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 2，添加有4.5 g / L的L-谷氨酰胺，葡萄糖，10％热灭活小牛血清，100单位/ ml青霉素，100μg/ ml的链霉素，约18小时。 HeLa细胞被感染后，O / N文化。 </li></ol> 2。 HIV-1感染与活细胞成像<ol><li>添加荧光细胞（红CMTPX 1微升/ 2毫升）跟踪到玻璃底文化菜（步骤1.3），盘在37℃下孵育10分钟，从而使要的荧光染料。 </li><li>用PBS清洗细胞，并添加50μl的预热的新鲜培养基中。 </li><li>将菜到活细胞室，在37℃，掠现场共聚焦扫描显微镜。选择多个字段（10-15位）包含1-3细胞/平方米，与两个有丝分裂阶段的细胞之间传播出来，平面（参见图2c和2d），因为冷冻蚀刻电镜时，需要比较薄的区域的样品。未来成像（步骤2.5）存储这些位置。 </li><li>感染细胞与VSV-G伪类型的HIV-1颗粒含有GFP-Vpr的（我们用40微升的样品含有40 ng / ml的p24的）。当添加病毒颗粒进入底部的菜，要小心，不要打扰EM电网，因为成像的位置已经被选择和存储。 </li><li>除了病毒颗粒后，立即收集TI我告失效高速3D共聚焦图像，在先前选定的位置（从第2.3步）20-40分钟。 </li><li>共聚焦图像被收购使用Metamorph Imaris里软件（ 见图1）单粒子动力学使用的自动3D粒子跟踪分析。由于我们是关联的动态行为衍射极限的HIV-1颗粒与细胞的超微结构，时间推移活细胞成像和3D粒子跟踪的结果至关重要。然而，对于一个大的和静态的结构中，一个简单的荧光图像（无活细胞）。将足够的相关性分析。 </li></ol> 3。冷冻水合EM样品制备为了尽量减少之间的延迟时间的最后共聚焦图像的采集和冷冻的样品固定，FEI Vitrobot的（或其他玻璃化冷冻设备）应发起并准备切入冻结期间收集的荧光共聚焦活细胞图像。<ol><li>打开的玻璃化的移动设备上，并设置其气候温度至22°C，湿度为100％的目标，的印迹7秒的时间，并在等待和漏到1秒的时间。 </li><li>将格收纳盒，并准备在柱塞杜瓦冷液态乙烷。安装杜瓦到玻璃化设备。 </li><li>共聚焦活细胞成像（第2节）后，立即将培养皿中，并将其转移到冰水冷铜块冷冻蚀刻电镜样品制备室。 EM电网加载到专门的镊子，并迅速对电网涂抹任何媒体。印迹手动完成，用滤纸，之后，4微升为15nm的金胎圈溶液混合，用0.2μm的荧光微球被立即放置在网格上。黄金珠被用作基准标记，以帮助断层数据收集和对应。荧光微球用于帮助荧光灯和冷冻蚀刻电镜图像之间的相关性（参见图2c-2f的 ）。 </li><li>镊子加载到玻璃化设备，并指示设备，涂抹和投身冻结液态乙烷16。印迹参数优化，以达到最好的结果是:印迹时间7秒;印迹偏移，0排放时间，1秒，温度22°C，湿度100％。 </li><li>冷冻水合电网传输到冷冻蚀刻电镜的持有立即cryoET成像，或液氮存储杜瓦供以后使用。 </li></ol> 4。低温荧光光学显微镜自己组装的，冷冻的荧光样品室和舞台系统（ 图3）须进行步骤4.1-4.10。的详细规格和描述的阶段，可能会发现在军等15。 <ol><li>填补自增压液氮的杜瓦瓶用液氮低温荧光显微镜（参见图3a）开始前至少2小时。 </li><li>安装自制的冷冻-F到一个倒置荧光显微镜，如奥林巴斯IX 71 luorescence试料台15（ 图3）。 </li><li>液体氮气入口的低温样品台连接的自我加压的杜瓦瓶，放入适当的容器中放置液氮溢出保护插座。干燥的氮气气体管线连接在物镜保持镜头温暖和无霜下袖子。 </li><li>进入低温载置台室，用于装载冷冻水合样品网格放置一个铜块平台（在图3b中的黑色箭头）。冷却下来，并通过入口管线从自我加压填充杜瓦瓶填充铜室用液氮的低温阶段。 </li><li>当在低温阶段达到液氮温度下（​​约4-6分钟），转移的格收纳盒（步骤3.5）的低温阶段。 </li><li>将冷冻水合样品电网EM标本盒上的铜块和夹克摆脱地方使用C型环（如果使用Polara镜盒），或放置预冷铜环之上（ 图3d黑箭头），保持电网的地方，也便于检索的网格后低温荧光成像（如果一个非Polara的低温电子显微镜）。 </li><li>将墨盒内室中的低温阶段（在图3b中的白箭头）（步骤4.6）。 </li><li>搜索，发现同样的病毒颗粒从活细胞成像数据。由于EM网格有一个索引，它是直接在活细胞图像和低温花期图像定位相同的粒子对电网。 </li><li>冷冻DIC和GFP图像采集在低温条件下，具有很长的工作（2.7-4毫米）的物镜中使用光学显微镜所识别的位置。在的低温荧光成像，定期检查液氮低温阶段和填充它，需要在-170以下，保持样品阶段℃。 </li><li>低温荧光成像完成后，仔细地返回标本盒铜块平台，将墨盒存放在液氮杜瓦未来cryoET分析与一个Polara系统。如果使用一个非Polara的低温电子显微镜，取出铜圈，检索样本网格，将其放置在一个网格中的收纳盒，并存储在液氮杜瓦直至试样由cryoET检查的试样。 </li><li>对数据进行分析，重叠冷冻DIC和GFP所获取的图像（从步骤4.9，参见图4b）和关联的GFP信号在低温与活细胞成像系列中得到的荧光图像中的位置。 </li></ol> 5。低温电子断层扫描<ol><li>装入样品盒，电子显微镜，场发射枪，如Polara G2显微镜配备到冷冻中转站。 </li><li>在低剂量搜索模式时，在140X的放大倍数下，IDEntify成舞台文件，并保存相关的方格（从步骤4.11）。 </li><li>以3500倍的倍率下的低剂量的搜索模式中，将物镜孔径为100μm，搜索和保存所有与GFP的信号转换成第二级文件的位置。 </li><li>在低剂量曝光模式下，调整束强度，剂量为1或2电子- 2埃/缩小的倾斜轴的空白区域，使用阶段y功能，通过燃烧掉冰在不重要的碳区域几个黄金珠。 </li><li>低剂量对焦模式下，调整焦距〜3微米，并调节角度对准焦点的方向是平行的倾斜轴。 </li><li>下低剂量搜索模式，记得保存的位置（5.3），调整标本eucentric高度，并检查该地区的利益倾斜范围。采集的投影图像具有非常低的电子剂量（1〜E - / A 2）在8至10微米的散焦，在博览会的URE模式，以确认相关的病毒颗粒在低温电子断层扫描。 </li><li>切换到低剂量对焦模式。在TEM自动功能菜单，之前自动eucentric的高度，重点区域和重点功能。 </li><li>收购倾斜系列的参数设置。对于图4在本文中，倾斜角度为±70°，低于和高于45°，在3°和2°倾斜递增，分别使用6微米散焦和大约70 - / A 2总剂量。 </li><li>重复步骤5.7和5.8所有保存的位置。批处理断层软件可用于自动数据采集的多个位置，以增加吞吐量。 </li></ol> 6。三维重建使用IMOD 17 <ol><li>检查原料系列倾斜调整的最小和最大密度值，并确定是否有任何要排除由于大的图像移位的视图。 </li><li>启动eTomo和安装断层扫描面板采用轴类型，像素大小，基准直径等 。然后创建COM脚本。 </li><li>配置的主窗口中的断层图像计算的几个阶段的同时可以调整/其次。修改必要的参数，并执行所要求的每一个加工步骤（查看eTomo教程， <a href="http://bio3d.colorado.edu/imod/doc/etomoTutorial.html" target="_blank">http://bio3d.colorado.edu/imod/doc/etomoTutorial.html</a> ）的具体方案。 </li><li>在最后阶段，创建一个完整的对齐堆栈（平均剩余误差小于0.6），并使用加权反投影算法在IMOD重建断层扫描。 </li><li>去噪潜在的感兴趣的领域使用3D非线性各向异性扩散边缘增强程序IMOD实施适当的参数，以提高对比度和清晰度。 </li></ol>

<ol>
	<li>Leis, A., Rockel, B., Andrees, L., Baumeister, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+cells+at+the+nanoscale.">Visualizing cells at the nanoscale.</a> Trends in Biochemical Sciences. 34, 60-70 (2009).</li><li>Maurer, U. E., Sodeik, B., Grunewald, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+3D+intermediates+of+membrane+fusion+in+herpes+simplex+virus+1+entry.">Native 3D intermediates of membrane fusion in herpes simplex virus 1 entry.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 10559-10564 (2008).</li><li>McDonald, D., 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=Visualization+of+the+intracellular+behavior+of+HIV+in+living+cells.">Visualization of the intracellular behavior of HIV in living cells.</a> The Journal of Cell Biology. 159, 441-452 (2002).</li><li>Arhel, N., 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=Quantitative+four-dimensional+tracking+of+cytoplasmic+and+nuclear+HIV-1+complexes.">Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes.</a> Nature Methods. 3, 817-824 (2006).</li><li>Gousset, K., 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=Real-time+visualization+of+HIV-1+GAG+trafficking+in+infected+macrophages.">Real-time visualization of HIV-1 GAG trafficking in infected macrophages.</a> PLoS Pathogens. 4, e1000015 (2008).</li><li>Jouvenet, N., Bieniasz, P. D., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+biogenesis+of+individual+HIV-1+virions+in+live+cells.">Imaging the biogenesis of individual HIV-1 virions in live cells.</a> Nature. 454, 236-240 (2008).</li><li>Koch, P., 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+fusion+of+pseudotyped+HIV-1+particles+in+real+time+by+live+cell+microscopy.">Visualizing fusion of pseudotyped HIV-1 particles in real time by live cell microscopy.</a> Retrovirology. 6, 84 (2009).</li><li>Zhang, P., 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+receptor+arrays+in+frozen-hydrated+sections+and+plunge-frozen+specimens+of+E.+coli+engineered+to+overproduce+the+chemotaxis+receptor+Tsr.">Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr.</a> Journal of Microscopy. 216, 76-83 (2004).</li><li>Wang, K., Strunk, K., Zhao, G., Gray, J. 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>

作者宣称没有竞争经济利益。

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, ...

correlative-microscopy-for-3d-structural-analysis-dynamic

Research

JoVE Journal

Bioengineering

13.9K 次观看.  University of Pittsburgh School of Medicine. 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。

视频: Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

科研

生物工程

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

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

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 ...

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

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

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

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

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 widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

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

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

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

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

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

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

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

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

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 cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

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

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

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

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

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

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data remains difficult because segmentation in all volumetric frames is time-consuming. We aimed to analyze joint kinematics using a sequential 3D-3D registration technique to provide the kinematics of the moving bone with respect to the fixed bone semiautomatically using 4DCT DICOM data and existing software. Surface data of the source bones are reconstructed from 3DCT. The trimmed surface data are respectively matched with surface data from the first frame in 4DCT. These trimmed surfaces are sequentially matched until the last frame. These processes provide positional information for target bones in all frames of the 4DCT. Once the coordinate systems of the target bones are decided, translation and rotation angles between any two bones can be calculated. This 4DCT analysis offers advantages in kinematic analyses of complex structures such as carpal or tarsal bones. However, fast or large-scale motions cannot be traced because of motion artifacts.

We analyzed joint kinematics from four-dimensional computed tomography data. The sequential 3D-3D registration method semiautomatically provides the kinematics of the moving bone with respect to the subject bone from four-dimensional computed tomography data.

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