Name: obtaining 3d chemical maps by energy filtered transmission electron microscopy tomography
Uploaded: 2018-06-09T12:30:00.000+00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography at JoVE.com

我们感谢法国高等教育和研究部、各公约 Industrielles 研究所 (CIFRE) 和为他们提供财政支助的新生力量。

1. 样品准备
<ol><li>在砂浆中粉碎样品, 并将其分散在酒精或蒸馏水中;将样品滴放在显微镜网格上, 让它干。 注: 如二氧化硅氧化铝或二氧化钛氧化铝可以是粉末或挤压材料, 并可通过超声波在溶液中粉碎和分散。一般情况下, 对于 ET 分析来说, 网格中的样本浓度很低, 避免了在大角度倾斜网格时的样本叠加和阴影。推荐了支持孔碳或莱西碳薄膜的200网格显微网格。</li>
	<li>使用吸管, 在样品上放入含有基准标记的胶体溶液滴。吸收任何多余的溶液, 让它干燥。 注意: 基准标记是经过良好校准的 Au 纳米粒子悬浮在溶液中。在添加示例之前, 基准标记也可以分散在网格上。举例来说, 如果样品是由与基准标记相似大小的纳米粒子制成的, 那么将基准标记放在网格上, 以便在数据分割和量化过程中将它们分离得很好。基准标记是对齐倾斜图像时使用的位置引用。</li>
</ol>2. 过滤式倾斜系列图像的记录
<ol><li>用电子显微镜, 在显微镜网格的中心找到一个孤立的样本。 注: 在电子显微镜下, X 轴沿样品架, Y 轴垂直于样品架, Z 轴沿电子束。为了能够倾斜的最大倾角的样品, 分析的样本, 尽可能接近 X 轴。</li>
	<li>一旦样品定位良好, 检查样品的化学成分。使用能量色散 X 射线光谱分析 (EDS) 或电子能量损耗谱 (鳗鱼) 进行化学分析, 将光束聚焦在所选样品上, 并记录光谱。如果该示例包含感兴趣的化学元素, 请从其上移开, 然后在附近的代表示例上运行下一次测试。</li>
	<li>检查电子束强度的样本, 过滤图像的能量窗口的宽度, 以及每个过滤图像的展示时间。找到最佳折衷的样本之间的光束损伤和化学信号记录在化学预测12,13,17。 注: 要记录过滤图像中的最大化学信号, 请使用最大光束强度。但是, 在进行任何分析之前, 必须进行辐照测试, 以检查电子束对样品的任何变化。要做到这一点, 计算的电子剂量在记录的倾斜系列。此外, 可以通过比较图像之前和图像后的实验来进行轻松测试。</li>
	<li>使用记录软件的 EFTEM 模式, 使用三窗口方法计算2D 化学图, 并检查是否记录了足够的化学信号。 注: 该软件记录三过滤图像;前两个用于估计第三个图像的背景。<ol>
<li>在软件中, 选择所研究的化学元素及其电离边缘。设置能量窗口和曝光时间的宽度。记录图像, 然后用幂定律计算化学图以提取背景。在具有 512 x 512 像素的32位环境中, 最小信号大约是在化学图像中记录的每个像素的300-400 计数。</li>
	</ol>
</li>
	<li>在 eucentric 高度设置样品, 并检查最小倾斜角度,即-70°或更小, 以及最大倾角,即+ 70°或更多。</li>
	<li>将样本分析回视图并记录图像 (这将是获取前的图像)。然后使用适当的软件记录倾斜的过滤图像系列。 注: 专用 EFTEM 层析成像插件可以同时录制多个倾斜系列。这意味着, 在每个倾斜角度, 可以记录几个连续的图像。第一个图像可以是以零损耗为中心的过滤图像, 该图像是典型的亮场图像。前边缘图像和后边缘图像的第一个化学元素, 其次是前边缘图像和后边缘图像的第二个化学元素。化学元素的继承是由它们所选择的电离边缘给出的。<ol>
<li>在记录 EFTEM 倾斜系列软件中, 选择每一个能量窗口的宽度及其曝光时间, 然后是最大和最小倾斜角和倾斜角步长。要在倾斜系列中的图像数量和样本到电子束的总曝光时间之间进行折衷, 请使用4°的倾斜步骤,即在71°之间的每个倾斜系列中的51幅图像;但是, 如果样品不在光束下降解, 则可以选择较小的倾斜步骤。</li>
		<li>对于每个化学元素, 记录三倾斜系列过滤图像, 使用三窗口方法计算化学投影。在记录过滤图像时, 要量化样品在每个倾斜角度的自然漂移 (样品可以保持在一定的倾斜角度超过1分钟), 第一个图像可以是在零损耗信号上的过滤图像, 以便最后记录的图像将是一个 unfil所有的电子在所有能量中形成的掌握图像。这两种图像可用于计算样品的厚度图。因此, 为观察两个元素在每个倾斜角度的化学分布, 记录7滤除的图像 (1 零损耗, 3 为第一化学元素, 3 为第二化学元素) 和1未过滤的图像 (总共, 8 个倾斜系列记录)。</li>
	</ol>
</li>
</ol>3. 倾斜系列的对准和重建
<ol><li>对齐每个倾斜角度对应于每个化学元素的三过滤图像, 并使用专用的 EFTEMTJ15、18插件 ImageJ 来计算化学图。在 ImageJ 软件中, 使用路径文件 |打开并选择对应于滤镜图像倾斜系列的文件。打开所有三过滤倾斜系列: 两个前边缘和一个后缘。<ol>
<li>打开专用的 EFTEMTJ 插件。点击+ |图像或堆栈, 然后选择已打开的倾斜系列。</li>
		<li>在出现的表中, 填充每个倾斜系列的能量移位,即记录每个倾斜系列的能量。此外, 填充狭缝宽度,即, 能量窗口。填充每个已过滤图像的"曝光时间" 。检查所有三倾斜系列映射。单击 "下一步"。</li>
		<li>已选择第一个图像作为参考图像, 然后单击 "应用"。单击下一页: 显示建议对齐的预览图像。直观地验证在同一倾角下记录的3幅图像是否叠加 (在图像之间不应有移位)。 注意: 此协议是在 EFTEMTJ 插件的版本0.9 上执行的。此时, 在同一倾斜角度记录的过滤图像将对齐。</li>
		<li>选择 EFTEMTJ 窗口, 图像对应的背景和化学边缘。选择信号抽取的模型作为电源, 然后单击 "创建地图"。在 ImageJ 上, 选择文件 |保存并找到保存此最后一个文件的路径。 注: 化学图的倾斜系列得到。有关如何使用插件的更多信息, 请在线提供。</li>
		<li>对所有化学倾斜系列重复步骤3.1。</li>
	</ol>
</li>
	<li>使用2009年发布的 Imod 软件19版本对齐倾斜系列, 对齐零损耗倾斜系列。该软件允许在倾斜系列上应用计算的对齐到另一个倾斜系列。 注意: 对齐软件在包含每个图像应用的所有位移的磁盘文件上写入。在其他地方审查使用 Imod20的对齐过程, 不属于本文的范围。</li>
	<li>使用为零损耗倾斜系列计算的对齐方式, 并将其应用于先前计算的化学倾斜系列。 注意: 在本软件版本中, 通过保留文件的名称并应用以前计算过的位移, 可以通过化学倾斜系列文件更改零损耗倾斜系列文件。否则, 对于软件, 该文件将具有相同的名称, 相同数量的图像相同的大小, 但它不会包含零损失图像, 但化学图像。</li>
	<li>通过交叉关联第一个记录的图像来量化漂移,即, 以零损耗为中心, 最后一个 (未过滤的图像)。该示例可以在每个倾斜角度花几秒钟时间, 同时记录所有筛选的图像。在此期间, 样品自然地漂移少量。<ol>
<li>在 ImageJ 软件上, 单击 "文件" | "打开并选择零损耗对准倾斜系列, 然后打开化学地图对齐倾斜系列。</li>
		<li>点击编辑 |颜色 |合并通道。选择对应于红色的零损耗的文件, 第一个绿色元素和蓝色的第二个化学元素, 按顺序排列。取消选中 "创建复合" 并检查 "保留源图像"。在每个倾斜角度为所有记录的图像创建一个堆栈。</li>
		<li>点击插件 |对齐 RGB 计划21。红色是参考图像。选择绿色并使用箭头, 将其与红色重叠。点击下一步, 并对所有角度重复。</li>
		<li>点击编辑 |颜色 |拆分通道和 RGB 堆栈将在三栈中拆分: 红色对应零损耗, 与绿色和蓝色对应的化学贴图与漂移校正。点击文件 |保存以保存倾斜系列。</li>
	</ol>
</li>
	<li>点击插件 |Tomoj22,23选择加载角度表单文件。因为所有倾斜系列都已对齐, 所以直接导航到重建。使用诸如艺术、SIRT、OS 艺术等重建算法计算零损耗量以及化学量. 注: 建议使用迭代算法进行化学量的重建。通过使用该软件, 可以使用 GPU 重建卷。</li>
	<li>计算所有卷后, 使用 "合并通道" 选项对获得的卷应用不同的颜色, 并在单个卷中重叠, 以获得3D 化学图。</li>
</ol>4. 3D 建模与量化
<ol><li>通过选择相应的灰度水平, Binarize 重建的 ZL 体积, 这将是以白色 (8 位, 强度为 255) 和黑色 (8 位, 强度为 0) 的体积。在 ImageJ 中, 使用 "选择阈值" 选项。选择与样本相对应的所有像素 (在 BF 图像中, 较暗的像素对应于样品质量), 并制作一个样品为白色且真空为黑色的体积。 注: 零损耗量提供分析样品的形态学信息,即样品的形状和尺寸。</li>
	<li>将零损耗的二进制体积除以 255, 并获得一个体积, 其中样本内的强度为 1, 其他地方为0。这是规范化的卷。</li>
	<li>每一个计算的化学卷 (步骤 4.1) 乘以规范化的体积, 以获取样本内的强度对应于化学信息的体积, 而这些强度在别处是0。 注: 化学信息来源于样品, 因此, 所有工件都被排除在外。</li>
	<li>在 ImageJ 中, 计算化学量的直方图, 并在制表软件中导入直方图的值。<ol>
<li>在制表软件中, 删除与强度盗窃计数为0的行 (这条线对应于真空)。</li>
		<li>在新列中, 计算卷中每个强度的比例。将每个强度的计数除以所有计数的总和, 并将其乘以100。</li>
		<li>在新列中, 通过增量添加当前计数的比例来计算与卷合计强度对应的比例, 先前计算的比例为以前的百分比。 注: 在化学量中, 高强度对应于化学信息。然而, 强度低, 噪音在音量高。阈值是通过选择最高强度来创建的。</li>
		<li>了解从鳗鱼谱计算的样品中化学元素的相对浓度, 选择盗窃强度。从255开始 (如果存在) 和降低强度对应的化学元素浓度。</li>
		<li>为了找到与化学浓度相对应的最低强度, 导航到在总容积中计算相应强度比例的柱, 100% 通常对应于255的强度。从 100%, 提取出计算的相对比例的化学元素 (从鳗鱼谱): 结果将对应于最低强度的门槛。这样, 化学二值化量得到, 素对应的化学元素与强度为 255, 其余的是0。重复第二个元素的过程并获得两个化学二值化卷。</li>
	</ol>
</li>
	<li>使用 "合并通道" 选项重叠二值化化学卷, 并为每个元素卷分配不同的颜色以制作 RGB 卷。 注: 通过重叠两个二值化化学卷, 叠加的素, 属于化学元素 (公社素) 和素不属于任何化学元素 (自由素) 突出。例如, 二值化的红色和绿色的化学卷创造公社素是黄色的。</li>
	<li>将 RGB 音量转换为8位音量;颜色将有不同的灰色强度。使用阈值选项, 选择属于两种化学物种的素 (RGB 卷中的黄色)。然后, 使用相同的阈值选项, 选择不属于任何化学元素的素 (它们的强度比前面选择的素低)。不要选择强度为0的真空度。</li>
	<li>规范公社素的数量和自由素的数量。将自由素的体积乘以化学量, 然后减去其他化学量。 注意: 这计算了每一个具有最高强度的体素中的化学元素。</li>
	<li>添加所属素的卷和不属于这些素中具有最高强度的化学元素体积的素。 注意: 以这种方式, 每个公社体素或自由素被区分并分配给具有最高强度的化学元素的体积。这可以由 "图像计算器" 选项执行:</li>
</ol><img alt="Equation 1" src="/files/ftp_upload/56671/56671eq1v3.jpg">
<ol start="9"><li>要量化示例的素数, 请将分段的卷导入到专门的曲面渲染软件中, 如3D 切片器。然后, 将3中的体素体积乘以 3D, 以获得样品的体积。</li>
	<li>使用 "查找边缘" 选项, 量化生成样本表面的素, 并以 nm2中的素体面积相乘, 以获得样品的表面。</li>
	<li>通过样品的质量除以试样的表面来计算特定表面积。 注: 样品的质量可以用样品的理论密度来估计。一般情况下, 由 ET 计算出的比表面积小于2吸附解吸的专用方法计算的比表面积小10。</li>
	<li>要计算孔径分布, 使用零损耗的二值化体积 (高炉容积)。BF 重建的二进制体积使用3D 工具箱/形态学扩张3D 插件扩张, 直到所有毛孔被覆盖, 然后使用3D 工具箱/形态学腐蚀3D 多次扩张而被侵蚀。然后, 得到的体积乘以二值化高炉体积的倒体积, 从而导致孔隙分布体积, 可以可视化使用专用的表面渲染软件。</li>
</ol>

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Microanal. 18 (05), 1118-1128 (2012).</li><li>Roiban, L., Sorbier, L., Hirlimann, C., Ersen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3+D+Chemical+Distribution+of+Titania-Alumina+Catalyst+Supports+Prepared+by+the+Swing-pH+Method.">3 D Chemical Distribution of Titania-Alumina Catalyst Supports Prepared by the Swing-pH Method.</a> ChemCatChem. 8 (9), 1651-1657 (2016).</li><li>Egerton, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electron Energy-Loss Spectroscopy in the Electron Microscope. , (2011).</li><li>Messaoudi, C., Aschman, N., Cunha, M., Oikawa, T., Sorzano, C. O. S., 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=Three-Dimensional+Chemical+Mapping+by+EFTEM-TomoJ+Including+Improvement+of+SNR+by+PCA+and+ART+Reconstruction+of+Volume+by+Noise+Suppression.">Three-Dimensional Chemical Mapping by EFTEM-TomoJ Including Improvement of SNR by PCA and ART Reconstruction of Volume by Noise Suppression.</a> Microscopy and Microanalysis. 19 (6), 1669-1677 (2013).</li><li>Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., Ferrin, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UCSF+Chimera-A+visualization+system+for+exploratory+research+and+analysis.">UCSF Chimera-A visualization system for exploratory research and analysis.</a> Journal of Computational Chemistry. 25 (13), (2004).</li><li>Roiban, L., Ersen, O., Hirlimann, C., Drillon, M., Chaumonnot, A., Lemaitre, L., Gay, A. S., Sorbier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Analytical+Surface+Quantification+of+Heterogeneous+Silica-Alumina+Catalyst+Supports.">Three-Dimensional Analytical Surface Quantification of Heterogeneous Silica-Alumina Catalyst Supports.</a> ChemCatChem. 9 (18), 3503-3512 (2017).</li><li>Kremer, J. R., Mastronarde, D. N., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+visualization+of+three-dimensional+image+data+using+IMOD.">Computer visualization of three-dimensional image data using IMOD.</a> J Struct Biol. 116 (1), 71-76 (1996).</li><li>Align RGB planes. ImageJ Available from: <a target="_blank" href="https://ImageJ.net/Align_RGB_planes">https://ImageJ.net/Align_RGB_planes</a> (2018)</li><li>MessaoudiI, C., Boudier, T., Sorzano, 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 Bioinf. 8 (1), 288 (2007).</li><li>Saxton, W. O., Baumeister, W., Hahn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+imperfect+two-dimensional+crystals.">Three-dimensional reconstruction of imperfect two-dimensional crystals.</a> Ultramicroscopy. 13 (1-2), 57-70 (1984).</li></ol>

作者没有什么可透露的。

本文的目的是描述如何利用 EFTEM 层析技术获得3D 化学地图。这个协议是完全原创的, 是由作者开发的。
此处描述的 EFTEM 层析成像有以下几个缺点: (i) 仅能分析电子束电阻的样品, 因为获得过滤图像所需的时间很长。(二) EFTEM 层析成像对衍射对比度敏感。(三) 许多对齐方式是手动执行的。为了获得3D 化学图, 零损耗量和化学量需要在一个单一的坐标系中。这就要求所有的倾斜系...

功能材料的性能取决于其3D 参数。为了充分理解其性质, 增强其功能, 对3D 的形态和化学分布进行分析是十分重要的。电子层析成像1 (ET) 是最好的技术提供这一信息在纳米级2,3。它包括旋转样本在一个大的角度范围和记录一个图像在每个角度的步骤。利用所得到的倾斜级数, 采用基于氡变换4、5的数学算法重建试样体积。选择卷中的灰度级别有助于在3D 中对样本进行建模, 并量化3D 参数, 如粒子定位6和尺寸分布7、孔位置和尺寸分布8等.
一般情况下, ET 是通过电子显微镜进行的, 将样品倾斜到最大可能的角度, 最好是在任一方向70°。在每个倾斜角度, 样本的投影被记录成图像倾斜系列。该倾斜系列是对齐的, 用于重建样本的体积, 将被分割和量化。由于样品不能从90°旋转到 + 90°, 因此由于盲记录角, 重建的体积在正交轴9上有一个各向异性的分辨率。
ET 可以在不同的成像模式下进行。采用光亮场 tem 模式 (BF tem) 研究了非晶态材料、生物样品、聚合物或具有复杂形状的催化剂支持。图像分析是基于对元件10密度的灰度级的区分 (一个稠密分量将比打火机更暗,即较轻的分量)。采用扫描 TEM 模式 (HAADF) 的高角度环形暗场对晶体样品进行分析。该信号提供化学信息作为原子数的函数;样品的一个重的组分将显示更加明亮的一个打火机9。其他模式, 如能量色散 x 射线光谱学 (EDX), 收集 x 射线发射的材料11, 和能量过滤成像模式 (EFTEM)12,13, 也可以评估3D 化学分布在样本中。
在 EFTEM 成像中, 2D 化学图可以用电子能量谱仪记录 TEM。光谱仪通过将电子作为能量的函数分散而充当磁性棱镜。由电子产生的图像取决于与特定原子相互作用而失去的能量。如果在不同倾角下计算出相同的2D 化学图, 则得到了倾斜系列的化学投影, 可用于重建3D 化学量。
并非所有的材料都可以通过 EFTEM 层析成像来分析。该技术是保留的样品与薄弱或无序的材料。然而, 它可以用于分析在使用其他成像技术时很难区分的光元素。此外, 为了获得可靠的2D 化学图, 材料的厚度需要小于平均自由路径的电子通过材料14。在这种情况下, 单电子与单个原子相互作用的概率最大。用两种方法计算2D 化学图。第一个, 最常用的是 "三窗法", 其中两个过滤的能量窗口被记录在分析的元素的电离边缘之前, 第三个在电离边缘13之后。前两个图像用于估计背景, 这是推断使用幂定律在第三个窗口的位置, 并从中减去。所得到的图像是样品体积中分析化学元素3D 分布的投影。第二种方法称为 "跳跃比";它只使用两个能量过滤的图像, 一个之前和一个后电离边缘。这种方法是定性的, 因为最终的图像只通过执行两个图像之间的比例, 并没有解释背景能量变化。
通过 EFTEM 与 ET 的结合, 可以得到过滤能量的解析层析成像。EFTEM 层析成像和原子探针层析成像 (APT) 是互补技术。与 APT 相比, EFTEM 层析成像是一种非破坏性的特征分析, 不需要复杂的样品制备。它可用于对独特的纳米颗粒进行各种表征。EFTEM 层析成像可以分析绝缘材料, 而易需要在最起码的激光协助下测量它们。APT 在原子尺度上运行, 而 EFTEM 层析成像则能以较低的分辨率进行充分的执行。EFTEM 层析成像仅适用于在实验期间抵抗光束降解的样品。为了在所有倾斜的角度记录所有的过滤图像, 样品可以暴露在电子束上长达2小时。此外, 为了在2D 地图上记录一个最大的化学信号, 在高光束强度下, 可能需要更长的博览会工期。在这种情况下, 光束敏感样品遭受剧烈的形态和化学变化。因此, 必须在实验前建立对电子束样品灵敏度的精确测量。此外, EFTEM 层析成像是根据需要记录尽可能多的断层的结果, 以确定样本中化学元素的空间位置和性质.然而, EFTEM 层析成像可以为样品的3D 化学分布提供重要的信息, 例如催化剂支持, 以便为其催化应用建模提出新的见解。
今天可以使用专门的软件来选择能量区间, 记录过滤的能量窗口图像, 并在不同的倾斜角度计算化学图。它们允许在 EFTEM 模式下倾斜采样、跟踪、聚焦和记录过滤图像。2D 化学图可以计算, 然后倾斜系列可以被排列, 化学容量用迭代算法计算, 并且最后系列可以被分割和定量15,16。

<table><tbody><tr><td>JEOL 2100f</td><td>JEOL</td><td></td><td>Electron microscope</td></tr><tr><td>Tridiem Gatan Imaging Filter (GIF)</td><td>Gatan</td><td></td><td>Post colum energy filter</td></tr><tr><td>Digital micrograph</td><td>Gatan</td><td></td><td>Software</td></tr><tr><td>Gatan EFTEM tomography plugin</td><td>Gatan</td><td></td><td>Dedicated software to record filtered tilt series for EFTEM tomograohy</td></tr><tr><td>Tomoj</td><td>Imagej plugin http://www.cmib.fr/en/download/softwares/</td><td></td><td>Free software developed by Currie Institute in Paris, France for electron tomography</td></tr><tr><td>EFTEM-Tomoj</td><td>Imagej plugin http://www.cmib.fr/en/download/softwares/</td><td></td><td>Free software developed by Currie Institute in Paris, France , for EFTEM imaging</td></tr><tr><td>Imod</td><td>http://bio3d.colorado.edu/imod/</td><td></td><td>Free software developed by University of Colorado, USA for electron tomography</td></tr><tr><td>Imagej</td><td>https://imagej.nih.gov/ij/</td><td></td><td>Free software developed by National Institute of Mental Health, Bethesda, Maryland, USA for images treatment</td></tr><tr><td>Merge channels</td><td>https://imagej.net/Color_Image_Processing</td><td></td><td>Fonction in Imagej allowing to give different colors to volumes while they are overlapped</td></tr><tr><td>3D Slicer</td><td>https://www.slicer.org/</td><td></td><td>Free software developed by a large consortium lead by Ron Kikinis , Harvard Medical School, Boston, MA, SUA</td></tr><tr><td>Chimera</td><td>https://www.cgl.ucsf.edu/chimera/</td><td></td><td>Free software developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco,for data segmentation, cuatification and visualisation of 3D models</td></tr><tr><td>silica alumina support of catalyst</td><td>IFPEN</td><td></td><td>sample prepared for eleboration of this protocol</td></tr><tr><td>titania alumina support of catalyst</td><td>IFPEN</td><td></td><td>sample prepared for eleboration of this protocol</td></tr><tr><td>alcohol</td><td></td><td></td><td></td></tr><tr><td>water</td><td></td><td></td><td></td></tr><tr><td>Au nanoparticles of 5 nm</td><td>BBI Solutions</td><td></td><td></td></tr><tr><td>Holey carbn film 200 mesh microscopy grid</td><td>Agar</td><td></td><td></td></tr><tr><td>EDX sepctrometer</td><td>Oxford Instruments</td><td></td><td></td></tr></tbody></table>

obtaining 3d chemical maps by energy filtered transmission electron microscopy tomography

能量过滤透射电子显微成像 (EFTEM 层析成像) 可以提供三维 (3D) 的材料化学地图的纳米尺度。EFTEM 层析成像可以分离出很难用其他成像技术区分的化学元素。这里描述的实验协议说明了如何创建3D 化学图来理解材料的化学分布和形貌。给出了数据分割的样本准备步骤。该协议允许在纳米样品中对化学元素进行3D 分布分析。然而, 应该指出的是, 目前, 3D 化学地图只能为不敏感的样品生成, 因为过滤图像的记录需要长时间的曝光时间到一个强烈的电子束。应用该协议对两种不同的异构催化剂的组分的化学分布进行了定量分析。在第一次研究中, 分析了钛-氧化铝支架中铝和钛的化学分布。用摆动 pH 法制备样品。第二, 用溶胶-粉末和机械混合法制备了硅-氧化铝支架中铝和硅的化学分布。

本协议的应用示例见参考13。EFTEM 层析技术用于分析二氧化钛催化剂的支持。为了提高 MoS2纳米粒子活性相的催化活性, 在加氢脱硫 (HDS) 等应用中, 重要的是在支撑面上的二氧化钛具有优势, 并且与活性相接触。众所周知, 二氧化钛比氧化铝具有更小的比表面积。该研究的目的是建立氧化铝支持的二氧化钛 (因此, 创建一个增强的特定表面), 然后测试...

Watch this Scientific Journal Video about Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography at JoVE.com

Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography

通过能量过滤透射电镜成像获得3D 化学图

本文介绍了一种结合能量滤波成像和电子层析成像实现3D 化学图的协议。研究了由其它成像技术难以区分的元素所形成的两种催化剂载体的化学分布。每个应用包括映射重叠的化学元素-分别间隔-电离边缘。

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a ...

obtaining-3d-chemical-maps-energy-filtered-transmission-electron

Research

JoVE Journal

Chemistry

6.4K 次观看.  Université Claude Bernard Lyon 1. 本文介绍了一种结合能量滤波成像和电子层析成像实现3D 化学图的协议。研究了由其它成像技术难以区分的元素所形成的两种催化剂载体的化学分布。每个应用包括映射重叠的化学元素-分别间隔-电离边缘。

视频: Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography

Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial resolution, and is even capable of providing atomically resolved elemental maps. In this technique, a highly focused electron beam is incident upon a thin sample and the energy of emitted X-rays is measured in order to determine the atomic species of material within the beam path. This elementally sensitive spectroscopy technique can be extended to three dimensional tomographic imaging by acquiring multiple spectrum images with the sample tilted along an axis perpendicular to the electron beam direction.
Elemental distributions within single nanoparticles are often important for determining their optical, catalytic and magnetic properties. Techniques such as X-ray tomography and slice and view energy dispersive X-ray mapping in the scanning electron microscope provide elementally sensitive three dimensional imaging but are typically limited to spatial resolutions of &gt; 20 nm. Atom probe tomography provides near atomic resolution but preparing nanoparticle samples for atom probe analysis is often challenging. Thus, elementally sensitive techniques applied within the scanning transmission electron microscope are uniquely placed to study elemental distributions within nanoparticles of dimensions 10-100 nm.
Here, energy dispersive X-ray (EDX) spectroscopy within the STEM is applied to investigate the distribution of elements in single AgAu nanoparticles. The surface segregation of both Ag and Au, at different nanoparticle compositions, has been observed.

The use of energy dispersive X-ray tomography in the scanning transmission electron microscope to characterize elemental distributions within single nanoparticles in three dimensions is described.

能量色散X射线断层摄影术对个人纳米3D元素映射

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial ...

科研

工程学

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

生物工程

Picometer-Precision Atomic Position Tracking through Electron Microscopy

The modern aberration-corrected scanning transmission electron microscopes (AC-STEM) have successfully achieved direct visualization of atomic columns with sub-angstrom resolution. With this significant progress, advanced image quantification and analysis are still at the early stages. In this work, we present the complete pathway for the metrology of atomic resolution scanning transmission electron microscopy (STEM) images. This includes (1) tips for acquiring high-quality STEM images; (2) denoising and drift-correction for enhancing measurement accuracy; (3) obtaining initial atomic positions; (4) indexing the atoms based on unit cell vectors; (5) quantifying the atom column positions with either 2D-Gaussian single peak fitting or (6) multi-peak fitting routines for slightly overlapping atomic columns; (7) quantification of lattice distortion/strain within the crystal structures or at the defects/interfaces where the lattice periodicity is disrupted; and (8) some common methods to visualize and present the analysis.
Furthermore, a simple self-developed free MATLAB app (EASY-STEM) with a graphical user interface (GUI) will be presented. The GUI can assist in the analysis of STEM images without the need for writing dedicated analysis code or software. The advanced data analysis methods presented here can be applied for the local quantification of defect relaxations, local structural distortions, local phase transformations, and non-centrosymmetry in a wide range of materials.

This work presents a workflow for atomic position tracking in atomic resolution transmission electron microscopy imaging. This workflow is performed using an open-source Matlab app (EASY-STEM).

通过电子显微镜进行精确原子位置跟踪

The modern aberration-corrected scanning transmission electron microscopes (AC-STEM) have successfully achieved direct visualization of atomic columns ...

化学

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

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.

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 ...

Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms

Efficient methods for the extraction of features of interest remain one of the biggest challenges for the interpretation of cryo-electron tomograms. Various automated approaches have been proposed, many of which work well for high-contrast datasets where the features of interest can be easily detected and are clearly separated from one another. Our inner ear stereocilia cryo-electron tomographic datasets are characterized by a dense array of hexagonally packed actin filaments that are frequently cross-connected. These features make automated segmentation very challenging, further aggravated by the high-noise environment of cryo-electron tomograms and the high complexity of the densely packed features. Using prior knowledge about the actin bundle organization, we have placed layers of a highly simplified ball-and-stick actin model to first obtain a global fit to the density map, followed by regional and local adjustments of the model. We show that volumetric model building not only allows us to deal with the high complexity, but also provides precise measurements and statistics about the actin bundle. Volumetric models also serve as anchoring points for local segmentation, such as in the case of the actin-actin cross connectors. Volumetric model building, particularly when further augmented by computer-based automated fitting approaches, can be a powerful alternative when conventional automated segmentation approaches are not successful.

Here, we present a protocol to place simplified volumetric models into noisy, complex, tomographic 3D volumes. This allows the fast segmentation of actin filament densities, detection of systematic filament bending and of gaps in hair bundle filaments, as well as convenient quantification of volumetric model properties, such as distances.

Efficient methods for the extraction of features of interest remain one of the biggest challenges for the interpretation of cryo-electron tomograms. ...

神经科学

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

通过冷冻软X射线断层扫描对细胞进行3D制图描述

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ,&#160;by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

This protocol presents a technique for high-resolution mapping of replication sites in structurally preserved chromatin in situ that employs a combination of pre-embedding EdU-streptavidin-Nanogold labeling and ChromEMT.

通过电子断层扫描对超构造保存的染色质中的复制结构域进行成像

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

冷冻电子断层扫描远程数据收集和断层扫描平均

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryogenic electron microscopy (cryo-EM) relies on the imaging of biological or organic specimens embedded in their native aqueous medium; water is solidified into a glass (i.e., vitrified) without crystallization. The cryo-EM method is widely used to determine the structure of biological macromolecules recently at a near-atomic resolution. The approach has been extended to the study of organelles and cells using tomography, but the conventional mode of wide-field transmission EM imaging suffers a severe limitation in the specimen thickness. This has led to a practice of milling thin lamellae using a focused ion beam; the high resolution is obtained by subtomogram averaging from the reconstructions, but three-dimensional relations outside the remaining layer are lost. The thickness limitation can be circumvented by scanned probe imaging, similar to the scanning EM or the confocal laser scanning microscope. While scanning transmission electron microscopy (STEM) in materials science provides atomic resolution in single images, the sensitivity of cryogenic biological specimens to electron irradiation requires special considerations. This protocol presents a setup for cryo-tomography using STEM. The basic topical configuration of the microscope is described for both two- and three-condenser systems, while automation is provided by the non-commercial SerialEM software. Enhancements for batch acquisition and correlative alignment to previously-acquired fluorescence maps are also described. As an example, we show the reconstruction of a mitochondrion, pointing out the inner and outer membrane and calcium phosphate granules, as well as surrounding microtubules, actin filaments, and ribosomes. Cryo-STEM tomography excels in revealing the theater of organelles in the cytoplasm and, in some cases, even the nuclear periphery of adherent cells in culture.

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryo-STEM tomography provides a means to visualize organelles of intact cells without embedding, sectioning, or other invasive preparations. The 3D resolution obtained is currently in the range of a few nanometers, with a field of view of several micrometers and an accessible thickness in the order of 1 &#181;m.

通过能量过滤透射电镜成像获得3D 化学图

摘要

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能量色散X射线断层摄影术对个人纳米3D元素映射

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

通过电子显微镜进行精确原子位置跟踪

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

准备厚生物样品的观察扫描透射电子断层扫描

Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms

通过冷冻软X射线断层扫描对细胞进行3D制图描述

通过电子断层扫描对超构造保存的染色质中的复制结构域进行成像

冷冻电子断层扫描远程数据收集和断层扫描平均

通过冷冻STEM断层扫描原位可视化细胞器

通过能量过滤透射电镜成像获得3D 化学图

摘要

探索更多视频

此视频中的章节

能量色散X射线断层摄影术对个人纳米3D元素映射

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

通过电子显微镜进行精确原子位置跟踪

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

准备厚生物样品的观察扫描透射电子断层扫描

Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms

通过冷冻软X射线断层扫描对细胞进行3D制图描述

通过电子断层扫描对超构造保存的染色质中的复制结构域进行成像

冷冻电子断层扫描远程数据收集和断层扫描平均

通过冷冻STEM断层扫描原 位 可视化细胞器

通过冷冻STEM断层扫描原位可视化细胞器