Name: liquid-cell transmission electron microscopy for tracking self-assembly of nanoparticles
Uploaded: 2017-10-16T13:00:00.000+00:00
Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles at JoVE.com

我们感谢 Prof. Alivisatos 在加州大学伯克利分校和 Prof. Taeghwan 在首尔国立大学进行了有益的讨论。这项工作得到了 IBS-R006-D1 的支持。W.C.L. 感谢汉阳大学研究基金 (HY-2015-N) 的支持。

1. 纳米颗粒的合成 <ol> <li> 合成铂纳米粒子 <ol> 组合17.75 毫克铵铂 (iv) (NH <li> 4 ) 2 Pt (iv) Cl 6 ), 3.72 毫克酸铵 (ii) (NH 4 ) 2 Pt (ii) Cl 4 ), 115.5 毫克四甲基溴化铵, 109 毫克聚 (基) (兆瓦: 2.9万), 10 毫升的乙二醇, 在100毫升 3-颈圆底烧瓶中搅拌棒配有橡胶隔膜. </li>
		<li> 将烧瓶与回流冷凝器一起装备, 并在真空下清洗。搅拌的反应混合物与磁性搅拌棒在 1000 rpm. </li>
		<li> 将反应混合物加热到180和 #176; 在加热地幔中, 以10和 #176 的速率; 在氩流动下的 c/分钟. </li>
		<li> 保持温度 (180 和 #176; C) 为20分钟。该溶液变成深褐色的颜色. </li>
		<li> 从加热地幔中取出烧瓶, 使其冷却到室温. </li>
		<li> 将产品转移到50毫升离心管。在产品中加入30毫升丙酮, 以沉淀纳米颗粒, 将样品在 2400 x g 离心10分钟. 
		注意: 为安全起见, 此过程应在通风罩中执行. </li>
		<li> 用10毫升乙醇丢弃上清和 re-disperse 黑沉淀. </li>
		<li> 沉淀的产品与30毫升的甲苯和离心机的悬浮在 2400 x g 10 分钟. 丢弃上清和 re-disperse 的黑色沉淀与10毫升乙醇。重复此过程3次. </li>
		<li> 将5毫升的油加入铂纳米颗粒, 并将分散体转移到100毫升的圆底烧瓶中。在 1000 rpm 的磁搅拌下过夜分散, 使配体交换反应发生. </li>
		<li> 将配体交换的纳米粒子在 1万 x g 离心30分钟, 将它们与溶液分离. </li>
		<li> 丢弃上清液, 在10毫升的疏水溶剂中分散铂纳米颗粒, 如甲苯、己烷或氯仿. </li>
	</ol> </li> <li> 合成的硒化铅纳米粒子 <ol> 制备油酸铅的758毫克醋酸铅 (铅 (Ac) <li> 2 <ol> 和 #183; 3H <li> 2 O), 2.5 毫升油酸性, 和10毫升的二苯基醚与一个搅拌酒吧在100毫升3颈圆底烧瓶配备了橡胶隔膜. </li>
			<li> 将烧瓶与回流冷凝器一起装备. </li>
			<li> 加气在70和 #176; C 在真空条件下为 2 h, 搅拌与磁性搅拌棒在 1000 rpm. </li>
			<li> 用氩气清洗烧瓶, 然后冷却到室温. </li>
		</ol> </li> <li> 在一个单独的容器中制备膦硒化物 (TOPSe) <ol> <li>, 在惰性气氛下结合474毫克硒和6毫升的膦 (顶), 使用手套防止试剂暴露在空气中. </li>
			<li> 将硒粉溶解在顶部, 超声波在 110 W, 40 赫, 混合使用旋流混合器, 直到溶液变得明显透明. </li>
		</ol> </li> <li> 在单独的250毫升3颈圆底烧瓶中, 在120和 #176 上加气15毫升的二苯基醚; C 在真空条件下与磁性搅拌在1000分钟的 30 min. </li>
		<li> 用氩气清洗烧瓶, 然后将二苯基醚加热到230和 #176; C. </li>
		<li> 将油酸铅和 TOPSe 溶液迅速注入预热二苯醚。注射时温度会下降到一定程度。将老化温度设置为170和 #176; C. </li>
		<li> 将混合物的温度保持在170和 #176; C 为10分钟, 并进行剧烈搅拌, 以使硒化铅纳米颗粒的生长发生. </li>
		<li> 从加热地幔中取出烧瓶, 使其冷却到室温. </li>
		<li> 准备两个50毫升离心管, 将产品分成相等的体积并转移到管子。在每根管子上加入30毫升乙醇, 沉淀纳米颗粒, 并将悬浮液在 2400 x g 上离心10分钟. </li>
		<li> 丢弃上清和 re-disperse 10 毫升的甲苯. </li>
		<li> 沉淀30毫升乙醇和离心悬浮在 2400 x g 10 min. 丢弃上清和 re-disperse 的黑色沉淀与10毫升甲苯。重复此过程3次. </li>
		<li> 丢弃上清液, 将硒化铅纳米微粒分散在10毫升的疏水溶剂中, 如甲苯、己烷或氯仿. </li>
	</ol> </li> </ol> 2。液芯制作 <ol> <li> 氮化硅液体电池 ( 图 3a ) <ol> 沉积氮化硅层 <li> <ol> 将低氮化硅薄膜存入100和 #181. 由低压化学气相沉积 (LPCVD) 在835和 #176; C 和 140 mTorr 的压力, 100 sccm 烷和 50 sccm 氨的流动, m 厚硅片 (4 英寸)。通过改变沉积时间来控制氮化硅层厚度为 25 nm。沉积率为 2.5-3.0 nm/分钟, 但每个 CVD 的轻微变化. </li>
		</ol> </li> <li> 顶部和底部芯片 注: 有关微细加工过程的详细说明, 请参阅参考 25 。
		<ol> <li> 自旋涂层10毫升的正光刻胶在氮化硅/硅片上的 3000 rpm 为三十年代, 使用足够的光刻胶完全润湿晶片. 
			注: 在纺纱过程中, 超薄硅片容易破碎。我们通常将超薄晶片贴在另一硅片上, 厚度为500和 #181; m 使用光刻胶以避免破损。经旋涂后, 将薄晶圆片与浓晶片分离, 浸入丙酮中。该技术还可用于超薄硅片上的自旋涂层. </li>
			<li> 在85和 #176 的热板上烘烤硅片; C. 六十年代. 
			注意: 不要在 #62 的温度下烘烤准备好的晶片; 110 和 #176; c. 在高温下烘烤光刻胶会使光刻胶从正极变成负光刻胶. </li>
			<li> 通过铬面膜 ( 图 1a ) 将光刻胶涂层硅片暴露于十年代的紫外线 (365 nm). </li>
			<li> 将光刻胶涂覆的硅片浸入到50毫升的显影液中, 四十年代. 在50毫升的去离子水中浸泡1分钟冲洗晶片。重复洗涤过程两次. </li>
			<li> 使用活性离子蚀刻蚀刻氮化硅1分钟, 其流量为50的六氟化硫的 sccm. </li>
			<li> 将顶部芯片和底部芯片浸入丙酮中2分钟以除去光刻胶。用手轻轻摇动碟, 有效去除光刻胶. </li>
			<li> 用氢氧化钾 (30 毫克/毫升) 的水溶液在85和 #176 上蚀刻硅; C 为 1.5-2 h. 使用水浴, 以确保蚀刻溶液具有均匀的温度剖面。不均匀的温度会导致一些区域过度的 etched, 而有些是不完全蚀刻. </li>
			<li> 当窗口看起来完全被肉眼蚀刻时, 停止蚀刻并将电池从蚀刻液中取出。将电池从蚀刻液中取出时, 将其倾斜, 以避免窗口因浮力而破裂的可能性, 如果水平提升, 则可能发生. </li>
			<li> 使用底部芯片的掩码重复这些过程 ( 图 1b ). 
			注: 由于顶部和底部芯片的窗口非常薄, 大约 25 nm, 它们非常脆弱。因此, 在处理时应格外小心。当芯片停留在表面上时, 窗口的一侧应始终朝向向上. </li>
		</ol> </li> <li> 粘接顶部和底部芯片 <ol> <li> 自旋涂层在氮化硅/硅片上的正光刻胶10毫升, 在三十年代为 3000 rpm. </li>
			<li> 在90和 #176 的热板上烘烤硅片; C. 六十年代. </li>
			<li> 通过铬面膜 ( 图 1c ) 将光刻胶涂层硅片暴露于十年代的紫外线 (365 nm). </li>
			<li> 将光刻胶涂覆的硅片浸入到50毫升的显影液中, 四十年代. 在50毫升的去离子水中浸泡1分钟冲洗晶片。重复洗涤过程两次. </li>
			<li> 使用热蒸发器将 100 nm 厚的铟层沉积到底部芯片上。铟层用作垫片和密封材料. </li>
			<li> 将芯片浸入丙酮中2分钟以除去光刻胶。用手轻轻摇动碟, 有效去除光刻胶. </li>
			<li> 使用器将底部和顶部的芯片对齐, 并在100和 #176 之间进行粘合. </li>
		</ol> </li> </ol> </li> <li> 硅液-单元格 ( 图 3b ) <ol> <li> 准备顶部和底部芯片 <ol> <li> 使用具有层厚度的 p 型硅 (SOI) 硅片100 nm, 400 nm, 600 和 #181; m 为顶部的硅, 被埋没的 2 , 和处理硅层, 分别. </li>
			<li> 在 950-1100 和 #176 的氧化炉中进行 SOI 晶圆片的湿氧化; C. 以 170 nm 的厚度生长一层氧化硅. </li>
			<li> 在室温下将 SOI 硅片浸入到缓冲氧化物蚀刻 (BOE) 溶液中, 将硅氧化物蚀刻为2分钟, 这是一个湿蚀刻的氧化硅。步骤2.2.1.2 和2.2.1.3 的目的是将 SOI 硅片的顶部硅层厚度降低到 25 nm. </li>
			<li> 使用与步骤2.1.1.1 相同的工艺条件, 在 SOI 晶片 (4 英寸) 上沉积一层低氮化硅, 厚度为 25 nm. </li>
			<li> 使用与步骤 2.1.2.1 2.1.2.4 相同的进程和掩码模式来对氮化硅进行图案处理. </li>
			<li> 将硅片浸入丙酮中2分钟以除去光刻胶。用手轻轻摇动碟, 有效去除光刻胶. </li>
			<li> 在三十年代使用京东方解决方案蚀刻氧化硅. </li>
			<li> 用氢氧化钾 (500 毫克/毫升) 的水溶液在80和 #176 上蚀刻硅; C 为 7-12 h. 三甲胺 (TMAH) 水溶液也可用于硅蚀刻. </li>
			<li> 用85% 磷酸在160和 #176 上蚀刻氮化硅; C 为 10 min. </li>
			<li> 在室温下用京东方溶液蚀刻硅氧化物3分钟. </li>
			<li> 使用底部芯片的掩码重复步骤 2.2.1.1 2.2.1.10 ( 图 1b ). </li>
		</ol> </li> <li> 粘接顶部和底部芯片 <ol> <li> 自旋涂层在氮化硅/硅片上的正光刻胶10毫升, 在三十年代为 3000 rpm. </li>
			<li> 在90和 #176 的热板上烘烤硅片; C. 六十年代. </li>
			<li> 通过铬面膜 ( 图 1c ) 将光刻胶涂层硅片暴露于十年代的紫外线 (365 nm). </li>
			<li> 将光刻胶涂覆的硅片浸入到50毫升的显影液中, 四十年代. 在50毫升的去离子水中浸泡1分钟冲洗晶片。重复洗涤过程两次. </li>
			<li> 使用热蒸发器将 100 nm 厚的铟层沉积到底部芯片上。铟层用作垫片和密封材料. </li>
			<li> 将芯片浸入丙酮中2分钟以除去光刻胶。用手轻轻摇动碟, 有效去除光刻胶. </li>
			<li> 使用器将底部和顶部的芯片对齐, 并在100和 #176 之间进行粘合. </li>
		</ol> </li> </ol> </li> </ol> 3。液-细胞 TEM <ol> <li> 加载解决方案 <ol> <li> 添加20和 #181; L 的纳米颗粒分散 (协议1.1 和 1.2) 变成5毫升的小瓶, 在空气中晾干10分钟. 将纳米粒子分散在溶剂混合物中 (1 毫升的邻二氯苯,250、#181、烷、10、#181、油的 l). 
		注: 在极端条件下干燥, 如高温、低压和延长时间, 可能导致微粒分散。由于 ligand-to-particle 相互作用是动态的, 在极端条件下, 在配体脱离后, 颗粒团聚的几率很高。由于邻二氯苯、烷和油具有不同的蒸气压, 在加载过程前应立即进行干燥过程, 以保持溶剂比恒定. </li>
		<li> 使用光学显微镜检查液体细胞. 
		注: 如果任何一个液体细胞的窗户被打破, 不要使用该细胞. </li>
		<li> 将纳米颗粒分散的 100 nL 载入储层 ( 图 2a 和 2b ) 的液体细胞。配备有超薄毛细管 ( 图 2c ) 的注射器可以有效地将少量的色散加载到液细胞的储层中. 
		注: 一般情况下, 纳米颗粒分散的注入量超过了储层的容量。如果分散物溢出从水库, 液体细胞可能不完全地密封。因此, 在储层外的任何溢出的分散体都必须被滤纸的尖端吸收为扇形形状。在吸收过程中避免与窗户接触. </li>
		<li> 将液体电池暴露在空气中10分钟以使邻二氯苯干燥. </li>
		<li> 将真空油脂应用于铜孔径网格的一侧, 其大小为 2 mm, hole-size 为600和 #181; m, 并将液体电池覆盖在孔径网格的润滑一侧, 以创建密封环境. 
		注: 在窗户上沉积的真空油脂大大降低了 TEM 的分辨率。因此, 在第一次尝试时, 不应更正铜孔径网格与单元格窗口的孔匹配问题;相反, 该单元格应被丢弃, 并准备了一个新的. </li>
	</ol> </li> <li> tem 测量 <ol> <li> 将液体电池置于标准 tem 支架中。该液体电池设计成符合标准的持有人. 
		注意: 使用手套避免 TEM 支架的污染. </li>
		<li> 在连续图像获取模式下设置 TEM。在 200 kV 的加速度电压下捕获 TEM 图像, 电流密度为 ~ 700 米/秒 2 . 
		注: 经常检查 TEM 压力水平。如果压力水平不正常, 立即停止成像, 并尽快从 TEM 室中取出支架。液胞 TEM 的低对比度使图像聚焦困难。良好的对准和对焦可以很容易地执行初始粗糙的焦点在窗口的边缘。通过改变电流密度可以控制干燥速率。2D 干燥速率可以通过跟踪不断变化的干燥补丁的大小来测量。然而, 很难数量按容积计算干燥率. </li>
		<li> 使用 ImageJ 软件包打开原始 TEM 图像。选择该程序的多点图标, 并分配图像中所有单个纳米粒子的中心, 然后提取所选粒子的 x 和 y 坐标. </li>
		<li> 计算径向分布函数 (RDF): 
		<img alt="Equation 1" src="/files/ftp_upload/56335/56335eq1.jpg"/> 其中, r 是间距离, N 是粒子数, A 是粒子所覆盖的区域, 和 #961; 是2D 的面积密度粒子 ( N / A ), <img alt="Equation 2" src="/files/ftp_upload/56335/56335eq2.jpg"/> 是从粒子 j 到粒子 k 的位置向量, 和 #948; ( r ) 是狄拉克δ函数。我们使用和 #160; <img alt="Equation 3" src="/files/ftp_upload/56335/56335eq3.jpg"/> where = 0.8 nm 作为狄拉克δ函数进行实际计算. </li>
	</ol>
	</li>
</ol>

<ol>
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作者没有什么可透露的。

以铂铵 (IV) 和酸铵 (II) 为原料, 用聚 (基) (PVP) 作为配体和乙二醇作为溶剂和还原剂, 合成了 7 nm 的铂纳米颗粒.27.用油进行配体交换反应, 在疏水溶剂中分散颗粒。以硒为原料, 将铅油酸酯配合物的热分解, 合成了铅化硫纳米颗粒,28 (参见参考29 , 用于详细合成的纳米晶)。由于合成的铅硒化物纳米颗粒已经被长链配体所覆盖, 这些微粒不需要配基交...

胶体纳米粒子的自组装是有兴趣的, 因为它提供了一个机会来获得单个纳米粒子的集体物理性质11。在实际的设备规模应用中, 自组装最有效的方法之一是在基板上通过挥发性溶剂的蒸发来自我组织,6,7,8,9,10,11. 这种溶剂蒸发法是一种非平衡过程, 主要受蒸发速率和纳米粒子-基体相互作用的变化等动力学因素的影响。然而, 由于难以估计和控制的动力学因素, 机械理解纳米粒子自组装的溶剂蒸发不完全成熟。虽然原位X 射线散射研究提供了非平衡纳米粒子自组装过程的集合平均信息,12,13,14, 此技术不能确定单个纳米粒子的运动, 它们与整体轨迹的联系不能轻易地被访问。
液体细胞 TEM 是一种新的工具, 跟踪单个纳米粒子的轨迹, 使我们能够理解纳米粒子运动的不均匀性及其对合奏行为的贡献15,16, 17,18,19,202122,2324,25 26。我们以前用液体细胞 TEM 跟踪单个纳米粒子在溶剂蒸发过程中的运动, 表明溶剂边界的运动是诱导纳米粒子自组装的主要推动力18,19. 本文介绍了利用液-细胞 TEM 观察纳米粒子自组装过程的实验。首先, 我们提供了合成铂和硒化铅纳米粒子的协议, 然后介绍了 TEM 中的液细胞的制备方法, 以及如何将纳米微粒加载到液细胞中。作为代表性的结果, 我们展示了由溶剂干燥驱动的纳米粒子自组装的 TEM 电影的快照图像。通过跟踪这些电影中的单个粒子, 我们可以了解在单个纳米粒子水平上的溶剂-干燥-介导的自组装的详细机制。在自组装过程中, 氮化硅窗口上的铂纳米粒子主要跟随蒸发溶剂前缘的运动, 因为在薄溶剂层上的强毛细管力作用。其他的纳米粒子 (硒化铅) 和基体 (硅) 也有类似现象, 这表明溶剂锋的毛细管力是粒子在基底附近迁移的一个重要因素。

<table><tbody><tr><td>ammonium hexachloroplatinate (IV)</td><td>Sigma-Aldrich</td><td>204021</td><td></td></tr><tr><td>ammonium tetrachloroplatinate (II)</td><td>Sigma-Aldrich</td><td>206105</td><td></td></tr><tr><td>tetramethylammonium bromide, 98%</td><td>Sigma-Aldrich</td><td>195758</td><td></td></tr><tr><td>poly(vinylpyrrolidone) powder</td><td>Sigma-Aldrich</td><td>234257</td><td>Mw ~29,000</td></tr><tr><td>ethylene glycol, anhydrous, 99.8%</td><td>Sigma-Aldrich</td><td>324558</td><td></td></tr><tr><td>n-hexane, anhydrous, 95%</td><td>Samchun Chem.</td><td>H0114</td><td></td></tr><tr><td>ethanol, anhydrous, 99.5%</td><td>Sigma-Aldrich</td><td>459836</td><td></td></tr><tr><td>oleylamine, 70%</td><td>Sigma-Aldrich</td><td>O7805</td><td>Technical grade</td></tr><tr><td>lead(II) acetate trihydrate, 99.99%</td><td>Sigma-Aldrich</td><td>467863</td><td></td></tr><tr><td>oleic acid, 90%</td><td>Sigma-Aldrich</td><td>364525</td><td>Technical grade</td></tr><tr><td>diphenyl ether, 99%</td><td>Sigma-Aldrich</td><td>P24101</td><td>ReagentPlus</td></tr><tr><td>selenium powder, 99.99%</td><td>Sigma-Aldrich</td><td>229865</td><td></td></tr><tr><td>tri-n-octylphosphine, 97%</td><td>Strem</td><td>15-6655</td><td>Air sensistive</td></tr><tr><td>Toluene, anhydrous, 99.9%</td><td>Samchun Chem.</td><td>T2419</td><td></td></tr><tr><td>acetone 99.8%</td><td>Daejung Chem.</td><td>1009-2304</td><td></td></tr><tr><td>potassium hydroxide, 95%</td><td>Samchun Chem.</td><td>P0925</td><td></td></tr><tr><td>p-type silicon-on-insulator wafers</td><td>Soitec</td><td>Power-SOI</td><td>for liquid cells with silicon windows</td></tr><tr><td>tetramethylammonium hydroxide, 25% in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>J.T.Baker</td><td>02-002-109</td><td></td></tr><tr><td>AZ 5214 E</td><td>AZ Electronic Materials</td><td>AZ 5214 E</td><td>Positive photorest</td></tr><tr><td>AZ-327</td><td>AZ Electronic Materials</td><td>AZ-327</td><td>AZ 5214 develper</td></tr><tr><td>indium pellets 99.98-99.99%</td><td>Kurt J. Lesker Company</td><td>EVMIN40EXEB</td><td>thermal evaporator target</td></tr><tr><td>1,2-dichlorobenzene, &gt;99%</td><td>TCI</td><td>D1116</td><td></td></tr><tr><td>pentadecane, &gt;99%</td><td>Sigma-Aldrich</td><td>P3406</td><td></td></tr><tr><td>buffered oxide etch 7:1</td><td>microchemicals</td><td>BOE 7-1 VLSI</td><td></td></tr><tr><td>phosphoric acid, 85%</td><td>Samchun Chem.</td><td>P0449</td><td></td></tr></tbody></table>

liquid-cell transmission electron microscopy for tracking self-assembly of nanoparticles

干燥纳米粒子的分散是建立纳米微粒自组装结构的一种通用方法, 但这一过程的机制还没有完全被理解。我们用液-细胞透射电镜 (TEM) 追踪了单个纳米粒子的运动轨迹, 以研究其组装过程的机理。在此, 我们提出的协议用于液体细胞 TEM 研究的自组装机制。首先, 我们介绍了详细的合成协议, 用于生产均匀大小的铂和硒化铅纳米颗粒。接下来, 我们介绍了用氮化硅或硅窗生产液态细胞的微细加工过程, 然后描述了液-细胞 TEM 技术的加载和成像过程。包括一些说明, 为整个过程提供有用的提示, 包括如何管理脆弱的细胞窗口。通过液-胞 TEM 跟踪的纳米粒子的单个运动表明, 蒸发引起的溶剂边界的变化影响了纳米粒子的自组装过程。溶剂的边界驱使纳米颗粒主要形成无定形的聚合体, 其次是聚集体的扁平化以产生2维 (2D) 自组装结构。这些行为也观察到不同的纳米颗粒类型和不同的液体细胞组成。

该液芯由一个顶部芯片和一个底部芯片组成, 它装有氮化硅窗口, 对电子束具有25纳米厚度的透明。顶部芯片有一个储存样品溶液和蒸发溶剂的储罐。这些芯片是通过传统的微细加工处理25。用于顶部和底部芯片的掩码分别显示在图 1a和 1b中。图 2a和 2b分别显?...

Watch this Scientific Journal Video about Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles at JoVE.com

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

用于跟踪纳米粒子自组装的液体细胞透射电子显微镜

在这里, 我们介绍了实验协议的 real-time 观察的自组装过程中使用液体细胞透射电镜。

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

liquid-cell-transmission-electron-microscopy-for-tracking-self

Research

JoVE Journal

Chemistry

12.6K 次观看.  Institute for Basic Science (IBS). 在这里, 我们介绍了实验协议的 real-time 观察的自组装过程中使用液体细胞透射电镜。

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

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 ...

科研

工程学

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

生物化学

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

就地 液体生物组件的 TEM

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

生物工程

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 ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Studying the Effects of Temperature on the Nucleation and Growth of Nanoparticles by Liquid-Cell Transmission Electron Microscopy

Temperature control is a recent development that provides an additional degree of freedom to study nanochemistry by liquid cell transmission electron microscopy. In this paper, we describe how to prepare an in situ heating experiment for studying the effect of temperature on the formation of gold nanoparticles driven by radiolysis in water. The protocol of the experiment is fairly simple involving a special liquid cell with uniform heating capabilities up to 100 &#176;C, a liquid-cell TEM holder with flow capabilities and an integrated interface for controlling the temperature. We show that the nucleation and growth mechanisms of gold nanoparticles are drastically impacted by the temperature in liquid cell. Using STEM imaging and nanodiffraction, the evolution of the density, size, shape and atomic structure of the growing nanoparticles are revealed in real time. Automated image processing algorithms are exploited to extract useful quantitative data from video sequences, such as the nucleation and growth rates of nanoparticles. This approach provides new inputs for understanding the complex physico-chemical processes at play during the liquid-phase synthesis of nanomaterials.

Temperature control during liquid-phase electron microscopy experiments opens up new perspectives of studying the dynamic of nanoparticles in liquid environments mimicking their formation or application media. Using recently developed heating liquid cells, we directly observed the influence of temperature on the nucleation and growth processes of gold nanoparticles in water.

研究温度对液细胞传输电子显微镜对纳米粒子核化和生长的影响

Temperature control is a recent development that provides an additional degree of freedom to study nanochemistry by liquid cell transmission electron ...

化学

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

推进液体和冰中病毒组件的高分辨率成像

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

聚合物拥挤脂质膜上的单分子扩散和组装

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

直接合成金纳米颗粒用于蛋白质定位分析

Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various physiological or pathological processes in all living organisms. Therefore, the development of clonable tags that can be used as electron microscopy probes is of great value, just as fluorescent proteins have played a crucial role in the field of optical imaging. The autonucleation suppression mechanism (ANSM) was recently uncovered, which allows for the specific synthesis of gold nanoparticles (AuNPs) on cysteine-rich tags, such as metallothionein (MT) and antifreeze protein (AFP).
Based on the ANSM, an electron microscopy labeling technology was developed, which enables the specific detection of tagged proteins in prokaryotic and eukaryotic cells with an unprecedented labeling efficiency. This study illustrates a protocol for the detection of MTn (an engineered MT variant lacking aldehyde-reactive residues) fusion proteins in mammalian cells with well-preserved ultrastructure. In this protocol, high-pressure freezing and freeze-substitution fixation were performed using non-aldehyde fixatives (such as tannic acid, uranyl acetate) to preserve near-native ultrastructure and avoid damage to the tag activity caused by aldehyde crosslinking.
A simple one-step rehydration was used prior to the ANSM-based AuNP synthesis. The results showed that the tagged proteins targeted various organelles, including the membranes and the lumen of the endoplasmic reticulum (ER), and mitochondrial matrices were detected with high efficiency and specificity. This research provides biologists with a robust protocol to address an enormous range of biological questions at the single-molecule level in cellular ultrastructural contexts.

作者聚焦：在细胞中直接合成 EM-Visible 金纳米颗粒，用于具有保存完好的超微结构的蛋白质定位分析  

The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique.

用于跟踪纳米粒子自组装的液体细胞透射电子显微镜

摘要

探索更多视频

此视频中的章节

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

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

就地液体生物组件的 TEM

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

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

研究温度对液细胞传输电子显微镜对纳米粒子核化和生长的影响

推进液体和冰中病毒组件的高分辨率成像

聚合物拥挤脂质膜上的单分子扩散和组装

在细胞中直接合成EM-可见金纳米颗粒，用于蛋白质定位分析，具有保存完好的超微结构

在细胞中直接合成EM-可见金纳米颗粒，用于蛋白质定位分析，具有保存完好的超微结构

用于跟踪纳米粒子自组装的液体细胞透射电子显微镜

摘要

探索更多视频

此视频中的章节

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

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

就地 液体生物组件的 TEM

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

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

研究温度对液细胞传输电子显微镜对纳米粒子核化和生长的影响

推进液体和冰中病毒组件的高分辨率成像

聚合物拥挤脂质膜上的单分子扩散和组装

在细胞中直接合成EM-可见金纳米颗粒，用于蛋白质定位分析，具有保存完好的超微结构

在细胞中直接合成EM-可见金纳米颗粒，用于蛋白质定位分析，具有保存完好的超微结构

就地液体生物组件的 TEM