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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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

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

Trace explosive vapors of TNT and RDX collected on sorbent-filled thermal desorption tubes were analyzed using a programmed temperature desorption system coupled to GC with an electron capture detector. The instrumental analysis is combined with direct liquid deposition method to reduce sample variability and account for instrumentation drift and losses.

定量检测痕量爆炸物蒸气通过程序升温脱附 - 气相色谱 - 电子捕获检测器

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive ...

科研

化学

1,3,5-Triphenylbenzene and Corannulene as Electron Receptors for Lithium Solvated Electron Solutions

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using two types of polyaromatic hydrocarbons (PAH), namely 1,3,5-triphenylbenzene and corannulene, as electron receptors. The solid PAHs were first dissolved in tetrahydrofuran (THF) to form a solution. Metallic lithium was then dissolved into these PAH/THF solutions to yield either blue or greenish blue solutions, colors which are indicative of the presence of solvated electrons. Conductivity measurements at ambient temperature carried out on 1,3,5-triphenylbenzene-based LiSES, denoted by LixTPB(THF)24.7 (x = 1, 2, 3, 4), showed an increase of conductivity with increase of Li:PAH ratio from x = 1 to 2. However, the conductivity gradually decreased upon further increasing the ratio. Indeed the conductivity of LixTPB(THF)24.7 for x = 4 is even lower than for x = 1. Such behavior is similar to that of the previously reported LiSES prepared from biphenyl and naphthalene. Conductivity versus temperature measurements on corannulene-based LiSES, denoted by LixCor(THF)247 (x = 1, 2, 3, 4, 5), showed linear relationships with negative slopes, indicating a metallic behavior similar to biphenyl and naphthalene-based LiSES.

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using 1,3,5-triphenylbenzene (TPB) and corannulene as electron receptors.

1,3,5-三苯和碗烯作为电子受体锂溶剂化电子解决方案

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using two types of polyaromatic ...

A Simple, Low-cost, and Robust System to Measure the Volume of Hydrogen Evolved by Chemical Reactions with Aqueous Solutions

There is a growing research interest in the development of portable systems which can deliver hydrogen on-demand to proton exchange membrane (PEM) hydrogen fuel cells. Researchers seeking to develop such systems require a method of measuring the generated hydrogen. Herein, we describe a simple, low-cost, and robust method to measure the hydrogen generated from the reaction of solids with aqueous solutions. The reactions are conducted in a conventional one-necked round-bottomed flask placed in a temperature controlled water bath. The hydrogen generated from the reaction in the flask is channeled through tubing into a water-filled inverted measuring cylinder. The water displaced from the measuring cylinder by the incoming gas is diverted into a beaker on a balance. The balance is connected to a computer, and the change in the mass reading of the balance over time is recorded using data collection and spreadsheet software programs. The data can then be approximately corrected for water vapor using the method described herein, and parameters such as the total hydrogen yield, the hydrogen generation rate, and the induction period can also be deduced. The size of the measuring cylinder and the resolution of the balance can be changed to adapt the setup to different hydrogen volumes and flow rates.

The study of methods to generate on-demand hydrogen for fuel cells continues to grow in importance. However, systems to measure hydrogen evolution from the reaction of chemicals with water can be complicated and expensive. This article details a simple, low-cost, and robust method to measure the evolution of hydrogen gas.

一种简单，低成本和强大的系统来测量氢气与水溶液化学反应放出的音量

There is a growing research interest in the development of portable systems which can deliver hydrogen on-demand to proton exchange membrane (PEM) ...

Determination of Carbonyl Functional Groups in Bio-oils by Potentiometric Titration: The Faix Method

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls cause an increase in viscosity (often referred to as &#39;aging&#39;) during storage of bio-oils. As such, carbonyl content has previously been used as a method of tracking bio-oil aging and condensation reactions with less variability than viscosity measurements. Additionally, carbonyls are also responsible for coke formation in bio-oil upgrading processes. Given the importance of carbonyls in bio-oils, accurate analytical methods for their quantification are very important for the bio-oil community. Potentiometric titration methods based on carbonyl oximation have long been used for the determination of carbonyl content in pyrolysis bio-oils. Here, we present a modification of the traditional carbonyl oximation procedures that results in less reaction time, smaller sample size, higher precision, and more accurate carbonyl determinations. While traditional carbonyl oximation methods occur at room temperature, the Faix method presented here occurs at an elevated temperature of 80 &#176;C.

Here we present a potentiometric titration technique for accurately quantifying carbonyl compounds in pyrolysis bio-oils.

该Faix方法:生物油羰基官能团通过电位滴定法测定

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls ...

Facile Preparation of (2Z,4E)-Dienamides by the Olefination of Electron-deficient Alkenes with Allyl Acetate

Direct cross-coupling between two alkenes via vinylic C-H bond activation represents an efficient strategy for the synthesis of butadienes with high atomic and step economy. However, this functionality-directed cross-coupling reaction has not been developed, as there are still limited directing groups in practical use. In particular, a stoichiometric amount of oxidant is usually required, producing a large amount of waste. Due to our interest in novel 1,3-butadiene synthesis, we describe the ruthenium-catalyzed olefination of electron-deficient alkenes using allyl acetate and without external oxidant. The reaction of 2-phenyl acrylamide and allyl acetate was chosen as a model reaction, and the desired diene product was obtained in 80% isolated yield with good stereoselectivity (Z,E/Z,Z = 88:12) under optimal conditions: [Ru(p-cymene) Cl2]2 (3 mol %) and AgSbF6 (20 mol %) in DCE at 110 &#186;C for 16 h. With the optimized catalytic conditions in hand, representative &#945;- and/or &#946;-substituted acrylamides were investigated, and all reacted smoothly, regardless of aliphatic or aromatic groups. Also, differently N-substituted acrylamides have proven to be good substrates. Moreover, we examined the reactivity of different allyl derivatives, suggesting that the chelation of acetate oxygen to the metal is crucial for the catalytic process. Deuterium-labeled experiments were also conducted to investigate the reaction mechanism. Only Z-selective H/D exchanges on acrylamide were observed, indicating a reversible cyclometalation event. In addition, a kinetic isotope effect (KIE) of 3.2 was observed in the intermolecular isotopic study, suggesting that the olefinic C-H metalation step is probably involved in the rate-determining step.

Facile Preparation of 2Z,4E-Dienamides by the Olefination of Electron-deficient Alkenes with Allyl Acetate

The ruthenium-catalyzed olefination of electron-deficient alkenes with allyl acetate is described here. By using aminocarbonyl as a directing group, this external oxidant-free protocol has high efficiency and good stereo- and regioselectivity, opening a novel synthetic route to (Z,E)-butadiene skeletons.

（2<em&gt;ž</em&gt;，4<em&gt;电子</em&gt;） - 通过电子缺陷烯烃与烯丙基乙酸酯的烯化反应制备二烯酰胺

Direct cross-coupling between two alkenes via vinylic C-H bond activation represents an efficient strategy for the synthesis of butadienes with high ...

Quantifying X-Ray Fluorescence Data Using MAPS

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical composition and elemental distribution within a material. Synchrotron-based XRF has become an integral characterization technique for a variety of research topics, particularly due to its non-destructive nature and its high sensitivity. Today, synchrotrons can acquire fluorescence data at spatial resolutions well below a micron, allowing for the evaluation of compositional variations at the nanoscale. Through proper quantification, it is then possible to obtain an in-depth, high-resolution understanding of elemental segregation, stoichiometric relationships, and clustering behavior.
This article explains how to use the MAPS fitting software developed by Argonne National Laboratory for the quantification of full 2-D XRF maps. We use as an example results from a Cu(In,Ga)Se2 solar cell, taken at the Advanced Photon Source beamline 2-ID-D at Argonne National Laboratory. We show the standard procedure for fitting raw data, demonstrate how to evaluate the quality of a fit and present the typical outputs generated by the program. In addition, we discuss in this manuscript certain software limitations and offer suggestions for how to further correct the data to be numerically accurate and representative of spatially resolved, elemental concentrations.

Here, we demonstrate the use of the X-ray fluorescence fitting software, MAPS, created by Argonne National Laboratory for the quantification of fluorescence microscopy data. The quantified data that results is useful for understanding the elemental distribution and stoichiometric ratios within a sample of interest.

利用地图量化 X 射线荧光数据

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

用于跟踪纳米粒子自组装的液体细胞透射电子显微镜

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

透射电镜 hFOB 1.19 和 Saos-2 细胞产生的能量色散 X 射线显微分析

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in liquid environments. This manuscript describes the process for making graphene liquid cells through the example of graphene liquid cell transmission electron microscopy (TEM) experiments of gold nanocrystal etching. The protocol for making graphene liquid cells involves coating gold, holey-carbon TEM grids with chemical vapor deposition graphene and then using those graphene-coated grids to encapsulate liquid between two graphene surfaces. These pockets of liquid, with the nanomaterial of interest, are imaged in the electron microscope to see the dynamics of the nanoscale process, in this case the oxidative etching of gold nanorods. By controlling the electron beam dose rate, which modulates the etching species in the liquid cell, the underlying mechanisms of how atoms are removed from nanocrystals to form different facets and shapes can be better understood. Graphene liquid cell TEM has the advantages of high spatial resolution, compatibility with traditional TEM holders, and low start-up costs for research groups. Current limitations include delicate sample preparation, lack of flow capability, and reliance on electron beam-generated radiolysis products to induce reactions. With further development and control, graphene liquid cell may become a ubiquitous technique in nanomaterials and biology, and is already being used to study mechanisms governing growth, etching, and self-assembly processes of nanomaterials in liquid on the single particle level.

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching

Graphene liquid cell electron microscopy can be used to observe nanocrystal dynamics in a liquid environment with greater spatial resolution than other liquid cell electron microscopy techniques. Etching premade nanocrystals and following their shape using graphene liquid cell Transmission Electron Microscopy can yield important mechanistic information about nanoparticle transformations.

用石墨烯液细胞透射电镜研究原位纳米晶蚀刻

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in ...

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. The versatility of the GSMLCs is demonstrated in the context of a study about etching and growth dynamics of gold nanostructures from a HAuCl4 precursor solution. GSMLCs combine the advantages of conventional silicon- and graphene-based liquid cells by offering reproducible well depths together with facile cell manufacturing and handling of the specimen under investigation. The GSMLCs are fabricated on a single silicon substrate which drastically reduces the complexity of the manufacturing process compared to two-wafer-based liquid cell designs. Here, no bonding or alignment process steps are required. Furthermore, the enclosed liquid volume can be tailored to the respective experimental requirements by simply adjusting the thickness of a silicon nitride layer. This enables a significant reduction of window bulging in the electron microscope vacuum. Finally, a state-of-the-art quantitative evaluation of single particle tracking and dendrite formation in liquid cell experiments using only open source software is presented.

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

石墨烯支持的微井液体细胞制备,用于原位透射电子显微镜

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...