This work was funded by two ANR grants (ANR-11-BSV8-016 and ANR-10-IDEX-0001-02). We also acknowledge the PICT-IBiSA for providing access to chemical imaging equipment.

注意:使用前请咨询各种试剂的材料安全数据表（MSDS）。几个样品制备过程中使用的化学品是有毒的，致癌，致突变，和/或生殖毒性。在处理样品使用个人防护装备（手套，实验室外套，全长裤，封闭趾鞋）和工作在通风橱内。切片与超微样本涉及使用锋利的仪器，并谨慎使用这些工具是强制性的。在下面描述的步骤中，作者认为样品是细胞培养物的样品。该协议可能需要根据样品类型，尤其是离心步骤进行修改。 1.缓冲器和混合物的制备<ol><li>制备磷酸盐缓冲盐水（PBS）溶液（137毫摩尔NaCl，2.7毫米氯化钾，10毫的 Na 2 HPO 4，和1.76毫摩尔KH 2 PO 4）。 </li><li>制备含有30％，50％，70％等的解决方案hanol在PBS样品脱水。通过逐步培养在增加乙醇浓度样品进行样品脱水。 </li><li>通过混合双酚A-（表氯醇）（20毫升），十二碳烯基琥珀酸酐（9毫升），甲基-5-降冰片烯-2,3-二羧酸（11毫升）和2,4,6-三制备环氧树脂（二甲基氨基甲基）苯酚（0.7毫升）;其它混合比率可根据树脂的所需的硬度使用。 </li><li>制备含有30％，50％，和70％的环氧树脂在乙醇中样本嵌入的解决方案。逐渐培养随着树脂浓度的样品进行树脂嵌入。 注:制备的溶液的体积取决于所研究的样品的类型。所需的组织比细胞培养更多的解决方案。 </li></ol> 2.固定与染色样品<ol><li>离心细胞培养5分钟，在5000×g的。执行所有使用相同的条件下离心的。</li><li>弃去上清液，并通过与4％多聚甲醛和2％戊二醛再悬浮它于PBS（1mL）中固定细胞沉淀。在室温下30分钟（ 图1A）孵育它。可替代地，固定于4℃将细胞过夜或过周末。 </li><li>离心后，除去上清，用PBS（1mL）中洗涤沉淀3次，以完全除去固定液剂。 </li><li>用1％OSO 4于PBS（1mL）中，在室温下30分钟后的固定样品。 </li><li>离心后，除去上清，用PBS（1mL）中洗涤沉淀3次，以完全除去OSO 4。停用OSO 4石油和丢弃。 </li><li>于PBS（1mL）中添加2％醋酸双氧铀作为造影剂和孵化的样品在室温下（ 图1B）30分钟。 </li><li>离心后，除去上清，用PBS（1mL）中洗涤沉淀3次以去除未结合的双氧铀的交流etate;醋酸铀中含有放射性元素，并应致力于放射性废物箱进行处理。 注:孵育时间可根据样品的大小进行修改。例如，固定和较厚的样品，如组织样品染色，需要较长的温育时间。较长的孵育时间还可以是用于脱水和嵌入步骤更好。醋酸铀可以通过包含钆盐一"铀少"的解决方案来代替。 </li></ol> 3.样品脱水<ol><li>离心后，弃去上清，在含有30％乙醇4℃在PBS（1mL）中（ 图1C）孵育样品30分钟。 </li><li>重复步骤3.1采用的解决方案与逐渐增加的乙醇浓度（50％，70％，和最多100％）。在这些脱水步骤中，保持在4℃样品保存目的样品。 </li><li>离心后，弃去supernat蚂蚁和悬浮在纯乙醇（1mL）中的小球;在4℃孵育过夜。 </li></ol> 4.树脂嵌入<ol><li>离心后，弃去上清，重悬，用30％的环氧树脂（1毫升）在乙醇中沉淀，在室温（ 图1D）30分钟。 </li><li>重复步骤4.1采用的解决方案与逐渐增加的环氧树脂浓度（50％，70％，和最多100％）。 </li><li>离心后，弃去上清，重悬在纯环氧树脂（1mL）中的小球;在室温孵育过夜。硬化DMP-30可能引发树脂的聚合的存在;如果发生这种情况，准备环氧树脂两批，其中一个和一个没有DMP-30。 </li><li>离心后，悬浮在纯环氧树脂的200微升（含硬化剂）在塑料胶囊中的小球;样品应在塑料胶囊的底部。 </li></ol> 5.树脂聚合<ol><li>一世ncubate含有48小时设定为60℃的培养箱样品塑料胶囊;其他种类的树脂可能会使用不同的设置聚合。 </li><li>从孵化器中取出胶囊并验证树脂聚合;如果按压硬表面的树脂要硬。 </li><li>加在包含样品的聚合树脂的顶（含硬化剂）纯的环氧树脂;树脂的这种额外的层将使它更容易切割样品。孵育在60℃的胶囊48小时。 </li></ol> 6.样品切片<ol><li>安装在试样保持器样品倒置，使样品朝向顶部并修复它的超薄切片的修剪块上。紧紧锁定杆修剪块固定在超薄。 </li><li>用刀片或类似的锋利切割器械，切出的塑料胶囊将其从聚合树脂分离。切割树脂，使所述样品是包含版在树脂在金字塔的大致形状。没有必要删除整个塑料胶囊;只能去除覆盖在聚合样品的部分。 </li><li>取的胶囊和试样保持关闭修剪块的和对超微的移动臂安装它们。 </li><li>刀块上安装一个修边刀，准确地修剪金字塔的面孔。使用双筒望远镜的建议给完美的体形金字塔;金字塔越好，越好的章节会。 </li><li>脱下修边刀并用，可用于切割是厚度大于0.5微米的部分组织学刀替换它。 </li><li>填充所述组织学刀与水的正确量的反应杯后，使刀的切割边缘到样品的表面上，并使用望远镜来调整胶囊型桨距角，使得刀将垂直切片的金字塔。 </li><li>根据所希望的调整切割速度截面厚度恢复均匀部分。 </li><li>让部浮在水中，并用一个循环鱼它。 </li><li>走向已放置在滤纸的顶部的电子显微镜铜网环路。 注意:网格应该已经处理以前，以增加其亲水性。这可以用一个网格涂布笔来执行。 </li><li>由于通过滤纸吸水率，让部分粘到电网。让它将其插入到电子显微镜前干燥几分钟。 </li></ol> 7.通过焦点倾斜系列的设计<ol><li>聚集在持续关注的时间间隔，通过焦图像。 注:通过焦点倾斜系列由一组通过焦图像被在倾斜系列的每个倾斜角度收集。整焦点振幅应足以覆盖整个样品厚度。 </li><li>确定聚焦间隔的值。如果该间隔等于对dEPTH的场，收集在焦点整个样本;这个设置代表了最高采样可能。如果该间隔比场深度的更大，样品的某些部分将不会在焦点获取，这意味着样品的某些部分将不会在焦点被收集。 </li><li>计算深度的领域，从电子的波长的电子束和电子束的会聚半角的。而电子的波长直接相关的加速电压，可使用通过电子显微镜的制造商提供的收敛半角度或从已知试样16的衍射图案实验确定它。因为电子在波长λ加速击中α的会聚半角样品中的电子显微镜，可使用以下公式来确定深度的领域（DOF）: <img alt="方程" src="/files/ftp_upload/55215/55215eq1.jpg" /></li><li>设计焦点系列，使整个焦点振幅将覆盖在其最大表观厚度的样品（即在其最高的倾斜）。 注:0.75微米厚的样品将具有2.19微米的表观厚度在70°倾斜，所以重点振幅应该大于2.19微米。对于此示例，如果上述确定的焦点间隔等于50纳米，这将需要44图（2.19微米/ 50纳米）以覆盖在其最大表观厚度的样品。整焦点幅度等于焦点时间间隔乘以焦步骤的数目的值。在最高倾斜（θ），将样品表观厚度被定义为以下: <img alt="方程" src="/files/ftp_upload/55215/55215eq2.jpg" /></li></ol> 8.样品成像注:这已经超出了协议的范围来描述如何设置在STEM模式电子显微镜;大部分信息都可以找到我n中的参考手册由电子显微镜的制造商提供的。然而，注意以下几点:i）该点尺寸应根据所希望的倍率进行选择; ii）该Ronchigram必须很好对齐;和iii）中的BF模式下，相机长度必须被调节以选择未散射电子，同时保持检测器的良好的照明。沉积在电子显微镜铜网上树脂切片应该图像采集前被点亮，以防止收缩。树脂收缩和充电的影响可能会在图像采集过程羞愧。发生充电效果时，使用一个客观的镜头光圈可以改善样品的稳定性。然而，在STEM设置的特定情况下，目标孔不与HAADF成像模式兼容，和非常小的目标孔是不与高炉成像模式兼容。 <ol><li>使用低倍率（约20000倍在STEM），浏览通过样品和定位感兴趣的区域。 </li><li>调整Ť他eucentric高度（Z轴），从而使在旋转样品的感兴趣的区域不漂移距离。 </li><li>验证感兴趣的对象将在整个倾斜范围仍然可见。 </li><li>因为通过焦倾斜系列中获取的图像的大量的，使用自动收集软件。无论是使用商业解决方案，或者如果需要设计一个自定义的数据采集软件。确保软件程序能够执行以下三个步骤: <ol><li>跟踪和集中在一个标准的倾斜系列采集方案。 </li><li>确保通过焦点的图像与样品的Z位置重叠。 </li><li>自动获取在每个倾斜角一系列通过焦图像。 </li></ol></li><li>给软件程序的通过焦点倾斜系列的主要参数（ 即，焦间隔，焦步骤，最小倾斜角度，最大倾斜角，倾斜步骤，和扫描速度）。 </li><li>开始图像采集处理，并等待软件程序来完成。 </li></ol> 9.图像处理注意:在这个协议中，使用ImageJ插件17进行通焦倾斜的一系列处理。补充数据1示出了用于对准步骤（Turboreg）和用于为通过焦点倾斜系列设计有三个焦点值合并的聚焦信息（扩展景深）的ImageJ的宏。 <ol><li>对齐在同一倾斜角（ 即，通焦图像）中收集的图像。由于图像不具有相同的对焦信息，他们不怀有对准相同的特性;通过调整相邻图像两两执行校准（ 即下一个金字塔形的层次结构，与最接近的焦点值第一）使用Turboreg 18，一个ImageJ的插件。 </li><li>在每个倾斜角度一致验证通过焦点的图像的对准。堆栈对齐通过焦点影像以改善目视检查。 注:只有该区域是焦点，而应该通过堆栈浏览移动。准确的定位是最重要的事情，因为下面的步骤包括的是将重点从不同的图像信息的恢复。 </li><li>合并使用字段19，ImageJ的插件扩展深度是焦点中的信息;这将最终生成的图像的堆叠的单个图像。数设置可以深度的场的恢复过程中被使用，但使用最高设置保存的图像信息。 注意:倾斜系列现由每倾斜角的单个图像的，从通焦图像中回收含在焦信息的每个图像。 </li><li>处理数据。 注意:用于对齐和重建标准工具可以使用，因为数据现在已经被减少到一个标准的倾斜系列。在这个协议中，TomoJ是我们ED数据比对和数据重建20,21。可以在原始出版物22中找到有关如何使用TomoJ更多细节。 </li><li>执行倾斜系列精细对准。 注意:一个好的策略是用黄金珠基准标记准确地跟踪图像之间变化。也可以使用局部极小如果金珠不能被添加到样品中。 </li><li>执行对齐数据的三维重建;几种算法可供在实空间或傅立叶空间中，每个与优点和局限性进行重建。 </li><li>如果倾斜系列的最后对准差，通过优化的初始步骤做一些改进。重申对准步骤，如果最终的三维重建显示模糊或变形;在实践中，收集的焦平面的数量越多，对比度和三维重建的细节就越好。 注:硒veral软件程序可以用来处理通过焦点倾斜系列。因为然而，在该协议中使用的工具被选择，根据我们的经验，他们表现最好。 Turboreg 18在调整图像时的焦点梯度存在方面表现较好。更多信息可在专用网站23上找到。现场插件19的扩展深度收回来自不同的图像对焦信息方面表现非常出色，而其他工具造成可见像素修改。更多的信息，尤其是关于剧本创作，可以在这个专用网站24上找到。 </li></ol>

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E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans Image Process. 7 (1), 27-41 (1998).</li><li>Forster, B., Van De Ville, D., Berent, J., Sage, D., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+wavelets+for+extended+depth-of-field:+a+new+method+for+the+fusion+of+multichannel+microscopy+images.">Complex wavelets for extended depth-of-field: a new method for the fusion of multichannel microscopy images.</a> Microsc Res Tech. 65 (1-2), 33-42 (2004).</li><li>Messaoudii, C., Boudier, T., Sanchez Sorzano, ., O, C., Marco, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TomoJ:+tomography+software+for+three-dimensional+reconstruction+in+transmission+electron+microscopy.">TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy.</a> BMC Bioinformatics. 8, 288 (2007).</li><li>Sorzano, C. O., 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=Marker-free+image+registration+of+electron+tomography+tilt-series.">Marker-free image registration of electron tomography tilt-series.</a> BMC Bioinformatics. 10, 124 (2009).</li><li>Kohl, L., Bastin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+flagellum+of+trypanosomes.">The flagellum of trypanosomes.</a> Int Rev Cytol. 244, 227-285 (2005).</li><li>Luft, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+in+epoxy+resin+embedding+methods.">Improvements in epoxy resin embedding methods.</a> J Biophys Biochem Cytol. 9, 409-414 (1961).</li><li>Mielanczyk, L., Matysiak, N., Michalski, M., Buldak, R., Wojnicz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closer+to+the+native+state.+Critical+evaluation+of+cryo-techniques+for+Transmission+Electron+Microscopy:+preparation+of+biological+samples.">Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples.</a> Folia Histochem Cytobiol. 52 (1), 1-17 (2014).</li><li>Hovden, R., Xin, H. L., Muller, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+depth+of+field+for+high-resolution+scanning+transmission+electron+microscopy.">Extended depth of field for high-resolution scanning transmission electron microscopy.</a> Microsc Microanal. 17 (1), 75-80 (2011).</li><li>Dahmen, T., 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=The+Ettention+software+package.">The Ettention software package.</a> Ultramicroscopy. 161, 110-118 (2016).</li><li>Murata, 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=Whole-cell+imaging+of+the+budding+yeast+Saccharomyces+cerevisiae+by+high-voltage+scanning+transmission+electron+tomography.">Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography.</a> Ultramicroscopy. 146, 39-45 (2014).</li><li>Kizilyaprak, C., Daraspe, J., Humbel, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+ion+beam+scanning+electron+microscopy+in+biology.">Focused ion beam scanning electron microscopy in biology.</a> J Microsc. 254 (3), 109-114 (2014).</li><li>Kubota, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+electron+microscopy+for+serial+image+acquisition+of+neuronal+profiles.">New developments in electron microscopy for serial image acquisition of neuronal profiles.</a> Microscopy (Oxf). 64 (1), 27-36 (2015).</li><li>Levin, B. 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=Nanomaterial+datasets+to+advance+tomography+in+scanning+transmission+electron+microscopy.">Nanomaterial datasets to advance tomography in scanning transmission electron microscopy.</a> Sci Data. 3, 160041 (2016).</li></ol>

We have no conflict of interest to disclose.

在这篇文章中，我们提出了一个常规的样品制备协议与一步一步的指导使用STEM通过焦点断层扫描厚的生物样品进行三维分析一起。生物标本的树脂嵌入已使用了几十年26，和适于不同种类的样品替代协议可以在整个文献27中找到。与此相反，成像厚的样品在STEM使用通过焦方法是一种新的任务，并当它变得更普遍的方法将最有可能得到改善。 <p class="jove_content"...

自从70年代初，断层扫描透射电子显微镜（TEM）已被广泛地用于生物标本1，2，3的结构表征方法。透射电子断层扫描的吸引力是，它可用于在纳米尺度，研究了广泛的生物结构的，从细胞的结构和细胞器的大分子复合物和蛋白质的结构的超微结构。然而，透射电子断层扫描不能用来研究非常厚的样品（大于0.5微米）。的确，厚的样品产生太多的散射电子，产生低信噪比（SNR）的图像。另外，断层摄影涉及倾斜标本的图像的集合，将样品与倾斜角度增加的表观厚度。即使非弹性散射可以被过滤出用能过滤器，需要的高信噪比图像电子的临界量在TEM是勉强达到。因此，厚的生物标本只用了4切片研究。 有些样品不能被切片:它们被切割时一些可能会降低，和其他需要的全部进行研究，以了解它们的复杂性。另一种方法是在扫描模式5，6，7，8使用的TEM。在扫描透射电子显微镜（STEM），电子的光路是与在常规的TEM不同。流过试样而不散射电子可以与亮场（BF）检测器9的光轴被收集，而那些弹性散射可以在从与暗场（DF）检测器的光轴以一定角度被收集。干的另一优点是，聚焦的电子束在所述样品的表面扫描，从而使该像素由像素集合的图像。即使通过样品10，而电子束变宽，这种特殊的收集方案是非弹性散射电子的比常规的TEM不太敏感。此外，不存在后透镜试样在STEM，从而避免可能发生在TEM色差。相机长度可以调整，以使高炉检测器主要检测未散射电子。使用DF探测器研究厚的样品不建议，因为多次散射，从而产生不准确的图像。取而代之的是，高炉检测器可以用来11。而干可以产生高信噪比的图像，其具有因为相比TEM相对高的电子束会聚的相对低的深度的领域中，降低，可以从厚要回收的的深入信息量标本。在像差校正STEM显微镜，其中该会聚角可高达30兆拉德的情况下，使得即在焦点中的信息，从只有几纳米的焦平面起源的深度的领域可以是足够低。设置在并行模式下的电子束增强了深度的领域的电子束的决议12的损害。然而，这种设置并不总是可能的。 每当有必要使用一个会聚电子束，人们必须使用学习厚样品时，可增强深度的领域的电子束的技术。最近的研究已经报道了采集在不同焦平面多个图像的整个样品回收的在焦信息13，14的最大量。两项研究描述不同的方式来处理来自不同的焦平面的信息。 Hovden 等。结合在傅立叶空间中被收集在不同的焦平面的图像，并且直接从3D逆傅立叶获得的最终重建变换13。与此相反，达门等。开发出会聚光束重建引擎在现实空间重构来自各种焦平面14中的3D体积。我们的实验室还开发了厚成像生物标本的方法。我们的策略是从在上述两种方法的不同，我们合并是在聚焦在各焦平面中的信息，并使用平行光束投影15重构了最终的3D体积在现实空间。我们的目的是开发，可以很容易在任何电子显微镜实验室中进行的方法。为此，我们的目的是收集焦点图像的时间也是有限的，与常规断层实验的时间框架。此外，我们提出的方法C乌尔德适于与不同类型的对齐和重建软件的使用。 在本刊从2015年15的背景下，我们想可视化和表征景深-的恢复，所以我们用25毫弧度大收敛半的角度。在这里，我们提出了一个一步一步的协议根据我们的实验室在2015年15开发的方法在STEM通过焦成像表现，以及我们提出从2015年数据是如何处理。这种方法恢复整个厚（750 nm）的生物样品中，重点从几个焦点平面信息，使高品质的3D重建。如果相关的，在这种方法与其他团体使用的方法的差异还提出。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy resin</td>
			<td>EMS</td>
			<td>14120</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Add paraformaldehyde powder to PBS heated at approximately 60 &#176;C. 
			Increase pH by adding 1 N NaOH until no PFA powder is visible.</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxyde</td>
			<td>EMS</td>
			<td>19150</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>73943</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin Capsule</td>
			<td>EMS</td>
			<td>70110</td>
			<td></td>
		</tr>
		<tr>
			<td>Triming and Histo knives</td>
			<td>LFG Distribution</td>
			<td>Diatome diamond knives</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy copper grid</td>
			<td>LFG Distribution</td>
			<td>G200-Cu</td>
			<td></td>
		</tr>
		<tr>
			<td>Grid Coating Pen</td>
			<td>LFG Distribution</td>
			<td>70624</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen Holder</td>
			<td>JEOL</td>
			<td>EM-21311 HTR</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscope</td>
			<td>JEOL</td>
			<td>JEM-2200FS</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and observation of thick biological samples by scanning transmission electron tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

在我们的研究中，电子被加速到在所述场发射枪透射电镜200千伏。图像用一个20微秒的停留时间收集在STEM BF模式。关于通过焦点倾斜系列的设计中，我们发现的150焦点间隔处产生令人满意的结果的750纳米厚的生物，即使样品虽然电子束的深度的领域只是超微结构的研究3纳米，因为我们使用了25毫弧度非常大的收敛半的角度。 ?...

Watch this Scientific Journal Video about Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography at JoVE.com

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

准备厚生物样品的观察扫描透射电子断层扫描

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

preparation-observation-thick-biological-samples-scanning

Research

JoVE Journal

Biology

9.2K 次观看.  Campus Universitaire d'Orsay. This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

视频: Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

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 ...

科研

生物学

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

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

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (&#62;100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.

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 ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

扫描电子显微镜（SEM）协议有问题的工厂，卵菌和真菌样本

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

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 ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

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 ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

阵列断层扫描工作流程，用于使用扫描电子显微镜有针对性地采集体积信息

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...

准备厚生物样品的观察扫描透射电子断层扫描

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ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

平山准备观察斑马鱼胚胎标本染色，并通过分析全山

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

扫描电子显微镜（SEM）协议有问题的工厂，卵菌和真菌样本

透射电镜下体的样品制备与成像

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

透射电镜的小型化样品制备方法

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

阵列断层扫描工作流程，用于使用扫描电子显微镜有针对性地采集体积信息

生物组织样本的串行块面扫描电子显微镜（SBF-SEM）

准备厚生物样品的观察扫描透射电子断层扫描

摘要

探索更多视频

此视频中的章节

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

平山准备观察斑马鱼胚胎标本染色，并通过分析全山

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

扫描电子显微镜（SEM）协议有问题的工厂，卵菌和真菌样本

透射电镜下体的样品制备与成像

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

透射电镜的小型化样品制备方法

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

阵列断层扫描工作流程，用于使用扫描电子显微镜有针对性地采集体积信息

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

生物组织样本的串行块面扫描电子显微镜（SBF-SEM）