Name: in situ tem of biological assemblies in liquid
Uploaded: 2013-12-30T20:00:00
Description: Watch this Scientific Journal Video about In situ TEM of Biological Assemblies in Liquid at JoVE.com

作者感谢弗吉尼亚理工大学卡里里昂研究所所长迈克尔·弗里德兰德博士鼓励我们的研究努力。该项目得到了S.M.M和D.F.K.的发展基金的支持，部分得到了弗吉尼亚理工大学关键技术和应用科学研究所的纳米生物倡议的支持。

1. 准备亲和力捕获设备16
<ol><li>清洁氮化硅E芯片，在15毫升乙酮中孵育2分钟，然后用15毫升甲醇2分钟（图1A）。允许芯片在层压气流下干燥。</li>
	<li>在加热搅拌板上（不搅拌）上孵化干屑，在 150 °C 下 1.5 小时，然后让它们在使用前冷却到室温。</li>
	<li>使用汉密尔顿注射器在小玻璃管中合成脂质混合物，以包含 25% 的氯仿， 55% DLPC （1，2-迪劳罗伊?...

<ol>
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U.S.A. 107, 6298-6303 (2010).</li><li>Dubochet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+microscopy+of+vitrified+specimens.">Cryo-electron microscopy of vitrified specimens.</a> Q. Rev. Biophys. 21, 129-228 (1988).</li><li>Unwin, P. N., Henderson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+structure+determination+by+electron+microscopy+of+unstained+crystalline+specimens.">Molecular structure determination by electron microscopy of unstained crystalline specimens.</a> J. Mol. 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Improved environmental chambers make it possible to examine wet specimens easily.</a> Science. 186, 407-414 (1974).</li><li>Parsons, D. F., Matricardi, V. R., Moretz, R. C., Turner, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+and+diffraction+of+wet+unstained+and+unfixed+biological+objects.">Electron microscopy and diffraction of wet unstained and unfixed biological objects.</a> Adv. Biol. Med. Phys. 15, 161-270 (1974).</li><li>Hui, S. W., Parsons, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+diffraction+of+wet+biological+membranes.">Electron diffraction of wet biological membranes.</a> Science. 184, 77-78 (1974).</li><li>Hui, S. W., Parsons, D. F., Cowden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+diffraction+of+wet+phospholipid+bilayers.">Electron diffraction of wet phospholipid bilayers.</a> Proc. Natl. Acad. Sci. U.S.A. 71, 5068-5072 (1974).</li><li>Ring, E. A., de Jonge, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+system+for+transmission+electron+microscopy.">Microfluidic system for transmission electron microscopy.</a> Microsc. Microanal. 16, 622-629 (2010).</li><li>Dukes, M. J., Ramachandra, R., Baudoin, J. P., Gray Jerome, W., de Jonge, N. <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+locations+of+gold-labeled+proteins+in+a+whole+mount+eukaryotic+cell+obtained+with+3nm+precision+using+aberration-corrected+scanning+transmission+electron+microscopy.">Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy.</a> J. Struct. Biol. 174, 552-562 (2011).</li><li>Yuk, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+EM+of+colloidal+nanocrystal+growth+using+graphene+liquid+cells.">High-resolution EM of colloidal nanocrystal growth using graphene liquid cells.</a> Science. 336, 61-64 (2012).</li><li>Peckys, D. B., de Jonge, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+gold+nanoparticle+uptake+in+live+cells+with+liquid+scanning+transmission+electron+microscopy.">Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy.</a> Nano Lett. 11, 1733-1738 (2011).</li><li>Klein, K. L., Anderson, I. M., de Jonge, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+electron+microscopy+with+a+liquid+flow+cell.">Transmission electron microscopy with a liquid flow cell.</a> J. Microsc. 242, 117-123 (2011).</li><li>Degen, K., Dukes, M., Tanner, J. R., Kelly, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+affinity+capture+devices-a+nanoscale+purification+platform+for+biological+in+situ+transmission+electron+microscopy.">The development of affinity capture devices-a nanoscale purification platform for biological in situ transmission electron microscopy.</a> Rsc. Adv. 2, 2408-2412 (2012).</li><li>Gilmore, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+viral+assemblies+in+a+nanoscale+biosphere.">Visualizing viral assemblies in a nanoscale biosphere.</a> Lab Chip. 13, 216-219 (2013).</li><li>Bican, P., Cohen, J., Charpilienne, A., Scherrer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+bovine+rotavirus+cores.">Purification and characterization of bovine rotavirus cores.</a> J. Virol. 43, 1113-1117 (1982).</li><li>Frank, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPIDER+and+WEB:+processing+and+visualization+of+images+in+3D+electron+microscopy+and+related+fields.">SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields.</a> J. Struct. Biol. 116, 190-199 (1996).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-atomic+resolution+using+electron+cryomicroscopy+and+single-particle+reconstruction.">Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 1867-1872 (2008).</li><li>Scheres, S. 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在我们的介绍工作中，我们采用亲和力捕获方法将轮状病毒 DLP 系在微流体平台上。这允许在液体微环境下对大分子复合物进行 原位 成像。捕获 方法对于其他微流体成像技术具有重要意义 ，因为它将生物标本定位到成像窗口，以否定在以液体中记录图像时出现的大型扩散问题。然而， 我们协议中最关键的步骤 之一是将脂质生物膜转移到微芯片表面。如果脂质单层...

生物学家和工程师的共同目标是了解分子机器的内部工作。传输电子显微镜 （TEM） 是以近原子分辨率1-2可视化这些复杂细节的理想仪器。为了维持TEM的高真空系统，生物样本通常嵌入在薄膜的微薄冰3，糖4，重金属盐5，或其中的一些组合6。因此，嵌入式标本的图像可能只显示动态过程的有限快照。
帕森斯及其同事利用差分泵送阶段，对环境液体室中水合的生物标本进行了早期维护尝试。未染色催化晶体的电子衍射模式在水合状态7-8中被成功记录到3的分辨率。此外，相分离的脂质域可以在人类红细胞9-10的水合膜中检查。然而，由于扩散液体及其对电子束的干扰引起的运动导致严重分辨率损失，直到最近才尝试使用生物标本进行进一步实验。
新开发的微流体标本持有者已引进，利用半导体微芯片形成微尺度环境室。这些设备可以在将样品放置在 TEM 列11-12中时，将样品保持在液体中。TEM成像技术上的这一突破使研究人员首次能够在分子水平13上看到渐进事件。我们称这种新模式为"原位 分子显微镜"，因为现在可以在EM列14-15的"内部"进行实验。该方法的总体目标是在液体中对生物组件进行成像，以便以纳米分辨率观察其动态行为。开发技术背后的理由是记录实时观测，并检查生物机械在溶液中的新特性。这种方法扩展了TEM在细胞和分子生物学12-16中用于更广泛的目的。
在当前的视频文章中，我们提出了一个全面的协议，以组装和使用市售的微流体标本支架。这些专业支架利用用集成垫片生产的氮化硅微芯片形成一个液体腔室，将微量溶液包围起来。薄而透明的窗户被蚀刻在微芯片中，用于成像目的12。我们演示了使用微流体支架使用 TEM 检查液体中的模拟轮状病毒双层颗粒 （DLP） 的正确用途。为了确保生物组件（如DLP）在成像时不会在很远的距离上快速扩散，我们采用亲和力捕获方法将它们系在微流体16的表面。与液体中生物标本成像 的替代技术而言 ，这种分子捕获步骤具有重大优势，因为它允许获取用于下游处理程序的图像。这个捕获步骤与微流体成像相结合，是我们程序17所独有的。使用 TEM 或微流体成像室使用结构生物学应用程序的读者可能会考虑在分子水平上的动态观测是最终目标时使用亲和力捕获技术。

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	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">E-chips, spacer chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPB-52TBD</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:237px;">E-chip, top chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPT-45W</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ni-NTA lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">790404P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">DLPC (12:0) lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">850335P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Volumetric flasks&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">20-812A; 20-812C</td>
			<td style="width:167px;">1 ml; 5 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Hamilton Syringes</td>
			<td style="width:183px;">Hamilton Co.</td>
			<td style="width:245px;">80300, 80400</td>
			<td style="width:167px;">1-10 &#956;l; 1-25 &#956;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Whatman #1 filter paper</td>
			<td style="width:183px;">Whatman</td>
			<td style="width:245px;">1001 090</td>
			<td style="width:167px;">100 pieces, 90 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Petri dishes</td>
			<td style="width:183px;">Corning&#160;</td>
			<td style="width:245px;">70165-101</td>
			<td style="width:167px;">100 mm x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Pasteur pipettes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">14673-010</td>
			<td style="width:167px;">14673-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass culture tubes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">47729-566</td>
			<td style="width:167px;">6 mm x 50 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Acetone</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A11-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Methanol</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A412-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Chloroform&#160;</td>
			<td style="width:183px;">Electron Microcopy Sciences</td>
			<td style="width:245px;">12550</td>
			<td style="width:167px;">100 ml&#160;</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">His-tagged Protein A</td>
			<td style="width:183px;">Abcam, Inc.</td>
			<td style="width:245px;">ab52953</td>
			<td style="width:167px;">10 mg</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Milli-Q water system</td>
			<td style="width:183px;">EMD Millipore Corp.</td>
			<td style="width:245px;">Z00QSV001</td>
			<td style="width:167px;">Ultrapure Water</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">HEPES</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">BP310-500</td>
			<td style="width:167px;">500 g</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Equipment&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:245px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Poseidon In situ specimen holder</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;FEI compatible</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">FEI Spirit BioTwin TEM</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>120 kV</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Eagle 2k HS CCD camera</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>10&#160;&#197;/pixel sampling at 30,000X</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:237px;">Gatan 655 Dry pump station</td>
			<td style="width:183px;">Gatan, Inc.</td>
			<td style="width:245px;"></td>
			<td>Pump holder tip to 10-6&#160;range</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">PELCO easiGlow, glow discharge unit</td>
			<td style="width:183px;">Ted Pella, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;Negative polarity mode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Isotemp heated stir plate</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;"></td>
			<td>Heat to 150 &#186;C for 1.5 hr</td>
		</tr>
	</tbody>
</table>

in situ tem of biological assemblies in liquid

研究人员定期使用透电电子显微镜（TEM）来检查生物实体和评估新材料。在这里，我们描述了这些仪器的附加应用 - 在液体环境中查看病毒组件。这种令人激动和新颖的生物结构可视化方法利用了最近开发的基于微流体的标本支架。我们的视频文章演示了如何组装和使用微流体支架在 TEM 内对液体标本进行成像。特别是，我们使用模拟轮状病毒双层颗粒 （DLP） 作为我们的模型系统。我们还描述了用亲和力生物膜覆盖液体室表面的步骤，这些生物膜将 DLP 与观看窗口绑在一起。这使我们能够以适合 3D 结构确定的方式对组件进行成像。因此，我们首次在原生液体环境中看到亚病毒粒子。

使用发光释放的电子芯片（图 3A）在液体中使用 DDLP 的代表性图像显示，与在亲和力捕获设备上浓缩的 DDLP相比，在给定观看区域（大概是由于扩散）中的 DLP 较少。在成像室中加入尿素用于增强标本的对比度，从而提高溶液中单个 DLP 的可见性（图 3C，顶部面板）。更好的对比度允许下游图像处理，如粒子平均（图3C，底部面板）和3D重建例程（<...

Watch this Scientific Journal Video about In situ TEM of Biological Assemblies in Liquid at JoVE.com

In situ TEM of Biological Assemblies in Liquid

就地 液体生物组件的 TEM

在这里，我们描述了一个程序，用传输电子显微镜以纳米分辨率对液体中的病毒复合物进行成像。

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In situ TEM of Biological Assemblies in Liquid

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Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

可扩展的，金属的高宽比纳米复合材料在生物液体中产生

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

在生物膜的力学性能原位制图粒子追踪Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

交变磁场敏感的混合明胶微凝胶药物控释

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

在生物环境的新技术的产生和观测化学发光

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

就地 液体生物组件的 TEM

就地液体生物组件的 TEM