这项工作得到了美国能源部、科学办公室、基础能源科学办公室、材料科学和工程司的支持, 合同号为。DE-AC02-05-CH11231 在无机纳米结构程序 (KC3103) 的物理化学中。

1. 制作石墨烯涂层 TEM 网格
<ol><li>切出大约2厘米2片的预制石墨烯-铜 (见材料表), 它适合约6到 8 TEM 网格。 注: 使用 3-5 层石墨烯代替单层石墨烯封装液体口袋, 成功率更高, 而不会失去分辨率。由于石墨烯是原子薄, 低 Z 材料, 大多数的分辨率损失是从液体厚度的石墨烯液体细胞。</li>
	<li>使用丙酮洗涤清洁石墨烯 (图 2A)。 注: 此步骤旨在去除在沉积过程中在石墨烯表面留下的任何残余 PMMA [聚甲基丙烯酸甲酯)]。如果用户确信他们的石墨烯是干净的, 这一步是不必要的。<ol>
<li>将石墨烯在铜片放在玻璃培养皿中, 然后用丙酮填充。 注: 使用丙酮, 因为 PMMA 溶解在丙酮。</li>
		<li>将丙酮溶液 (约50°c) 轻轻加热5分钟, 周期性地旋转溶液。 注意: 确保观察丙酮和温度以避免起火。这应该在通风罩里做。</li>
		<li>用镊子从丙酮洗涤中取出石墨烯在铜片上, 用新的清洁丙酮代替丙酮。 注意: 小心不要刮或以其他方式损坏石墨烯表面与镊子。</li>
		<li>重复洗涤过程共3次。</li>
		<li>在继续下一步之前, 让石墨烯在铜上彻底风干。</li>
	</ol>
</li>
	<li>平滑的石墨烯在铜片, 以消除任何宏观皱纹 (图 2B)。 注意: 这个平滑过程是为了确保穿孔支持箔-TEM 网格 (见材料表) 能够正确地与石墨烯表面粘结。铜石墨烯上的凹凸和折痕使得保持良好的接触变得困难。<ol>
<li>采取两个干净的玻璃幻灯片, 并放置一个折叠的擦拭 (见材料表) 在底部玻璃幻灯片。在擦拭的顶端, 放置石墨烯在铜片上。最后, 把第二个玻璃滑梯放在上面。 注: 将石墨烯在铜片上与石墨烯一侧向上 (触摸玻璃滑块), 以防止刮伤组织擦拭。折叠组织是用来逐步推出皱纹和防止折叠新的折痕。</li>
		<li>按下顶部的幻灯片, 逐步平滑的任何皱纹, 石墨烯在铜片上。减少组织中的褶皱数量, 并重复按压过程。继续这个过程, 直到在两个玻璃幻灯片之间的最后挤压没有组织擦拭。</li>
	</ol>
</li>
	<li>在铜石墨片上放置 TEM 网格 (图 2C)。<ol>
<li>将孔无定形碳支持箔-TEM 网格 (参见材料表) 放在石墨烯上, 与石墨烯接触的非晶碳。 注意: 小心不要弯曲或变形 TEM 网格时, 拿起镊子。弯曲的 TEM 网格不能正确地绑定到石墨烯。通过网格边缘拾取网格可防止网格变形。在这里, 金 TEM 网格被用来避免蚀刻的网格在步骤中删除铜从石墨烯-铜。</li>
		<li>在网格上放置几滴异丙醇。 注: 如果任何网格重叠, 在将异丙醇放到网格上后, 轻轻地将其与镊子的尖端一起移动。小心不要损坏石墨烯表面。</li>
		<li>让干燥 2 + h, 以确保网格是正确的粘合。这种干燥过程使孔非晶碳与石墨烯接触更好。 注: 要检查网格是否粘附在石墨烯上, 请轻轻地拾起石墨烯上的铜片并将其翻转。如果重力不移除栅格, 它们应该得到适当的粘合。</li>
	</ol>
</li>
	<li>使用过硫酸钠溶液蚀刻铜 (图 2D)。<ol>
<li>在10毫升的去离子水中, 用1克过硫酸铵做溶液。</li>
		<li>使用镊子, 小心地把石墨烯在铜片上的过硫酸铵溶液与铜侧向下。让棋子浮在过硫酸铵溶液的顶端 (图 2D)。</li>
		<li>用石墨烯涂层的网格在一夜之间保持溶液。请注意, 该解决方案将成为蓝色的铜蚀刻, 并将没有可见的铜后, 石墨烯片后蚀刻完成 (图 2E)。</li>
	</ol>
</li>
	<li>清洗网格以清除过硫酸钠。<ol>
<li>从解决方案中移除浮动网格, 并将它们放在清洁、去离子水的顶端 (请参阅第二个培养皿中的过滤器的材料表)。 注意: 传输网格最简单的方法是使用玻璃滑块来拾起网格, 然后将它们放在充满水的第二个培养皿中。在转移过程中, 有些网格会落到培养皿的底部。这通常是在网格上的石墨烯破裂或其他损坏的迹象。</li>
		<li>重复这个过程3次, 以去除所有的过硫酸铵残渣从石墨烯涂层的网格。</li>
		<li>用镊子拿起网格, 把网格石墨烯端到滤纸上, 让它们干。 注: 这最后的转移水清洗可能是困难的, 因为网格经常坚持的镊子由于毛细力从残余的水。</li>
	</ol>
</li>
</ol>2. 制作液体电池袋
<ol><li>采取两个石墨烯涂层 TEM 网格, 并把他们的石墨烯一侧上的玻璃幻灯片。使用小型外科手术刀刀片, 切断石墨烯涂层 TEM 网格的边缘, 大约1/4 到1/8 的网格区域 (图 3A)。 注: 切割其中一个网格是假设, 使两个网格上的石墨烯更接近接触, 以提供更好的石墨烯-石墨烯相互作用, 形成口袋。</li>
	<li>准备要封装的解决方案。 注: 该溶液为纳米晶蚀刻实验所特有。<ol>
<li>使三缓冲 HCl 溶液与去离子水浓度在10-100 毫米之间。 注: 我们发现, 为制备含水金属纳米粒子溶液, 三个缓冲 hcl 导致稳定袋的成功率更高, 虽然需要更多的研究来了解为什么三个缓冲盐酸有助于使稳定的口袋。在这种情况下, 使用三个缓冲基座或无三个缓冲器似乎有很低的成功率的口袋形成。每一个溶剂和样品可能会要求优化, 以找到条件, 创造稳定的口袋, 而不是破坏化学正在研究。对文献的简要调查表明, 与邻二氯苯/油胺 (9:1 比值), 23 0.5x 三硼酸-EDTA () 和200毫米 nacl 溶液, 33 和水性0.15 米氯化钠溶液30以及水下三缓冲 HCl 的成功 系统在这里提出。</li>
		<li>在溶液中做一个40毫米 FeCl3溶液, 用1.8 µL 的盐酸每毫升水。 注意: FeCl3是此蚀刻实验的蚀刻。其他实验可能会根据正在进行的实验添加不同的解决方案。</li>
		<li>在清理1、34后, 制作金纳米棒并集中纳米样品。</li>
		<li>混合0.15 毫升 0.01-0. 1 毫米三缓冲 hcl, 0.1 毫升40毫米 FeCl3在 HCl 和10µL 的纳米棒。</li>
	</ol>
</li>
	<li>将0.5 µL 液滴溶液封装在非切割石墨烯涂层 TEM 网格上。在放置水滴时, 使用镊子将 tem 栅格的边缘按住, 这样毛细管力就不会拾取 tem 网格 (图 3B)。 注意: 小心地使水滴尽可能小, 并把它尽可能靠近网格的中心。</li>
	<li>快速、小心地将石墨烯涂层 TEM 栅格置于液滴顶端的切角上;目标是让第二个网格在第一个网格的顶部休息, 没有液体被挤出 (图 3C)。 注: 有第二个网格已经放置在自闭镊子可以使这个过程更快, 更容易。这可以说是液态细胞形成过程中最棘手的一步, 可能会发生许多潜在的故障。当镊子在两个网格之间被卡住时, 在取下镊子时设置顶部栅格是一个挑战。一般情况下, 将顶部 TEM 网格的一个边缘向下, 然后逐步放开网格效果最好。请注意, 如果在玻璃滑梯上看到液体, 那么口袋里可能没有正确的密封。</li>
	<li>等待5分钟, 让石墨烯液体细胞口袋形成。 注: 当口袋成形时, 液体的某些蒸发可能会发生, 但一旦形成密封密封, 就不会有额外的液体损耗。溶液中每个物种的相对浓度应保持恒定。</li>
	<li>将样品带到透射电镜进行成像。 注: 为封口预留的时间从研究员到研究员不等。对于蚀刻实验, 在将液态电池带到 TEM 之前, 要避免预蚀刻的时间更短。</li>
</ol>3. 石墨烯液体电池的加载和成像
注: 透射电子显微镜的操作遵循用户手册中的标准程序。每一个 TEM 将有不同的校准程序。
<ol><li>将石墨烯液体电池放置在传统的 TEM 单倾角支架上 (图 4)。 注: 其他标准持有人, 如双倾角持有人或取暖持有人也可以使用。使用螺旋状机构来保护 TEM 网格的持有者可能会施加一个破坏石墨烯液体细胞的剪切力。</li>
	<li>将 tem 支架装入 tem 柱上。 注: 由于石墨烯液体细胞含有少量的液体, 没有储层, 并且有单独的口袋, 因此无需严格检查像氮化硅液细胞实验这样的泄漏。即使石墨烯液体电池口袋爆裂, 只有很小数量的液体被释放, 从而不会崩溃的 TEM 真空系统。</li>
	<li>使用样品中的纳米粒子和非晶碳适当地对准 TEM 光束 (枪倾斜, 冷凝孔径对准, 冷凝器 stigmation), 图像 (Z 高度调整, 目标 stigmation, 旋转中心对准, 和畸变校正器调整如果适用)。然后从光束路径中取下支架, 校准电子束剂量率。<ol>
<li>在校准前将 TEM 灯丝打开至少20分钟, 以使其稳定以重现的剂量率;根据 TEM 系统和电子枪类型, 这种等待时间可能会有所不同。 注: 选举 microscopists 经常使用剂量率来指每单位时间 (e-/Å2s) 每单位面积传递的电子数量。在辐射化学群落中, 这称为通量密度, 剂量率定义为单位时间单位面积吸收的能量量。由于计算样品所吸收的能量对液体细胞中发现的复杂几何很难, 并且保持与 TEM 群落的一致性, 我们选择使用剂量率来指单位时间内单位面积的电子。</li>
		<li>使用查看屏幕 (图 5A) 将光束压缩为最浓缩的量, 最高剂量率。读出并保存凝聚光束的透镜电流。 注: 对于3.3.2 到3.3.5 的步骤, 编写了一个自定义数字显微图像脚本, 它控制了 TEM 的冷凝器系统, 以提供的电子剂量率校准第二个冷凝器 (C2) 透镜电流。这使得研究人员可以重现性在实验中将电子剂量率设定为任意值。</li>
		<li>使用查看屏幕 (图 5B) 将光束传播到最大传播量, 最低剂量率。读出并保存传播光束的透镜电流。</li>
		<li>将冷凝器透镜电流的范围划分为10个相等的间距值, 并使用 CCD 摄像机收集每个冷凝器透镜值的图像。</li>
		<li>使用 ccd 灵敏度和放大校准为每个镜头电流转换 ccd 计数为剂量率。</li>
		<li>在不同的透镜电流下使用电子通量数据进行标定曲线。使用这个校准曲线, 其余的实验, 以控制电子束的期望通量。</li>
		<li>将该样本重新插入光束路径。</li>
	</ol>
</li>
	<li>开始在液体口袋中寻找纳米微粒, 同时保持剂量率低 (通常约 20 e-/Å2s)。 注意: 保持剂量率低防止纳米粒子在寻找纳米粒子时被蚀刻。</li>
	<li>当一个纳米微粒被发现在液体口袋里, 微调的焦点在纳米粒子, 同时保持低剂量率。 注: 确定纳米微粒是否在液体口袋中可能是棘手的, 但气泡或粒子运动的存在往往是一个稳定的液体口袋的良好迹象。有时, 而不是液体, 口袋像一个非常致密的凝胶与气泡移动非常缓慢。这些情况是由液体的蒸发引起的, 这可能是由于石墨烯中未密封的口袋或裂纹造成的。这是相当容易区分的凝胶与没有运动和液体环境与气泡迅速移动和改变形状。在形成良好的液体细胞口袋时可能会有一些蒸发, 但反应物之间的相对浓度保持不变。</li>
	<li>使用校准曲线 (请参见步骤 3.3), 为所需的剂量率设置冷凝器透镜电流 (图 5D)。 注: 内部脚本用于设置冷凝器镜头电流和图像采集参数</li>
	<li>开始收集图像文件中嵌入的剂量率和时间戳元数据的 TEM 图像时间序列。</li>
	<li>粒子完成蚀刻后, 展开光束, 开始寻找液体口袋中的其他纳米粒子。</li>
	<li>当收集到足够数量的纳米粒子蚀刻视频时, 将透射电镜持有者从 tem 中取出, 并遵循标准 tem 程序。把石墨烯液细胞从 TEM 架上取出。 注意: 一个典型的成像会话持续大约 2-3 h 与大约30个录影采取了。可用数据的视频数量取决于口袋的质量和蚀刻实验的类型。</li>
</ol>4. 基于计算软件的 TEM 视频图像分析
注意: 由于 TEM 视频是对3维形状的2维投影, 需要仔细地进行图像分析以提取蚀刻速率或形状变化。
<ol><li>使用 ImageJ 将本机 DM3 视频文件转换为 avi 格式, 并将 avi 视频导入计算软件 (请参见材料表)。</li>
	<li>分析视频每个帧中的每个纳米。<ol>
<li>通过对图像进行阈值确定纳米的轮廓 (图 7A)。 注: 金属纳米粒子的高对比度使图像分析更容易。为了研究对比度较低的系统, 在阈值前可能需要额外的过滤器。</li>
		<li>从纳米的大纲中, 确定最接近的拟合椭圆的主要和次要轴 (图 7B)。 注意: 内置图像分析软件确定主要和次要轴假定形状是椭圆。对于不是椭圆的纳米, 在对纳米粒子施胶时不应使用这些值。</li>
		<li>使用主轴将纳米轮廓剪切成两半 (图 7C)。</li>
		<li>对于这些半部分, 通过在主轴周围旋转该轮廓, 确定所包含形状的体积和表面积。 注: 这种微积分方法有时称为环的方法。这种分析方法仅在纳米在主轴周围对称时才有效。有两个一半来比较体积和表面积提供了一些保证, 纳米是真正的旋转对称。</li>
	</ol>
</li>
	<li>在提取视频的每个帧的纳米的体积和表面积后, 编译和解释数据。 注意: 此大纲方法还允许分析具有定义形状的纳米晶的小平面。</li>
</ol>

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作者没有什么可透露的。

石墨烯液体电池电子显微镜能提供纳米晶生长和蚀刻的机械信息, 具有较高的空间分辨率, 但由于石墨烯液体细胞难以和细腻, 因此该技术需要注意细节, 以提取可用数据。即使经过广泛的实践制作石墨烯液体细胞, 只有大约半到一季度的液态电池成功地封装液体溶液。形成液态细胞的关键步骤是将第二个网格放在液滴的顶端。常见的错误包括将镊子夹在两个网格之间, 将第二个网格放在离中心太远的位置, 然后从一个太大的水滴开始。由于石墨烯液体细胞的组装是微妙的, 需要良好的运动技能, 它通常需要实践成功地使液体口袋。由于石墨烯涂层 TEM 网格的花费, 强烈建议新的石墨烯液态电池用户首先在传统的铜、非晶碳 TEM 网格上实践液态电池制造过程, 以节省资金。
确定液体细胞失效的原因是很有挑战性的, 因为研究人员可能不知道每一个步骤都是成功的, 直到成像样品的最后, 和错误, 如挠石墨烯, 可能会被忽视。最容易识别的错误是不正确的装配, 因为研究人员将立即看到液体从石墨烯液体细胞泄漏出来。在铜网格上制造石墨烯的问题, 比如石墨烯的开裂, 可以更难精确地指出。石墨烯的质量可以用拉曼光谱对 TEM 网格进行前后检查, 但在这种测试之后, 石墨烯通常是不可用的。另外, 使用直接转移石墨烯是很重要的, 因为石墨烯的两个面被放在一起需要清洁, 以适当地形成一个密封通过范德华力。通过聚合物转移方法制造石墨烯涂层的网格可能会在石墨烯的侧面留下聚合物残留物, 而这些残渣有望结合在一起。如果正确的程序是遵循正确的 TEM 网格, 缺乏成功的石墨烯液体细胞通常是由于处理不当的石墨烯和网格在组装和制造。
石墨烯液相细胞透射电镜技术通过使用一个更薄的封装材料, 可以在任何传统 TEM 持有人, 使高分辨率和小关节弹道跟踪实验更容易。随着商用氮化硅膜液细胞的分辨率的解决, 石墨烯液体电池中蚀刻纳米晶所能获得的许多小平面和动力学信息将会丢失。石墨烯液细胞 TEM 实验也可以对现有的单倾角 TEM 持有者否定昂贵的新专门持有人的需要。此外, 石墨烯液体电池可以放在任何接受标准 TEM 网格样品的持有者, 允许在氮化硅液体的先进的持有者 (加热, 双倾斜, 冷却, 低温, 阴极射线) 进行液体细胞实验。尚未设计单元格。此外, 如果口袋破裂像其他的液体细胞 TEM 技术, 石墨烯液体细胞不构成崩溃真空的 TEM 专栏的风险。虽然石墨烯液态电池在纳米晶领域中不是无处不在的技术, 但它的易用性和空间分辨率将使其在未来得到更广泛的应用。
尽管石墨烯液体细胞 TEM 具有许多优点, 但它对可以进行的实验类型有一定的限制。一些液体确实蒸发成口袋形式, 所以很难确切地确定物种在溶液中的浓度, 即使不考虑电子束效应。石墨烯液体细胞也有随机大小, 高度和小口袋的分布, 因此氮化硅流动细胞有更多可量化的前束浓度和大, 均匀的液体层的好处。如本工作所述, 只有预加载的样品可以在 TEM 中使用石墨烯液体细胞来观察, 所以不可能在其他溶液中流动以引发化学反应。由电子束与液体溶液相互作用产生的辐物种是唯一可以用来启动反应的触发器。虽然尚未证明, 热启动过程可能会触发石墨烯液体细胞使用标准的加热持有者。电子束诱发的辐效应还没有得到充分的理解, 难以控制。研究人员已经开发出动力学模型来确定在光束相互作用后的液体细胞口袋的内容31,32, 但它们的精确度受模型中包含的反应数和任何未知浓度的限制。因干燥而发生的变化。复杂的初始口袋内容与许多反应物种如 FeCl3, 三缓冲器, 甚至石墨烯30, 可能很难完全理解使用一个动力学模型。液体细胞电子显微镜的另一个缺点是, 很难表征在动态过程中形成的晶体的组成。例如, 在多组分系统的生长实验中, 如果新的纳米晶是无定形的或不在区域轴上, 则可能无法区分哪些阶段或物种正在生长。这也是为什么在已知的区域轴线上蚀刻预先形成的纳米晶的另一个原因是可取的。最后, 还有一些论点认为, 在石墨烯液体细胞中的光束引起的反应并不代表瓶中的原位反应的条件。
未来的石墨烯液体细胞实验将有助于缓解这些担忧, 同时也使用新的 TEM 的进步来进一步探索纳米晶的潜在奥秘。相关的前原位纳米晶合成和蚀刻实验对于确证液细胞 TEM 实验中的机制至关重要。此外, 研究人员已经开始致力于增加流动能力的石墨烯液态电池 TEM35和使更多的控制口袋36 , 包括石墨烯液体细胞阵列使用 lithographically 准备孔37。电子显微分辨率和相机速度的进步将使石墨烯液体细胞进一步能够研究纳米晶变换过程中的原子动力学。在电子显微镜下用原子化的薄石墨材料包装小口袋里的液体有许多潜在的应用, 无疑将成为未来纳米研究的主要内容。

Controllably 合成纳米晶1并将纳米粒子组装成较大的结构2,3需要了解有关原子和纳米粒子如何相互作用和绑定的基本机制。一起。理想的情况下, 这些纳米过程的研究将在其原生液体环境中进行, 并需要相应的空间分辨率来观察感兴趣的现象, 但这些要求由于纳米长度而构成挑战。这些系统运行的刻度。研究人员一直希望利用电子显微术的空间分辨率来成像这些过程, 但是电子显微镜柱的高真空要求液体溶液的封装4。一些早期的液态细胞电子显微镜实验在两个氮化硅膜之间封装液体5,6,7,8, 此方法现在已成为商业上可用的研究动态纳米尺度过程的技术。
商业上可用的氮化硅液体电池 TEM 持有者提供了必要的分辨率, 以查看和理解在纳米尺度9,10,11,12的各种有趣现象,13,14,15,16. 一些商用液体电池 TEM 持有者有额外的能力, 如加热, 流量和电气连接, 进一步扩大纳米级的过程, 可以调查的领域。然而, 所有这些功能, 商业系统没有优化, 以达到最高的空间分辨率。对于需要改进空间分辨率的研究人员来说, 降低窗口厚度和降低液体厚度是两条可能的途径, 以减少电子束散射和更好的分辨率17。一些使用氮化硅液体细胞的组织制造自己的窗户, 从而对窗户和液体厚度产生更大的控制。18这些自制液体细胞的散射减少, 使电子显微学研究具有更大的空间分辨率, 包括原子分辨率研究19,20,21。
由于封装材料的厚度是影响液体细胞实验空间分辨率的一个方面, 原子化薄, 低 Z 材料, 如石墨烯将是理想的封装材料22, 23. 石墨烯片仍然足够坚固, 可以保护液体的口袋不受柱的压力差。此外, 这些石墨烯液体细胞袋通常含有较薄的液体层, 进一步提高了可实现的空间分辨率。研究了许多有趣的纳米石墨烯液体细胞, 包括纳米粒子的小关节轨迹和纳米粒子动力学, 原子分辨率为23,24,25 ,26,27。石墨烯液体细胞技术的一个意想不到的好处是, 这种高空间分辨率可以实现, 而无需购买不同的 TEM 持有人或专门的硅制造。使用具有高分辨率的氮化硅细胞进行的实验还需要由重原子组成的大型纳米粒子, 而石墨烯液体电池所获得的分辨率可以为 sub-2 纳米粒子25提供原子分辨率。此外, 石墨烯液体电池为研究生物样品提供了机会, 因为石墨烯用于封装28、29和石墨烯能够减轻电子束的一些破坏性作用30。由于这些优势, 石墨烯液态细胞电子显微镜有潜力成为一个标准技术在纳米社区一旦更多的研究人员更了解这项技术是否有助于他们的研究和如何应用这项技术。
研究人员在化学, 纳米材料, 生物, 和其他领域渴望空间分辨率的原位转换可以受益于使用石墨烯液细胞电子显微技术。对于需要在转换期间进行可视化的非平衡过程, 此就地方法特别有用。液细胞透射电镜技术的一个重要缺点是由扰电子束31产生的辐物种, 它可以诱发微妙样品中的不良变化。研究人员已经开发出模型来尝试量化光束驱动化学的31,32, 并正在开发策略来减轻这些影响30,32。石墨烯液体细胞透射电镜有额外的挑战, 是脆弱的, 往往很难作出, 特别是对新技术的研究人员。本文的目的是分享有关如何进行石墨烯液细胞 TEM 实验的细节 (图 1), 用一个例子实验观察纳米晶的单粒子蚀刻, 并希望表明, 石墨烯液体细胞几乎所有的组都可以使用电子显微镜进行实验。该协议将覆盖石墨烯涂层的网格, 液态细胞的形成, TEM 使用的石墨烯液细胞蚀刻实验, 和图像分析技术。在制造液体细胞的关键步骤, 如熔滴的大小, 仔细考虑液体溶液的内容, 并使用只直接转移石墨烯将包括更多的建议, 如何避免重复的陷阱以前的研究人员。石墨烯液体细胞透射电镜是纳米尺度研究的新兴技术, 本文将使新进入者能够开始利用这一技术。

<table><tbody><tr><td>2-propanol (Isopropanol)</td><td>Sigma Aldrich</td><td>190764-4L</td><td></td></tr><tr><td>Acetone</td><td>Fisher Chemical</td><td>A949-4 HPLC Grade</td><td></td></tr><tr><td>FeCl3</td><td>Sigma Aldrich</td><td>44944-250g</td><td></td></tr><tr><td>Gold Quantifoil, Amorphous Carbon TEM Grids</td><td>SPI Supplies</td><td>4230G-XA</td><td>300 Mesh Gold, R1.2/1.3- Often extensively on back-order</td></tr><tr><td>Graphene</td><td>ACS Materials</td><td>GnVCu3~5L-4x2in</td><td>We special order this to get graphene only on one side. The double sided product number is CVCU3022. Usually, we use 3-5 layer graphene for making Graphene Liquid Cells.&amp;nbsp; If researchers need single layer graphene for their liquid cells, we have been using Grolltex recently</td></tr><tr><td>Hot Plate</td><td>IKA</td><td>C-MAG HS 7 Digital</td><td></td></tr><tr><td>Hydrochlorid Acid</td><td>Fisher Chemical</td><td>7647-01-0</td><td></td></tr><tr><td>Kimwipe Tissues</td><td>Kimberly-Clark</td><td>34120</td><td></td></tr><tr><td>Matlab</td><td>Mathworks</td><td></td><td></td></tr><tr><td>Millipore Water Filter</td><td>Millipore</td><td>F4NA85846D</td><td></td></tr><tr><td>Sodium Persulfate</td><td>Sigma Aldrich</td><td>71890-500g</td><td></td></tr><tr><td>Surgical Scalpel Blade</td><td>Swann-Morton</td><td>No. 6</td><td></td></tr><tr><td>TEM</td><td>FEI</td><td>Tecnai T20 S-Twin</td><td>TEM needs to be linked to camera acquisition software to allow for dose rate calibration procedures.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>TEM Cameara for in situ data collection</td><td>Gatan</td><td>Orius SC200&amp;nbsp;</td><td>Custom digital micrograph scripts (written in house) for calibrating the C2 lens value to dose rate and collect in situ datasets</td></tr><tr><td>TEM Single Tilt Sample Holder</td><td>FEI</td><td></td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane hydrochloride (Tris Buffer HCl)</td><td>Fisher Biotech</td><td>1185-53-1</td><td></td></tr><tr><td>Tweezers</td><td>Excelta</td><td>7-SA</td><td></td></tr></tbody></table>

using graphene liquid cell transmission electron microscopy to study in situ nanocrystal etching

石墨烯液体细胞电子显微镜提供了观察纳米化学转化和动力学的能力, 因为反应发生在液体环境中。本文介绍了石墨烯液细胞透射电镜 (TEM) 在金纳米晶蚀刻实验中的制备方法。制作石墨烯液体电池的协议涉及用化学气相沉积石墨烯涂覆金、孔碳 TEM 网格, 然后使用这些石墨烯涂层的网格将液体封装在两个石墨烯表面之间。这些有兴趣的纳米材料的液体在电子显微镜中被成像, 以观察纳米过程的动力学, 在这种情况下, 金纳米棒的氧化蚀刻。通过控制电子束剂量率, 调节液体细胞中的蚀刻种类, 可以更好地理解原子从纳米晶中除去以形成不同的面和形状的基本机制。石墨烯液体电池 TEM 具有空间分辨率高、与传统 TEM 持有者相容、研究组启动成本低的优点。目前的限制包括精致的样品制备, 缺乏流动能力, 并依赖电子束产生的辐产品来诱导反应。随着进一步的发展和控制, 石墨烯液体电池可能成为一种无处不在的技术在纳米材料和生物, 并已被用来研究的机制, 控制生长, 蚀刻和自组装过程中纳米材料的液体上单一粒子水平。

在 800 e-/Å2s 的电子束剂量率下, 纳米蚀刻的代表性视频中的帧显示在图 6中。在纳米开始进行氧化蚀刻之前, 解决方案需要大约二十年代的光束照明。纳米开始蚀刻后, 原子的去除率保持稳定, 而纳米也保持恒定长宽比。在视频中, 纳米棒通常没有显著的运动, 这与以前的液体细胞 TEM 工作相一致, 这种大小的纳米粒子为24。由于纳米粒子的移动不多, 气泡生成和气泡运动通常是确定纳米粒子是否在液体口袋中的最佳方法。当纳米变小时, 纳米开始旋转, 进出焦平面, 确认纳米在液体环境中。
石墨烯液态细胞最常见的故障是无法封装稳定的液体。有时, 这可能导致完全干燥的口袋的特点是没有气泡, 没有微粒的运动或大小的变化。此外, 口袋可以开始与液体和气泡, 但后来干燥出来之前, 纳米颗粒完全蚀刻。通常为一个好的液体细胞, 每个口袋是稳定的约2-3 分钟, 在蚀刻剂量率, 和口袋干燥只成为一个问题, 大型纳米粒子或缓慢的蚀刻过程。有时, 液体可以从口袋里蒸发出来, 留下一个非常高盐浓度的凝胶状溶液。这些凝胶通常是显而易见的, 当成像由于高反差的解决方案和极慢的运动的气泡和粒子。在这些凝胶样的解决方案中收集的数据不能被信任。
在采集了液细胞 TEM 数据后, 分析了纳米粒子蚀刻的视频。可以进一步提取和评估卷、曲面区域和方面 (如果适用) (图 7)。干燥袋的一个征兆是大量减慢蚀刻速度随着时间的推移, 所以绘制的体积与时间可以是一个有效的方法检查的口袋稳定性和数据的可靠性。其他次优结果包括非对称蚀刻指示不均匀的口袋内容和不必要的沉淀的氢氧化铁的种类从氯化铁蚀刻。总的来说, 成功的石墨烯液态细胞的最重要的关键是一个稳定的液体环境, 导致在多个纳米粒子和液体袋上可重现的纳米晶动力学。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57665/57665fig1.jpg"> 图 1.石墨烯液细胞透射电镜技术示意图。(A)组装石墨烯液体电池, 将一滴溶液放在石墨烯涂层的孔碳 TEM 网格上。第二个石墨烯涂层网格放在水滴的顶端, 形成一个口袋。请注意, 此图像不缩放, 液滴约33% 太大。(B)在金纳米棒的 TEM 成像过程中, 在液体口袋中放大的示意图。这卡通也不算规模。<a href="/files/ftp_upload/57665/57665fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57665/57665fig2.jpg"> 图 2.石墨烯涂层 TEM 网格的制作工艺 (A)在温丙酮中清洗石墨烯在铜片上(B)在两个玻片之间平涂石墨烯, 以消除宏观皱纹。一个组织被放置在石墨烯的铜片下面, 以不折叠新的皱纹。(C)将非晶孔碳透射电镜网格置于铜石墨烯上, 其接触石墨烯的 TEM 网格的非晶碳侧。(D)在过硫酸铵蚀刻上的浮动铜/石墨烯/TEM 网格。这将从网格中移除铜。(E)石墨烯涂层的 TEM 网格在铜蚀刻后。溶液是蓝色的, 石墨烯涂层的网格上没有铜。尺寸参考, 玻璃培养皿的直径约为6厘米, 玻璃滑块7.5 厘米2.5 厘米.<a href="/files/ftp_upload/57665/57665fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57665/57665fig3.jpg"> 图 3.石墨烯液体电池制造工艺 (a)两个石墨烯涂层 TEM 网格在玻璃滑梯上制备, 边缘切断其中之一。用于切割网格的手术手术刀位于图像的右上方。在石墨烯涂层网格上的封装溶液的(B)水滴。顶端栅格上的水滴是正确的尺寸, 并在石墨烯上做了一个漂亮的珠子。底部网格上的水滴流过石墨烯, 可能是由于石墨烯中的裂纹。(C)第二个石墨烯涂层网格放在第一栅格顶部的溶液液滴上。这种石墨烯液态电池现在可以加载到 TEM 中了。对于尺寸参考, 玻璃滑块是7.5 厘米2.5 厘米.<a href="/files/ftp_upload/57665/57665fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57665/57665fig4.jpg"> 图 4.将石墨烯液体电池装入标准单倾角透射电镜支架。石墨烯液体电池适合于标准的单倾角 tem 持有者, 其方式与普通 tem 网格适合于持有者相同。对于尺寸参考, TEM 网格的直径为3毫米.<a href="/files/ftp_upload/57665/57665fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57665/57665fig5.jpg"> 图 5.透射电镜光束控制。 (A)使用荧光屏查看剂量率校准的压缩电子束。(B)扩展电子束, 用于使用荧光屏查看剂量率校准。强度随着每次面积的电子的减少而减小, 这就是电子束非常微弱的原因。(C)将电子束剂量率与冷凝器透镜电流相关的校准曲线。该标定曲线用于控制成像过程中的光束剂量率。在石墨烯液体电池中收集纳米粒子的 TEM 视频时使用的(D)参数。每个参数使用的特定值可能会根据所映像的材料和所需的分辨率而变化。<a href="/files/ftp_upload/57665/57665fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57665/57665fig6.jpg"> 图 6.金纳米蚀刻在石墨烯液体细胞口袋.在 800 e-/Å2s 的剂量率下, 金纳米蚀刻的典型 TEM 视频帧。在没有蚀刻的初始时期后, 纳米蚀刻以恒定的速率。<a href="/files/ftp_upload/57665/57665fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57665/57665fig7v2.jpg"> 图 7.分析视频帧的方法(A)在图像分析软件中使用阈值概述纳米。(请参阅材料表)这将纳米微粒与背景分离, 并提供了定量分析的形状。(B)确定纳米的主要轴和次要坐标轴。(C)提取沿主轴切割的2维轮廓的每一半。使用这些轮廓, 通过围绕 x 轴旋转轮廓来重建3维形状。<a href="/files/ftp_upload/57665/57665fig7v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching at JoVE.com

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

石墨烯液细胞电子显微镜可用于观察液体环境中的纳米晶动力学, 其空间分辨率比其它液态细胞电子显微镜技术更大。用石墨烯液细胞透射电镜对预制纳米晶及其形状进行蚀刻, 可以产生纳米粒子转化的重要机械信息。

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 ...

using-graphene-liquid-cell-transmission-electron-microscopy-to-study

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

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

17.0K 次观看.  University of California-Berkeley. 石墨烯液细胞电子显微镜可用于观察液体环境中的纳米晶动力学, 其空间分辨率比其它液态细胞电子显微镜技术更大。用石墨烯液细胞透射电镜对预制纳米晶及其形状进行蚀刻, 可以产生纳米粒子转化的重要机械信息。

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

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

科研

化学

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.

在液体中使用液体池透射电子显微镜揭示材料的动态过程

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted ...

工程学

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

合成与盘状磷酸盐锆剥落获取胶体液晶

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline.

This protocol describes the operation of a liquid flow specimen holder for scanning transmission electron microscopy of AuNPs in water, as used for the observation of nanoscale dynamic processes.

使用扫描透射电子显微镜研究纳米大小的物体的动态过程中的液体

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a ...

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 ...

Preparation of Graphene Liquid Cells for the Observation of Lithium-ion Battery Material

In this work, we introduce the preparation of graphene liquid cells (GLCs), encapsulating both electrode materials and organic liquid electrolytes between two graphene sheets, and the facile synthesis of one-dimensional nanostructures using electrospinning. The GLC enables in situ transmission electron microscopy (TEM) for the lithiation dynamics of electrode materials. The in situ GLC-TEM using an electron beam for both imaging and lithiation can utilize not only realistic battery electrolytes, but also the high-resolution imaging of various morphological, phase, and interfacial transitions.

Here, we present a protocol for the fabrication and preparation of a graphene liquid cell for in situ transmission electron microscopy observation, along with a synthesis of electrode materials and electrochemical battery cell tests.

锂离子电池材料用石墨烯液位的制备

In this work, we introduce the preparation of graphene liquid cells (GLCs), encapsulating both electrode materials and organic liquid electrolytes between ...

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. ...

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Presented here is a protocol for labeling membrane proteins in mammalian cells and coating the sample with graphene for liquid-phase scanning transmission electron microscopy. The stability of the samples against the damage caused by radiation can also studied with this protocol.

用于液相电子显微镜的化学固定哺乳动物细胞的石墨烯外壳

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using ...

生物学

用石墨烯单层涂覆 TEM 网格

Fabrication of Monolayer Graphene-Coated Grids for Cryoelectron Microscopy

Cryogenic electron microscopy (cryoEM) has emerged as a powerful technique for probing the atomic structure of macromolecular complexes. Sample preparation for cryoEM requires preserving specimens in a thin layer of vitreous ice, typically suspended within the holes of a fenestrated support film. However, all commonly used sample preparation approaches for cryoEM studies expose the specimen to the air-water interface, introducing a strong hydrophobic effect on the specimen that often results in denaturation, aggregation, and complex dissociation. Further, preferred hydrophobic interactions between regions of the specimen and the air-water interface impact the orientations adopted by the macromolecules, resulting in 3D reconstructions with anisotropic directional resolution. 
Adsorption of cryoEM specimens to a monolayer of graphene has been shown to help mitigate interactions with the air-water interface while minimizing the introduction of background noise. Graphene supports also offer the benefit of substantially lowering the required concentration of proteins required for cryoEM imaging. Despite the advantages of these supports, graphene-coated grids are not widely used by the cryoEM community due to the prohibitive expense of commercial options and the challenges associated with large-scale in-house production. This paper describes an efficient method for preparing batches of cryoEM grids that have nearly full coverage of monolayer graphene.

作者聚焦：使用石墨烯涂层网格提高 CryoEM 分辨率 

Here, we describe a protocol for applying a single monolayer of graphene to electron microscopy grids and how to prepare them for use in cryoEM structure determination.