我们感谢安德烈Kaech（苏黎世大学）为他的扫描电子显微成像有用的建议。这项工作是由美国 - 以色列两国农业研究和发展基金（批准号:US-4334-10，ZR），以色列科学基金会（批准号:1034年至1012年，ZR），和人类前沿科学计划支持（RGP0005 / 2013，ZR）。透射电镜研究在欧文和Cherna莫斯科维茨中心纳米和生物纳米成像在魏茨曼科学研究所进行。

叶片组织1.超低温固定用高压冻结注意:本节介绍了如何进行叶片组织的高压冻结的冻结破裂实验。对于与植物样品的考虑看29。这可以适用于其他类型的组织或样品与一些修改。 <ol><li>使用刀片的角，挠0.1 / 0.2 mm铝血小板的0.1毫米空腔的底部（ 图1）。至少准备一打血小板（6个样本）。采取小心不要在盘的圆周创建刀痕。 <ol><li>搔抓后，洗碟无水乙醇，让他们干，保持一个干净的菜。 </li></ol></li><li>根据制造商的说明制备高压冷冻机。填充用异丙醇高压冷冻机的酒精室。 </li><li>制备自制真空辅助infiltratioÑ​​设备来替换与液体的叶组织中发现的气体。 <ol><li>紧紧一个200μl的移液管尖端连接到10毫升注射器的端部。切断〜1厘米尖。使在1.5ml微量离心管的盖，以一个孔紧密地配合切割尖端。真空可在管内通过拉动注射器的柱塞被创建。 </li></ol></li><li>从使用刀片的植物切一小片叶子并用镊子放置一个离心管中一半填充有1-十六内的叶。通过轻轻拉动柱塞创建真空渗入与液体叶子的细胞间隙，保持约30秒，然后缓慢释放柱塞。 </li><li>删除用镊子管中的叶片。修剪用刀片的情况下，它是0.2mm以上厚的叶和切一小片，将适合成血小板。 </li><li>将一个洁净的血小板与抓伤的一面朝上放入冻机的支架，然后将切下的乐AF成血小板。填充周围用1-十六叶的剩余空间。使用其他血小板关闭三明治，但面向叶划痕。 </li><li>关闭并拧紧固定器，并将其插入到机器的腔。然后按&#39;喷气机&#39;按钮冻结（〜2100条300 - 500毫秒），并迅速持有者转移到液氮中。使用预冷却的镊子，小心不要折断夹心从支架小心取出冷冻的三明治。 注意事项:密闭冷冻三明治可以冻结裂隙立即或保存在供以后使用液氮。液氮操作时，使用的防护服和护目镜。 </li></ol> 2.冷冻断裂和双层涂层27,28 <ol><li>制备冷冻断裂机的使用，包括枪为铂/碳（铂/ C）和碳的蒸发，根据制造商的说明。放置的Pt / C枪在45度的次在90度的碳枪。 <ol><li>预编程的Pt / C（涂敷速度0.02 - 0.04纳米/秒）的2纳米层的一个涂覆方案和碳的6纳米层（在〜0.1纳米/秒）。 <ol><li>进入冷冻断裂机的屏幕上的安装。选择在哪里，该涂层方案将被保存在用户数。 </li><li>选择的Pt / C为"1层"。选择一个时间框架， 例如 ，5分钟（超过完成涂层所需的必要的时间）。使用上下箭头来定义的厚度为2纳米。 </li><li>按"层"按钮移动到2层重复步骤2.1.1.2，只不过选择碳和定义，厚度为6纳米。 </li></ol></li><li>测试和调整喷枪作业。 <ol><li>选择"图层1"。按"GUN1"按钮选择的Pt / C枪，然后按冷冻断裂机的蒸发器上的"打开"按钮。监视蒸发速率和使用的电压和发射旋钮UNT调整它IL达到0.02的速率 - 0.04纳米/秒。按"OFF"关闭的Pt / C的枪。 </li><li>选择"2层"。重复2.1.2.1，除了选择&#39;GUN2&#39;并调整为0.1nm /秒的〜的蒸发速率。 </li></ol></li></ol></li><li>酷（分别为水合或脱水叶）冷冻断裂系统的阶段-140℃或-160˚C。装上真空冷冻班车服务的机器，并用液氮填补其室。毫巴-7 8×10 -压在冷却冷冻断裂室通常在1之间。 </li><li>插入冷冻压裂试样保持器（适合于3毫米血小板）到装载站。洪水用液氮装载站室中。 </li><li>采用高精度等级镊子一个装载两个冷冻三明治到持有人之一。拧紧第一块三明治到支架，然后翻转持有人过来检查到位牢固地安装。重复第二个三明治。 </li><li> ðetach从冷冻断裂机冷却班车和它连接到装载站。打开梭阀，引进收回臂插入支架，并将其锁定到位。重新引入与保持到穿梭臂以及使用该装载站旋钮关闭梭阀。附加穿梭于冷冻断裂机和引入保持件与所述收回臂的腔室。从分离机的班车。 </li><li>对准冷冻断裂切片刀的高度，使得其将敲落三明治的顶端血小板。 </li><li>随着切片刀，迅速突破了三明治，然后立即提出来，以防止样品污染的碎片执着刀，并把新暴露的平面上方的冷却快门由水分子免受可能的污染样品室中。 </li><li>大衣搭配铂和碳样本。 <ol><li>输入"图层1"的SC颖。按"GUN1"按钮，然后按"ON"按钮打开的Pt / C的枪。审查的蒸发速率和一旦达到0.02 - 0.04纳米/秒，同时暴露出样品，并按下"测量"按钮来监测沉积层的厚度。 注:当厚度已经达到了预选值枪会自动关闭。 </li><li>盖与快门样本时完成的Pt / C的蒸发。 </li><li>按"层"按钮移动到"2层"。重复步骤2.8.1和2.8.2，以选择"GUN2&#39;（碳）和0.1纳米/秒的〜的蒸发速率的异常。 </li></ol></li><li>温冷冻断裂系统的阶段-120˚C。 </li></ol> 3.超低温扫描电子显微镜<ol><li>制备用于低温工作的扫描型电子显微镜。 <ol><li>从主菜单，确认选择舞台/舞台初始化。 </li><li>选择工具/ GOTO面板，并从列表中选择打开"气密室"面板。点击"关闭柱箱阀门"按钮。通过点击"真空"选项卡下的通风排气按钮，在显微镜室。 </li><li>选择工具/转到面板，打开"阶段点列表"面板。选择低温交换位置向载物台移动到正确的坐标用于接收试样架。注意:"低温交换位置"被预设为在对准与真空传送单元的位置。 </li><li>开用干净的一对手套的试样腔室和插入低温阶段到显微镜的主要阶段。关闭室，然后点击"真空"标签下泵按钮。 </li><li>酷显微镜-120˚C。填写显微镜用杜瓦瓶液氮。激活低温传送控制单元上的热作用到-120˚C一旦阶段的温度低于-120˚C。 </li></ol></li><li> ATTACH的低温传送梭显微镜并使用收回臂进入显微镜低温阶段（如在步骤2.5）传送样品架。分离从显微镜的班车。 </li><li>点击气密室面板中的"打开柱箱阀门"按钮。 </li><li>小心移动样品支架接近物镜（工作距离1 - 2mm）的使用操纵杆。选择在"孔径"选项卡10微米的孔径。在"枪"选项卡中双击EHT领域。输入10（千伏）的EHT目标，然后单击确定。点击底部EHT标签，选择"EHT开"。 </li><li>在"探测器"选项卡中选择从检测器下拉列表"InLens"。优化成像条件（对焦，亮度和对比度，光束对准，散光）为宜。 </li><li>点击"扫描"选项卡。选择一个快速扫描（"扫描速度= 1&#39;，对应于100纳秒每像素的驻留时间），以最小化光束的损坏，并1,024×768像素一个"存储分辨率"。在"降噪"下拉列表中选择"平均线"。 N的值调整为20 - 30线搜索。使用"中心站"的功能（通过按下键盘上的控制标签）来搜索与裂隙细胞和叶绿体（ 图2A和2B）的区域移动样品。 </li><li> （ - 70的kX 50）（ 图2C和2D）在低放大倍率获得裂缝叶绿体的图像。使用（按键盘上的控制移选项卡）的&#39;中心功能&#39;功能放大到有趣叶绿体裂缝面，在高放大倍率获取图像（100 - 200的kX， 图3和图4）。使用相同的参数，在步骤3.6图像采集，但改线平均价值N&gt; 100降噪30。 <ol><li>主显微镜控制单元上或在"扫描"选项卡按冻结，停止扫描并保存按"保存TIFF"按钮图像。 </li></ol></li></ol> 4.图像分析注意:本节介绍使用斐济31开源包冷冻断裂的SEM图像颗粒膜分割一小段程序。类似的结果可以与其他图像分析软件来获得。 <ol><li>通过拖动图像文件到主程序窗口斐济打开图像。通过选择图像/类型/ 8位图像转换为8位。选择图像/调整/亮度/对比度和根据需要调整（ 图5A）。 </li><li>采用直线工具画一条线嵌入图像比例尺的大小。选择分析/设置比例。输入正确的档位信息（长度已知的距离和单位），然后单击确定。保存此调整缩放后的图像。 </li><li>选择Process /过滤器/高斯模糊（1.5纳米半径），并单击OK（ 图5B）。 </li><li>选择图像/调整/ AUTØ局部阈值（使用Niblack法）并单击OK（ 图5C）。 </li><li>选择编辑/翻转，然后再处理/二进制/流域（ 图5D）。 </li><li>绘制手绘选择工具感兴趣区域（ROI）的区域周围的边框。通过选择编辑/选择添加选择的投资回报率经理/添加到管理器或通过点击"T"。 </li><li>选择编辑/清除外（ 图5E）。 </li><li>选择分析/集测量，选择合适的参数 （ 如面积，配合椭圆形）。 </li><li>选择分析/分析颗粒。输入一个适当的范围颗粒的尺寸和标记"显示结果"，"添加到管理器"，如果有必要，"排除在边缘"选项，然后单击确定。该结果示于图5F。 </li><li>打开保存的缩放成像文件（步骤4.2）。选择"显示所有"，并在投资回报率经理取消选择"标签"。查看所选partic莱放置到原始图像和手动添加或使用"添加"或"删除"的投资回报率经理的按钮移除选项。更正通过使用写意选择工具和投资回报率经理（ 图5G和5H）的"更新"按钮，任何必要的选择。 </li><li>分析使用所获得的测量数据。在结果窗口中，单击结果/总结获得，例如平均面积，平均主要和投资回报率管理器中列出的粒子的短轴。点击结果/配送，选择参数（例如面积），然后单击确定以查看该参数的直方图。 </li></ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoprotective+Energy+Dissipation+Involves+the+Reorganization+of+Photosystem+II+Light-Harvesting+Complexes+in+the+Grana+Membranes+of+Spinach+Chloroplasts.">Photoprotective Energy Dissipation Involves the Reorganization of Photosystem II Light-Harvesting Complexes in the Grana Membranes of Spinach Chloroplasts.</a> Plant Cell. 23 (4), 1468-1479 (2011).</li><li>Kirchhoff, H., Tremmel, I., Haase, W., Kubitscheck, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supramolecular+photosystem+II+organization+in+grana+thylakoid+membranes:+evidence+for+a+structured+arrangement.">Supramolecular photosystem II organization in grana thylakoid membranes: evidence for a structured arrangement.</a> Biochemistry. 43 (28), 9204-9213 (2004).</li><li>Belgio, E., Ungerer, P., Ruban, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V+Light-harvesting+superstructures+of+green+plant+chloroplasts+lacking+photosystems.">V Light-harvesting superstructures of green plant chloroplasts lacking photosystems.</a> Plant Cell Environ. , (2015).</li><li>Goral, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-harvesting+antenna+composition+controls+the+macrostructure+and+dynamics+of+thylakoid+membranes+in+Arabidopsis.">Light-harvesting antenna composition controls the macrostructure and dynamics of thylakoid membranes in Arabidopsis.</a> Plant J. 69 (2), 289-301 (2012).</li><li>Charuvi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoprotection+Conferred+by+Changes+in+Photosynthetic+Protein+Levels+and+Organization+during+Dehydration+of+a+Homoiochlorophyllous+Resurrection+Plant.">Photoprotection Conferred by Changes in Photosynthetic Protein Levels and Organization during Dehydration of a Homoiochlorophyllous Resurrection Plant.</a> Plant Physiol. 167 (4), 1554-1565 (2015).</li><li>Farrant, J. M., Brandt, W., Lindsey, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Overview+of+Mechanisms+of+Desiccation+Tolerance+in+Selected+Angiosperm+Resurrection+Plants.">An Overview of Mechanisms of Desiccation Tolerance in Selected Angiosperm Resurrection Plants.</a> Plant Stress. 1 (1), 72-84 (2007).</li><li>Walther, P., Schatten, H., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+cryoscanning+electron+microscopy+of+biological+samples.">High-resolution cryoscanning electron microscopy of biological samples.</a> Biological Low-Voltage Scanning Electron Microscopy. , 245-261 (2008).</li><li>Walther, P., M&#252;ller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-layer+coating+for+field-emission+cryo-scanning+electron+microscopy--present+state+and+applications.">Double-layer coating for field-emission cryo-scanning electron microscopy--present state and applications.</a> Scanning. 19 (5), 343-348 (1997).</li><li>Walther, P., Wehrli, E., Hermann, R., M&#252;ller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-layer+coating+for+high-resolution+low-temperature+scanning+electron+microscopy.">Double-layer coating for high-resolution low-temperature scanning electron microscopy.</a> J. Microsc. 179, 229-237 (1995).</li><li>Hess, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreparation+methodology+for+plant+cell+biology.">Cryopreparation methodology for plant cell biology.</a> Cell. Electron Microsc. 79, 57-100 (2007).</li><li>Schertel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo+FIB-SEM:+Volume+imaging+of+cellular+ultrastructure+in+native+frozen+specimens.">Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens.</a> J. Struct. Biol. 184 (2), 355-360 (2013).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat. Methods. 9 (7), 676-682 (2012).</li><li>Walther, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+freeze-fracturing+of+high-pressure+frozen+samples.">Recent progress in freeze-fracturing of high-pressure frozen samples.</a> J. Microsc. 212 (1), 34-43 (2003).</li><li>Nevo, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+Thylakoid+Membrane+Networks.">Architecture of Thylakoid Membrane Networks.</a> Lipids Photosynth. 30, 295-328 (2009).</li></ol>

The authors declare they have no competing financial interests.

本文中所描述的技术允许保存完好的高压冷冻的植物组织的由低温扫描电子显微镜的范围内冻结，割断膜调查。使用这些程序的主要优点是样品制备是纯物理;涉及化学品或脱水任何步骤是必要的。因此，它可以在一个近乎原生状态26,32研究生物结构。用叶组织的好处是，可以得到在整体蜂窝组织的信息，以及作为研究特定的膜，如类囊体膜，其天然生理环境， 即内，叶绿体内。另?...

氧光合作用，原产于古代的蓝藻，是由内共生事件，导致叶绿体细胞器的开发继承了藻类和陆生植物。在所有现代的产氧光养，光合作用电子传输和质子动力的产生，并减少功率被称为"类囊体"膜扁平囊样小泡内进行。这些膜容纳该进行光合作用的光驱动反应和提供能量转换介质中的蛋白复合物。植物和（部分）藻类的类囊体膜分化成两个不同的形态结构域:紧紧贴伏膜的区域称为"基粒"和互连基粒未叠加膜，称为"基质片层1。植物和藻类类囊体膜的各种冷冻断裂研究已经进行，在70年代初开始。当冷冻断裂，膜沿着它们的疏水芯2分割，生成exoplasmic面（EF）和原生质面（PF），这取决于蜂窝隔室其中半膜的边界，如最初由Branton 等人提出的。 。在1975年3植物和藻类类囊体有四种不同的裂缝面:EF文件，EFU，PFS和PFU，用&#39;s&#39;和&#39;U&#39;表示&#39;堆积&#39;和&#39;拆散&#39;膜的区域，分别为。膜蛋白复合物，这是不分裂或破裂，必须保持与E或膜的P一侧的倾向。最初的意见，即类囊体的不同裂缝面包含不同大小的颗粒和密度4，以及随之而来的大量调查，导致了开展光反应所观察到的颗粒和膜蛋白复合物的鉴定和相关5-13 （见还回顾14,15）。 FRE类囊体膜的结-断裂试验通常进行于叶绿体或分离的类囊体膜的制剂（但见16,17），在任何改变的在结构和/或超分子组织的分离过程期间可能发生的危险。继骨折，复制是铂/炭（PT / C）的蒸发法制备，再由碳（C）厚厚的一层，最后是生物材料18的消化。复制是透射电子显微镜（TEM）观察。传统的冷冻断裂-复制技术继续作为用于研究光合膜的超分子组织和它们适应于不同的一个重要的工具， 例如 ，光，条件19-23。 在我们最近的homoiochlorophyllous复苏植物Craterostigma细叶 24的研究中，我们的目的是探讨在超分子组织为O的变化˚F类囊体膜，以及在整体蜂窝组织，脱水和复水过程中。该homoiochlorophyllous复活的物种的独特性在于它们能够生存在他们的营养组织（叶子）干燥的条件下，同时保持其光合机构。一旦水可用时，这些植物恢复和恢复小时内光合活性几天25。在这项研究中，冻结，割断叶样品的冷冻扫描EM（SEM）成像用高压冷冻样品冷冻固定组合。这些程序提供了在接近其原生状态26的状态可视化冷冻水合生物样品的方法。一个主要的好处是，样品冷冻断裂和涂层，没有连续的步骤后直接检查。这是在不同的相对含水量（RWC）植物的调查尤为重要，因为他们的水合状态，制备过程中保持不变。怎么样以往，人们关键缺点是，当在高放大倍率，光合复合物的大小的精确测量需要扫描冷冻水合样品可从光束损坏成像期间遭受，更是如此。为了克服这一点，一种方法被称为"双层涂层&#39;（DLC）27,28与分别利用特定冷冻SEM成像条件相结合。这导致那些显著光束敏感较少样本和允许的有价值的信息，对光合蛋白质超分子组织和复苏植物C.其他细胞成分的阐明细叶 在原地高放大倍率。

<table><tbody><tr><td>Ethanol abs</td><td>Bio-Lab</td><td>052505</td><td></td></tr><tr><td>Isopropanol</td><td>Bio-Lab</td><td>162605</td><td></td></tr><tr><td>1-hexadecene</td><td>Sigma-Aldrich</td><td>H7009</td><td></td></tr><tr><td>0.1/0.2 Platelets</td><td>Engineering Office M. Wohlwend GmbH, Switzerland</td><td>241</td><td>Platelets are of 3-mm diameter and 0.5-mm-thick (Type A) with 0.1/0.2-mm-deep cavities (of diamater 2 mm). Similar platelets can be obtained from Leica Microsystems.</td></tr><tr><td>High-precision-grade tweezers</td><td>Electron Microscopy Sciences</td><td>72706-01</td><td>Dumont (Switzerland) Durostar style #5 tweezers; Can be substituted with other high-precision tweezers.</td></tr><tr><td>High-pressure freezing machine</td><td>Bal-Tec</td><td>HPM 010</td><td>High-pressure freezing alternatives: 1. HPF Compact 02, Wohlwend GmbH; 2. HPM 010, RMC Boeckeler; 3. EM PACT2, Leica Microsystems; 4. EM HPM 100, Leica Microsystems; 5. EM ICE, Leica Microsystems.</td></tr><tr><td>Freeze-fracture system</td><td>Leica Microsystems</td><td>EM BAF 060</td><td></td></tr><tr><td>Cryo preparation loading stage</td><td>Leica Microsystems</td><td>16770228</td><td></td></tr><tr><td>Specimen holder for univeral freeze fracturing</td><td>Leica Microsystems</td><td>16LZ04746VN</td><td>Clamp holder for specimen carriers of diameter 3 mm</td></tr><tr><td>Vacuum cryo-transfer shuttle</td><td>Leica Microsystems</td><td>EM VCT 100</td><td></td></tr><tr><td>Scanning electron microscope</td><td>Zeiss</td><td>Ultra 055</td><td></td></tr><tr><td>Cryo SEM stage</td><td>Leica Microsystems</td><td>16770299905</td><td></td></tr><tr><td>Image acquisiton software</td><td>SmartSEM, Carl Zeiss Microscopy GmbH</td><td></td><td></td></tr><tr><td>Image analysis software</td><td>Fiji/Image J, National Institute of Health</td><td>http://fiji.sc/Fiji</td><td></td></tr></tbody></table>

studying the supramolecular organization of photosynthetic membranes within freeze-fractured leaf tissues by cryo-scanning electron microscopy

冷冻断裂样品的冷冻扫描电子显微镜（SEM）允许在接近天然条件的生物结构的调查。在这里，我们描述了叶片样品中学习光合（类囊体）膜的超分子组织的技术。这是由叶组织的高压冷冻，冷冻压裂，双层涂层和最后低温SEM成像实现的。双层涂覆方法的使用允许获得高倍率（&gt; 100000）与到冷冻水合样品最小光束损害以及最小充电效果的图像。使用所描述的过程，我们在采取复苏植物Craterostigma细叶脱水过程中进行，现场光系统和捕光天线蛋白复合物超分子分布调查改变。

图1显示了包含高压冻结，冻结，割断Craterostigma细叶叶片血小板冷冻SEM图像。在一些样品中，裂缝单元的大区域被获得（ 图1A）。在其他情况下，叶片保持紧密结合到上盘并与它一起（ 图1B）脱落。然而，即使在第二种情况下，某些叶组织可保持附着在血小板（ 图1B，箭头）和数据的相当数量的刀槽仍可以通过成?...

Watch this Scientific Journal Video about Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy at JoVE.com

通过冷冻扫描电子显微镜研究冻裂叶组织内光合膜的超分子组织生物学 |视频

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.

通过低温扫描电子显微镜研究光合膜内冻结，割断叶片组织的超分子组织

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

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12.1K 次观看.  Weizmann Institute of Science. Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.

视频: Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although X-ray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells.

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

科研

生物化学

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography.

使用双光束扫描电子显微镜从玻璃体生物样品制备薄片进行冷冻电子断层扫描

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

冷冻电子断层扫描 的糖核 酸的准备和冷冻-FIB微加工

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

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

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

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 ...

生物学

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

冷冻断裂/冷冻蚀刻的生物电子显微技术的基本要素

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

This manuscript describes an easy-to-use and low-cost cryofixation method for visualizing suspension cells by transmission electron microscopy.

暴跌冻结:在透射电子显微镜的超微结构和免疫组化研究工具悬架细胞

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

闪存和冻结:一种新的技术来捕获膜动力学与电子显微镜

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

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 ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

通过低温扫描电子显微镜研究光合膜内冻结，割断叶片组织的超分子组织

摘要

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此视频中的章节

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

使用双光束扫描电子显微镜从玻璃体生物样品制备薄片进行冷冻电子断层扫描

冷冻电子断层扫描的糖核酸的准备和冷冻-FIB微加工

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

冷冻断裂/冷冻蚀刻的生物电子显微技术的基本要素

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

暴跌冻结:在透射电子显微镜的超微结构和免疫组化研究工具悬架细胞

闪存和冻结:一种新的技术来捕获膜动力学与电子显微镜

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

通过低温扫描电子显微镜研究光合膜内冻结，割断叶片组织的超分子组织

摘要

探索更多视频

此视频中的章节

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

使用双光束扫描电子显微镜从玻璃体生物样品制备薄片进行冷冻电子断层扫描

冷冻电子断层扫描 的糖核 酸的准备和冷冻-FIB微加工

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

冷冻断裂/冷冻蚀刻的生物电子显微技术的基本要素

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

暴跌冻结:在透射电子显微镜的超微结构和免疫组化研究工具悬架细胞

闪存和冻结:一种新的技术来捕获膜动力学与电子显微镜

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

冷冻电子断层扫描的糖核酸的准备和冷冻-FIB微加工