Name: correlative microscopy for 3d structural analysis of dynamic interactions
Uploaded: 2013-06-24T13:00:00
Description: Watch this Scientific Journal Video about Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions at JoVE.com

笔者想感谢特拉维斯·惠勒和细胞生物学系的机加工车间，生理学，美国匹兹堡大学的低温荧光样品阶段，长芦涛和徐诚在中国科学技术大学建设的技术帮助和批判性阅读的手稿的邓丽君Brosenitsch博士。这项工作是由美国国立卫生研究院（GM082251 GM085043）的支持。

1。文化对HeLa细胞碳包覆，黄金EM搜索网格<ol><li>辉光放电的碳 - 侧的200目R2 / 2的Quantifoil合金EM取景网格下25毫安，持续25秒。 </li><li> EM网格纤维连接蛋白，漂浮，碳端下来，到50微克/毫升纤维连接蛋白溶液40微升滴外套，然后消毒，在紫外光下2小时，在组织培养罩。 </li><li>铠到网格密度为2×10 4个细胞/毫升（总共有2毫升培养）在玻璃底培养皿的HeLa细胞和生长的细胞在DMEM，在37℃，5％CO ...

<ol>
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D., Simon, S. 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L., Zhang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+structure+determination+of+native+mammalian+cells+using+cryo-FIB+and+cryo-electron+tomography.">3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography.</a> Journal of Structural Biology. 180, 318-326 (2012).</li><li>Sartori, A., 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=Correlative+microscopy:+bridging+the+gap+between+fluorescence+light+microscopy+and+cryo-electron+tomography.">Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography.</a> Journal of Structural Biology. 160, 135-145 (2007).</li><li>Schwartz, C. L., Sarbash, V. I., Ataullakhanov, F. I., McIntosh, J. R., Nicastro, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-fluorescence+microscopy+facilitates+correlations+between+light+and+cryo-electron+microscopy+and+reduces+the+rate+of+photobleaching.">Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching.</a> Journal of Microscopy. 227, 98-109 (2007).</li><li>van Driel, L. F., Valentijn, J. A., Valentijn, K. M., Koning, R. I., Koster, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+correlative+cryo-fluorescence+microscopy+and+cryo-electron+tomography+applied+to+whole+mitochondria+in+human+endothelial+cells.">Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells.</a> European Journal of Cell Biology. 88, 669-684 (2009).</li><li>Rigort, A., 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=Micromachining+tools+and+correlative+approaches+for+cellular+cryo-electron+tomography.">Micromachining tools and correlative approaches for cellular cryo-electron tomography.</a> Journal of Structural Biology. 172, 169-179 (2010).</li><li>Gruska, M., Medalia, O., Baumeister, W., Leis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+tomography+of+vitreous+sections+from+cultured+mammalian+cells.">Electron tomography of vitreous sections from cultured mammalian cells.</a> Journal of Structural Biology. 161, 384-392 (2008).</li><li>Jun, S., 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=Direct+visualization+of+HIV-1+with+correlative+live-cell+microscopy+and+cryo-electron+tomography.">Direct visualization of HIV-1 with correlative live-cell microscopy and cryo-electron tomography.</a> Structure. 19, 1573-1581 (2011).</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> Quarterly Reviews of Biophysics. 21, 129-228 (1988).</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> Journal of Structural Biology. 116, 71-76 (1996).</li><li>Whitfield, M. 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=Identification+of+genes+periodically+expressed+in+the+human+cell+cycle+and+their+expression+in+tumors.">Identification of genes periodically expressed in the human cell cycle and their expression in tumors.</a> Molecular Biology of the Cell. 13, 1977-2000 (2002).</li><li>Agronskaia, A. V., 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=Integrated+fluorescence+and+transmission+electron+microscopy.">Integrated fluorescence and transmission electron microscopy.</a> Journal of Structural Biology. 164, 183-189 (2008).</li><li>Marko, M., Hsieh, C., Schalek, R., Frank, J., Mannella, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused-ion-beam+thinning+of+frozen-hydrated+biological+specimens+for+cryo-electron+microscopy.">Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy.</a> Nature Methods. 4, 215-217 (2007).</li></ol>

我们已经提出了一个简单的协议，提供了先进的方法来分析相关动态的细胞活动，使用时间推移共聚焦活细胞荧光成像由cryoET其次。 3D活细胞成像高分辨率cryoET的关联我们的方法学的发展是至关重要的调查许多具有挑战性的生物学问题，如可视化与宿主细胞的罕见的，动态的（不是一成不变的，如前期报告），衍射极限的病毒粒子及其相互作用。在活细胞中的特定颗粒的空间和时间的行为，通过?...

低温电子断层扫描（cryoET）是一个功能强大的成像技术，可以使细胞和组织的三维（3D）的可视化，并提供洞察组织在接近生理状态1的原生细胞器和细胞结构分子分辨率。然而，固有的低对比度冷冻水合未染色的标本，结合其辐射敏感性，使得它很难感兴趣的区域的单元格内，并随后执行倾斜系列的不破坏目标区域的情况下成功。为了克服这些问题，相关的方法，结合光学和电子显微镜是必要的。强调的荧光标记的特定的功能是确定和荧光光镜位于，然后它们的坐标被传输到采集的高分辨率三维结构数据的电子显微镜。此相关方法有助于定位目标相对的利益加以解决。由于与冷冻蚀刻电镜（小于300 nm）对样品厚度的限制，目前仅在小区的周边区域是合适的三维结构分析CryoET的。由冷冻水合标本的厚度进一步减少玻璃体切片或冷冻聚焦离子束（FIB）铣9将扩大相关成像能力。 此前，相关的方法进行主要用于促进cryoET数据采集，大和静态结构10-13。在这些研究中，低温阶段已经实施，接受冷冻蚀刻电镜电网和适合到一个堂堂正正的显微镜倒置显微镜10,11,14。虽然开关网格似乎相当简单，在他们的设计中，还有额外的步骤为EM网格传输，提高电网可能会变形，损坏和污染的机会。我们最近展示了技术的进步相关显微镜，使我们能够直接可视化动态本质上是很难捕捉到的事件，如HIV-1和宿主细胞的相互作用，在感染的早期阶段15。我们做到了这一点，设计和实施低温光镜样品阶段，适应的盒系统，以尽量减少由于网格处理的试件破坏，从而促进相关。我们的设计包括一个集成的试​​样盒保持器，使冷冻光低温电子显微镜进行，顺序，在相同的试样夹持器，没有样品的传输，从而简化相关工艺。此外，我们还实施了准确，可靠的相关程序，使用荧光乳胶珠作为基准标记。

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>DMEM 4.5 g/L Glucose w/ L-Glutamine</td>
			<td>Atlanta Biologicals, Inc. Lawrenceville, GA</td>
			<td>12-604F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma, St. Louis, MO</td>
			<td>F1141-1MG</td>
			<td>HeLa cell culture on EM grids</td>
		</tr>
		<tr>
			<td>Cell tracker</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>C34552</td>
			<td>Red CMTPX</td>
		</tr>
		<tr>
			<td>Protein A gold conjugates</td>
			<td>Ted Pella, Inc., Redding, CA</td>
			<td>15822-1</td>
			<td>15 nm diameter</td>
		</tr>
		<tr>
			<td>0.2 &mu;m fluorescent microspheres</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>F8811</td>
			<td>Yellow-green fluorescent</td>
		</tr>
		<tr>
			<td>Heat-inactivated fetal calf bovine serum</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>10082-139</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass-bottom culture dish</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gold quantifoil finder EM grids</td>
			<td>Quantifoil Micro Tools, Jena, Germany</td>
			<td>R2/2 Au NH2 200 mesh</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>70011-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Glow-discharge device 100X</td>
			<td>EMS, Hatfield, PA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tecnai Polara G2 electron microscope with a Field Emission Gun</td>
			<td>FEI, Hillsboro, OR</td>
			<td>&nbsp;</td>
			<td>300 keV</td>
		</tr>
		<tr>
			<td>Vitrobot Mark III</td>
			<td>FEI, Hillsboro, OR</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus IX 71 microscope</td>
			<td>Olympus America Inc., Center Valley, PA</td>
			<td>&nbsp;</td>
			<td>LUCPlanFLN 40X/0.6 NA (2.7-4 mm working distance) objective lens</td>
		</tr>
		<tr>
			<td>Nikon TiE microscope</td>
			<td>Nikon Instruments, Melville, NY</td>
			<td>&nbsp;</td>
			<td>using a 60X/1.35 NA oil immersion objective lens</td>
		</tr>
		<tr>
			<td>Sweptfield confocal microscope</td>
			<td>Prairie Technologies, Middleton, WI</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tokai Hit live cell chamber</td>
			<td>Tokyo, Japan</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryo-fluorescence sample stage</td>
			<td>Home-made</td>
			<td>&nbsp;</td>
			<td>Homebuilt by machine shop, see reference 15 for the design.</td>
		</tr>
	</tbody>
</table>

correlative microscopy for 3d structural analysis of dynamic interactions

低温电子断层扫描（cryoET）允许蜂窝结构的三维可视化分子分辨率在接近生理状态1。然而，直接可视化的个别病毒复合物在宿主细胞环境与cryoET是具有挑战性的，由于病毒进入的偶发性和动态特性，特别是在HIV-1的情况下。虽然时间推移活细胞成像已经取得了很大的许多方面的信息生命周期的HIV-1 3-7，所提供活细胞显微镜的分辨率是有限的（约200纳米）。我们工作的目的是允许直接可视化的HIV-1感染的早期事件的相关方法相结合，活细胞荧光光学显微镜，低温荧光显微镜，cryoET的发展。在这种方式下，活细胞和低温荧光信号可以被用来准确地引导在cryoET采样。此外，结构从cryoET可以得到的信息ê辅以动态功能数据获得通过活细胞成像的荧光标记的目标。 在这个视频文章中，我们提供了详细的方法和协议结构的研究HIV-1和宿主细胞的相互作用，使用3D相关高速活细胞成像和高分辨率cryoET的结构性分析。 HeLa细胞感染HIV-1的颗粒，其特征在于第一活细胞共聚焦显微镜，并含有相同的病毒颗粒的区域，然后通过低温电子断层扫描三维结构的细节。使用两套成像数据，光学成像和电子成像之间的相关性达到了一个家庭建造的低温荧光光学显微镜阶段。这里详细的做法将是有价值的，不仅为研究病毒宿主细胞的相互作用，同时也为在细胞生物学的更广泛的应用，如细胞信号传导，膜受体贩运，和许多其他动态细胞过程。/ P&gt;

为特征的病毒颗粒的动态行为，用HIV-1感染的HeLa细胞中，通过高速活细胞共聚焦显微镜成像和粒子运动由自动化的三维粒子跟踪的（ 图1）进行了分析。几分钟之间可能发生的收集，最后的活细胞共聚焦图像和小型冷冻（其可以是足够长的时间失去相关性的HIV-1颗粒），低温荧光显微镜阶段（ 图3中 ，以避免时间的推移）的设计和构造成像冷冻水合样品15，冷冻蚀?...

Watch this Scientific Journal Video about Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions at JoVE.com

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

我们描述了相关显微镜方法，结合高速的3D活细胞荧光显微镜和高分辨率低温电子断层扫描。我们的相关方法，小HIV-1颗粒与宿主相互作用的HeLa细胞成像动态演示的能力。

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Research

JoVE Journal

Bioengineering

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

科研

生物工程

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

模拟卵巢癌多细胞球体行为动态三维微型腹膜

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

低温固定化细胞跟踪和成像病毒蛋白相关结构的相关光电子显微镜 (克莱)

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

3D 动态培养系统构建多层间充质干细胞片材

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

预测神经植入物软化行为的环境动力力学分析

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Four-Dimensional CT Analysis Using Sequential 3D-3D Registration

使用顺序 3D-3D 注册进行四维 CT 分析

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data ...

相关显微镜的动力相互作用的三维结构分析