趣的区域被确定为低倍率（1500倍），用以下1 C的电子剂量- /纳米2（搜索模式）。该图像，然后集中在一个更高的放大倍率，关利益（对焦模式）的区域。被获取的图像，只有当中，感兴趣的区域上照射具有更高的电子剂量（曝光模式）。 这里，我们提出了如何收集和处理电子冷冻断层图像，利用ATP合酶的二聚体在线粒体内膜，例如一个概观。以下方案描述了如何制备线粒体为低温的ET，如何设置并收集与一个特定的电子总剂量，以及如何处理该倾斜系列以获得感兴趣区域的三维体积的倾斜系列。该过程的概述示于图1。

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			<td height="21" style="height:21px;width:231px;">Sucrose</td>
			<td style="width:235px;">ROTH</td>
			<td style="width:119px;">4621.2</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trehalose</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9449</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">HEPES</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Zymolase</td>
			<td style="width:235px;">biomol</td>
			<td style="width:119px;">Z1000.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Density gradient medium</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1556 or P1644</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Protein A gold</td>
			<td style="width:235px;">Aurion</td>
			<td style="width:119px;">810.111</td>
			<td>10 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Ethane</td>
			<td style="width:235px;">Air Liquide</td>
			<td style="width:119px;">P0502S02R0A001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Nitrogen</td>
			<td style="width:235px;">Air Liquide</td>
			<td style="width:119px;">I4150RG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Consumables</td>
			<td style="width:235px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Filter paper</td>
			<td style="width:235px;">Whatman, GE Healthcare</td>
			<td style="width:119px;">1004 090</td>
			<td>Nr 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Tweezers</td>
			<td style="width:235px;">Dumont</td>
			<td style="width:119px;">T506</td>
			<td>Non-magnetic, pattern #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EM combined test grid</td>
			<td style="width:235px;">Plano GmbH</td>
			<td style="width:119px;">S142</td>
			<td>grapite and gold islands on holey carbon film</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Holey carbon grids</td>
			<td style="width:235px;">Quantifoil Micro Tools GmbH or C-flat</td>
			<td style="width:119px;"></td>
			<td>R2/2 300 Cu mesh&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Cryo grid boxes</td>
			<td style="width:235px;">Plano GmbH</td>
			<td style="width:119px;">160-42</td>
			<td>Alternative home-made metal boxes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Equipment</td>
			<td style="width:235px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Potter</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7984</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blender</td>
			<td style="width:235px;">Waring</td>
			<td style="width:119px;">8011S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Glass bead mill - bead beater</td>
			<td style="width:235px;">BioSpec Products</td>
			<td style="width:119px;">1107900-105</td>
			<td>Including 0.5 mm beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Ball-bearing (Balch) homogeniser</td>
			<td style="width:235px;">isobiotec</td>
			<td style="width:119px;">H8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:235px;">Thermo Scientific</td>
			<td style="width:119px;">46915</td>
			<td>Sorvall RC6+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Glow-discharge apparatus</td>
			<td style="width:235px;">Bal-Tec A.G.</td>
			<td style="width:119px;">CTA 010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Grid vitrification device</td>
			<td style="width:235px;">FEI, Gatan or Leica</td>
			<td style="width:119px;"></td>
			<td>Alternative home-made plunger</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Transmission electron microscope</td>
			<td style="width:235px;">FEI or Jeol</td>
			<td style="width:119px;"></td>
			<td>300 keV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Direct electron detector (CMOS)</td>
			<td style="width:235px;">Gatan</td>
			<td style="width:119px;"></td>
			<td>4k x 4k pixels, counted mode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Charge coupled device</td>
			<td style="width:235px;">Gatan</td>
			<td style="width:119px;"></td>
			<td>4K x 4K pixels (alternative to Direct electron detector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Energy filter</td>
			<td style="width:235px;">Gatan or Jeol</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Software</td>
			<td style="width:235px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Image acquisition software</td>
			<td style="width:235px;">Gatan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Tomogram acquisition software</td>
			<td style="width:235px;">Gatan, FEI, USCF, or UC-Boulder</td>
			<td style="width:119px;"></td>
			<td>4 alternatives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Tomogram processing software</td>
			<td style="width:235px;">FEI, UCSF or UC-Boulder</td>
			<td style="width:119px;"></td>
			<td>3 alternatives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Subtomogram averaging software</td>
			<td style="width:235px;">UC-Boulder, or University of Basel</td>
			<td style="width:119px;"></td>
			<td>2 alternatives</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Tomogram visualisation software</td>
			<td style="width:235px;">UC-Boulder, FEI, or University of Utah</td>
			<td style="width:119px;"></td>
			<td>3 alternatives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">visualisation software plugin</td>
			<td style="width:235px;">Goethe university</td>
			<td style="width:119px;"></td>
			<td>http://www.biophys.uni-frankfurt.de/frangakis/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Structure visualisation software</td>
			<td style="width:235px;">UCSF</td>
			<td style="width:119px;"></td>
			<td>http://www.cgl.ucsf.edu/chimera/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Numerical calculation software</td>
			<td style="width:235px;">MathWorks</td>
			<td style="width:119px;"></td>
			<td>http://www.mathworks.de/academia/</td>
		</tr>
	</tbody>
</table>

visualization of atp synthase dimers in mitochondria by electron cryo-tomography

电子冷冻断层扫描是在结构生物学的有力工具，能够可视化的​​生物样品，如细胞，细胞器，膜小泡或病毒在分子细节的三维结构。为了实现这一点，所述水性样品迅速玻璃化在液体乙烷，它保留它在近距离到本地的，冷冻的水合状态。在电子显微镜下，倾斜系列被记录在液氮温度下，从该三维断层图像进行重建。断层容积的信号 - 噪声比是固有地低。辨认，经常性的功能是由subtomogram平均，其中个别子卷切出，对准，平均降低噪音提高。以这种方式，三维映射为2纳米或更能够得到解决。一件合身可用的高分辨率结构的三维体积，然后产生蛋白质复合物的原子模型在他们的天然环境。在这里，我们将展示我们如何使用电子低温tomograpHY研究现场组织大型的膜蛋白复合物在线粒体中。我们发现，ATP合成酶的组织中的行沿内膜嵴高度弯曲的顶点的二聚体，而复合物I中随机地分布在膜的区域上的行的任一侧。通过subtomogram平均我们得到的嵴膜中的线粒体ATP合酶的二聚体的结构。

电子冷冻断层线粒体清楚地揭示了细胞器的三维形态（ 图2）。在断层容积膜的手工分割示出了在线粒体的嵴结构。通过影像从缺乏某些蛋白质成分不同的酵母基因敲除株线粒体，这些蛋白质对嵴形态的影响可以通过评估。 图3显示了从酵母菌株缺乏ATP合成酶亚基E A线粒体。是必需的线粒体ATP合酶的二聚化的ATP合成酶复合物的这一部分。从该菌株中线粒体缺乏野生型线粒体的正常层状嵴（ 图2），而是包含了若干内部隔膜室中。这些车厢或者是缺乏嵴或包含小气球状的膜内陷（ 图3）。 汤姆ograms具有良好的对比度，大线粒体的蛋白质复合物，在这种情况下，ATP合酶的二聚体，很容易看到（ 图4;电影1）。 （; 电影2图5）配合物的结构，可以在由subtomogram平均2-3纳米的分辨率来确定。的平均体积可放回断层图像，以评估相对于彼此和其他蛋白复合物在膜个别复合物的组织（ 图6;电影3）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51228/51228fig1highres.jpg" width="500" /> 图1流程图显示低温电子断层扫描的阶段 。 <a href="https://www.jove.com/files/ftp_upload/51228/51228fig1highres.jpg" target="_blank">点击这里查看大图。</a> 图2：从形态野生型啤酒酵母线粒体中。通过野生S的断层量环片酵母线粒体（左）和对应的表面呈现体积（右）。外膜的分割体积显示为灰色和内边界的体积和嵴膜在淡蓝色。从戴维斯等人 8改编。 <a href="https://www.jove.com/files/ftp_upload/51228/51228fig2highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51228/51228fig3highres.jpg" width="500" /> 图3线粒体从啤酒酵母株缺乏所需的亚基ATP合成酶的二聚化。通过切片断层容积（左）和相应的从个线粒体表面呈现量（右）。 酵母菌株缺乏所需的ATP合酶二聚体蛋白亚基ê。当与图2相比较，从突变株的线粒体缺乏野生型线粒体的正常层状嵴。相反，线粒体有很多内膜舱，要么没有嵴或气球形嵴。因此，电子低温断层凸显了膜的形态，由于基因缺失的改变。从戴维斯等人 8改编。 <a href="https://www.jove.com/files/ftp_upload/51228/51228fig3highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51228/51228fig4highres.jpg" width="500" /> 造型玩具从木耳体育 4线粒体蕨麻 。切片通过线粒体的从丝状真菌P.断层容积（左）和伴随的表面呈现体积（右） 鹅绒委陵菜 。在此断层图像，行10纳米粒子（黄色箭头）位于上述内层膜嵴高度弯曲的膜隆起部（参照电影1）。这些颗粒被确定为ATP合酶的二聚体通过subtomogram平均。从戴维斯等人 9。 <a href="https://www.jove.com/files/ftp_upload/51228/51228fig4highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51228/51228fig5highres.jpg" width="500" /> 图5结构中的线粒体ATP合成酶和组织。边和表示从S中的ATP合酶的二聚体的电子密度的顶视图ç<html> erevisiae由subtomogram平均连装的原子模型（左）来确定。线粒体内膜泡出的ATP合成酶二聚体行（右）的组织。该图是通过定位subtomogram平均的ATP合酶的二聚体到膜囊泡的分割量，采用平均值中计算出的坐标生成的。从戴维斯等人 8改编。原子模型：F 1 /转子环[PDB：2WPD] 39（蓝色和紫色）;寡霉素敏感相关蛋白-OSCP [PDB：2BO5] 40（绿色）;周边秸秆片段[PDB：2CLY] 1与N端残基[PDB：2WSS] 2（黄色和红色）（见电影2） <a href="https://www.jove.com/files/ftp_upload/51228/51228fig6highres.jpg" target="_blank">点击此处查看大图。</a> <html> _upload / 51228 / 51228fig6highres.jpg“WIDTH =”500“/&gt; 从P.图6隔离嵴泡蕨麻线粒体。片从P的嵴泡的断层容积（左）和相应的表面呈现量（右） 鹅绒委陵菜 。蛋白浓度从膜突出都清晰可见。按黄色箭头指示的密度是ATP合酶二聚体，所确定的subtomogram平均。绿色箭头指向确定抗体标记为NADH脱氢酶浓度（复1;详见9）。蛋白质浓度的分割揭示其在嵴组织，与ATP合酶的二聚体（红色和黄色）沿高度弯曲的脊突脊形成行和NADH脱氢酶复合物（绿色）中的行的任一侧上的膜的区域（见电影3）。从戴维斯等人 9。28 / 51228fig7highres.jpg“目标=”_ blank将“&gt;点击这里查看大图。 电影1电子低温断层图像P的线粒体鹅绒委陵菜的影片通过从丝状真菌P.采取了线粒体的断层量显示连续切片鹅绒委陵菜 。 ATP合酶的行由黄色箭头表示。表面渲染，分段体积显示ATP合酶（黄色球）相对于所述三维嵴结构的位置。外膜，灰色;内界膜，透明的蓝色;嵴膜，不透明的蓝色。从戴维斯等人 9。参见图4， <a href="https://www.jove.com/files/ftp_upload/51228/movie1.mp4" target="_blank">点击此处查看视频。</a> 电影2 Subtomogram平均从S线粒体ATP合酶二聚体。 酵母与拟合的原子模型，平均为从121子体积计算。密度显示在三个轮廓层次：1秒 - 网，2S - 浅灰色和3S - 深灰色。原子模型，使用嵌合体的顺序拟合例程装入密度。原子模型：F 1 /转子环[PDB：2WPD] 39（蓝色和紫色）;寡霉素敏感相关蛋白-OSCP [PDB：2BO5] 40（绿色）;周边秸秆片段[PDB：2CLY] 1与N端残基[PDB：2WSS] 2（黄色和红色） <a href="https://www.jove.com/files/ftp_upload/51228/movie2.mov" target="_blank">点击此处查看视频。</a> 从戴维斯等人 8改编。参见图5。 从体育电影3。隔离嵴泡通过断层容积蕨麻线粒体。连续切片示，随后经分割表面呈现体积。蛋白密度从卷材胶粘突出e为清晰可见。红色和黄色的密度是ATP合酶的二聚体。绿色密度NADH脱氢酶（复合体I）通过抗体标记来确定。从戴维斯等人 9。参见图6。 <a href="https://www.jove.com/files/ftp_upload/51228/movie3.mpg" target="_blank">点击此处查看视频。</a>

Watch this Scientific Journal Video about Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography at JoVE.com

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

我们提出了如何收集和处理电子低温断层整个线粒体的协议。该技术提供了详细分析上市的结构，功能和组织的大的膜蛋白复合物中天然生物膜。

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

visualization-atp-synthase-dimers-mitochondria-electron-cryo

Research

JoVE Journal

Biology

29.4K 次观看.  Max Planck Institute of Biophysics. 我们提出了如何收集和处理电子低温断层整个线粒体的协议。该技术提供了详细分析上市的结构，功能和组织的大的膜蛋白复合物中天然生物膜。 

视频: Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

科研

生物学

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

快速分离纯化线粒体移植用组织分离和鉴别过滤

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

在联合电子能谱成像miniSOG标记的DNA修复蛋白可视化（ESI）

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

研究内质网和线粒体相互作用通过<em&gt;原位</em&gt;接近连接分析在固定细胞

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...