Name: advancing high-resolution imaging of virus assemblies in liquid and ice
Uploaded: 2022-07-20T13:30:00
Description: Watch this Scientific Journal Video about Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice at JoVE.com

作者感谢Luk H. Vandenberghe博士（哈佛医学院眼科）提供纯化的AAV-3。这项工作得到了美国国立卫生研究院和国家癌症研究所（R01CA193578，R01CA227261，R01CA219700至DFK）的支持。

1. 装载液体电磁的试样支架
<ol><li>通过将每个芯片在 150 mL 丙酮中孵育 2 分钟，然后在 150 mL 甲醇中孵育 2 分钟来清洁氮化硅 （SiN） 微芯片。让芯片在层流气流中干燥。</li>
	<li>使用辉光放电仪器使用辉光放电仪器清洁干燥的芯片，使用氩气在 30 W、15 mA 的标准条件下运行 45 秒。</li>
	<li>将干燥的基础微芯片装入样品支架的尖端。向基础芯片中加入 ~0.2 μL 样品（在 50 mM HEPES、pH 7.5、1...

<ol>
	<li>Deng, W., 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=Assembly,+structure,+function+and+regulation+of+type+III+secretion+systems.">Assembly, structure, function and regulation of type III secretion systems.</a> Nature Reviews Microbiology. 15 (6), 323-337 (2017).</li><li>Oikonomou, C. M., Chang, Y. -. W., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+view+into+prokaryotic+cell+biology+from+electron+cryotomography.">A new view into prokaryotic cell biology from electron cryotomography.</a> Nature Reviews Microbiology. 14 (4), 205-220 (2016).</li><li>Murata, K., Wolf, M. <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+for+structural+analysis+of+dynamic+biological+macromolecules.">Cryo-electron microscopy for structural analysis of dynamic biological macromolecules.</a> Biochimica et Biophysica Acta. General Subjects. 1862 (2), 324-334 (2018).</li><li>DiMaio, F., 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=Atomic+accuracy+models+from+4.5+&#197;+cryo-electron+microscopy+data+with+density-guided+iterative+local+refinement.">Atomic accuracy models from 4.5 &#197; cryo-electron microscopy data with density-guided iterative local refinement.</a> Nature Methods. 12 (4), 361-365 (2015).</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=A+model+of+protein+synthesis+based+on+cryo-electron+microscopy+of+the+E.+coli+ribosome.">A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome.</a> Nature. 376 (6539), 441-444 (1995).</li><li>Dukes, M. J., Gilmore, B. L., Tanner, J. R., McDonald, S. M., 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=In+situ+TEM+of+biological+assemblies+in+liquid.">In situ TEM of biological assemblies in liquid.</a> Journal of Visualized Experiments. (82), e50936 (2013).</li><li>Dearnaley, W. 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=Liquid-cell+electron+tomography+of+biological+systems.">Liquid-cell electron tomography of biological systems.</a> Nano Letters. 19 (10), 6734-6741 (2019).</li><li>Park, 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=Direct+observation+of+wet+biological+samples+by+graphene+liquid+cell+transmission+electron+microscopy.">Direct observation of wet biological samples by graphene liquid cell transmission electron microscopy.</a> Nano Letters. 15 (7), 4737-4744 (2015).</li><li>Chen, Q., 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=3D+Motion+of+DNA-Au+nanoconjugates+in+graphene+liquid+cell+electron+microscopy.">3D Motion of DNA-Au nanoconjugates in graphene liquid cell electron microscopy.</a> Nano Letters. 13 (9), 4556-4561 (2013).</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 (6077), 61-64 (2012).</li><li>Wang, X., Yang, J., Andrei, C. M., Soleymani, L., Grandfield, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomineralization+of+calcium+phosphate+revealed+by+in+situ+liquid-phase+electron+microscopy.">Biomineralization of calcium phosphate revealed by in situ liquid-phase electron microscopy.</a> Communications Chemistry. 1, 80 (2018).</li><li>Jonaid, G., 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+imaging+of+human+viruses+in+liquid+droplets.">High-resolution imaging of human viruses in liquid droplets.</a> Advanced Materials. 33 (37), 2103221 (2021).</li><li>Pohlmann, E. 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=Real-time+visualization+of+nanoparticles+interacting+with+glioblastoma+stem+cells.">Real-time visualization of nanoparticles interacting with glioblastoma stem cells.</a> Nano Letters. 15 (4), 2329-2335 (2015).</li><li>Jonaid, G. 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=Automated+tools+to+advance+high-resolution+imaging+in+liquid.">Automated tools to advance high-resolution imaging in liquid.</a> Microscopy and Microanalysis. , 1-10 (2022).</li><li>Varano, A. C., 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=Customizable+cryo-EM+chips+improve+3D+analysis+of+macromolecules.">Customizable cryo-EM chips improve 3D analysis of macromolecules.</a> Microscopy and Microanalysis. 25, 1310-1311 (2019).</li><li>Alden, N. 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=Cryo-EM-on-a-chip:+Custom-designed+substrates+for+the+3D+analysis+of+macromolecules.">Cryo-EM-on-a-chip: Custom-designed substrates for the 3D analysis of macromolecules.</a> Small. 15 (21), 1900918 (2019).</li><li>Tanner, J. R., 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-SiN+&#8211;+An+alternative+substrate+to+visualize+active+viral+assemblies.">Cryo-SiN &#8211; An alternative substrate to visualize active viral assemblies.</a> Journal of Analytical and Molecular Techniques. , (2016).</li><li>Solares, M. 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=Microchip-based+structure+determination+of+disease-relevant+p53.">Microchip-based structure determination of disease-relevant p53.</a> Analytical Chemistry. 92 (23), 15558-15564 (2020).</li><li>Casasanta, M. 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=Microchip-based+structure+determination+of+low-molecular+weight+proteins+using+cryo-electron+microscopy.">Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy.</a> Nanoscale. 13 (15), 7285-7293 (2021).</li><li>Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+data+acquisition+from+electron+microscopes+with+SerialEM.">Advanced data acquisition from electron microscopes with SerialEM.</a> Microscopy and Microanalysis. 24, 864-865 (2018).</li><li>Scheres, S. H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RELION:+Implementation+of+a+Bayesian+approach+to+cryo-EM+structure+determination.">RELION: Implementation of a Bayesian approach to cryo-EM structure determination.</a> Journal of Structural Biology. 180 (3), 519-530 (2012).</li><li>Punjani, A., Rubinstein, J. L., Fleet, D. J., Brubaker, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cryoSPARC:+algorithms+for+rapid+unsupervised+cryo-EM+structure+determination.">cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination.</a> Nature Methods. 14 (3), 290-296 (2017).</li><li>Pettersen, E. F., 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=UCSF+Chimera&#8212;A+visualization+system+for+exploratory+research+and+analysis.">UCSF Chimera&#8212;A visualization system for exploratory research and analysis.</a> Journal of Computational Chemistry. 25 (13), 1605-1612 (2004).</li><li>Goddard, T. D., Huang, C. C., Ferrin, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+density+maps+with+UCSF+Chimera.">Visualizing density maps with UCSF Chimera.</a> Journal of Structural Biology. 157 (1), 281-287 (2007).</li><li>Lerch, T. F., Xie, Q., Chapman, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+adeno-associated+virus+serotype+3B+(AAV-3B):+Insights+into+receptor+binding+and+immune+evasion.">The structure of adeno-associated virus serotype 3B (AAV-3B): Insights into receptor binding and immune evasion.</a> Virology. 403 (1), 26-36 (2010).</li><li>Sharma, G., 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=Affinity+grid-based+cryo-EM+of+PKC+binding+to+RACK1+on+the+ribosome.">Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome.</a> Journal of Structural Biology. 181 (2), 190-194 (2013).</li><li>Kiss, G., 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=Capturing+enveloped+viruses+on+affinity+grids+for+downstream+cryo-electron+microscopy+applications.">Capturing enveloped viruses on affinity grids for downstream cryo-electron microscopy applications.</a> Microscopy and Microanalysis. 20 (1), 164-174 (2014).</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&#8212;a+nanoscale+purification+platform+for+biological+in+situ+transmission+electron+microscopy.">The development of affinity capture devices&#8212;a nanoscale purification platform for biological in situ transmission electron microscopy.</a> Rsc Advances. 2 (6), 2408-2412 (2012).</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 (4132), 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> Proceedings of the National Academy of Sciences of the United States of America. 71 (12), 5068-5072 (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+object.">Electron microscopy and diffraction of wet unstained and unfixed biological object.</a> Advances in Biological and Medical Physics. 15, 161-270 (1974).</li><li>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=Structure+of+wet+specimens+in+electron+microscopy.+Improved+environmental+chambers+make+it+possible+to+examine+wet+specimens+easily.">Structure of wet specimens in electron microscopy. Improved environmental chambers make it possible to examine wet specimens easily.</a> Science. 186 (4162), 407-414 (1974).</li><li>Matricardi, V. R., Moretz, R. C., 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+proteins:+Catalase.">Electron diffraction of wet proteins: Catalase.</a> Science. 177 (4045), 268-270 (1972).</li></ol>

通过使用从冷冻电镜领域改编的新的自动化工具和技术，为简化当前的液电镜工作流程提供了新的机会。与其他方法相比，涉及新的微芯片夹层技术的应用具有重要意义，因为它们能够在液体或玻璃体冰中进行高分辨率成像分析。该协议中最关键的步骤之一是生产具有理想液体厚度的标本，以可视化纳米级的精美细节。在实施高通量例程进行自动数据收集之前，通过筛选整个样品，以较低的放大?...

生物医学研究通过开发新技术提高了我们对人类健康和疾病的了解。高分辨率成像正在改变我们对纳米世界的看法 - 使我们能够以精致的细节研究细胞和分子1，2，3，4，5。动态组件（如软聚合物、蛋白质组装或人类病毒）的静态信息仅揭示了其复杂叙述的有限快照。为了更好地了解分子实体如何运作，必须共同研究它们的结构和功能。
原子薄石墨烯或硅基微芯片等材料生产的最新进展为使用透射电子显微镜（TEM）进行实时结构函数分析提供了新的机会。这些材料可以创建用于实时EM成像的密封室6，7，8，9，10，11。液电镜的新领域，室温与冷冻电镜相关，提供了溶液中硬质或软质材料的前所未有的视图，使科学家能够同时研究其样品的结构和动力学。液体电镜应用包括治疗性纳米颗粒与癌症干细胞相互作用的实时记录，以及病毒病原体12，13，14分子复杂性的变化。
正如方法学的进步刺激了冷冻电镜领域的分辨率革命一样，需要新的技术和方法来扩展液电镜作为科学界高通量工具的使用。本文介绍的方法的总体目标是简化液电镜样品制备方案。所开发技术背后的基本原理是采用新的微芯片设计和自动加载器设备，适用于液体和冷冻电镜数据收集（图1）7，14，15，16，17。组件使用自动化仪器的标准网格夹进行机械密封，例如 Krios，每个会话可容纳多个样品或 F200C TEM（图 2）。这种方法将高分辨率成像的使用扩展到标准冷冻电镜应用之外，展示了实时材料分析的更广泛用途。
在当前的视频文章中，介绍了在有或没有市售标本支架的情况下在液体中制备病毒组件的实验方案。使用用于液电镜的专用试样支架，薄液体试样可以提供与冷冻电镜样品相当的结构信息，以及试样的动态见解。还展示了使用自动进样器工具制备液体标本的方法，用于高通量常规。与其他技术相比，自动化样品生产的主要优点是允许用户在数据收集之前快速评估样品的最佳厚度和电子剂量。这种筛选技术可快速识别液体或冰12，14，18，19中实时记录的理想区域。出于 3D 结构测定的目的，液电镜可以补充冷冻电镜中实施的长期建立的冷冻电镜方法。采用传统TEM或冷冻电镜技术的读者可以考虑使用液电镜工作流程，以补充其当前策略的方式提供新的样品动态观察结果。
本协议中使用的病毒样品包括作为礼物获得并在标准条件下培养的纯化的腺相关病毒亚型3（AAV）12。还使用了从COVID-19患者血清中提取的非传染性SARS CoV-2亚病毒组件12，并从商业来源获得。最后，从维克森林大学的Sarah M. McDonald Esstman博士的实验室获得纯化的猿猴轮状病毒（SA11菌株）双层颗粒（DLP），并使用标准条件 6，17进行培养。此处描述的软件包是免费提供的，链接已在 “材料表 ”部分中提供。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>&#160;A11-1</td>
			<td>1 Liter</td>
		</tr>
		<tr>
			<td>Autoloader clipping tool</td>
			<td>ThermoFisher Scientific</td>
			<td>N/A</td>
			<td>Also SubAngstrom supplier</td>
		</tr>
		<tr>
			<td>Autoloader grid clips</td>
			<td>ThermoFisher Scientific</td>
			<td>N/A</td>
			<td>top and bottom clips</td>
		</tr>
		<tr>
			<td>Carbon-coated gold EM grids</td>
			<td>Electron Microcopy Sciences</td>
			<td>CF400-AU-50</td>
			<td>400-mesh, 5-nm thickness</td>
		</tr>
		<tr>
			<td>COVID-19 patient serum</td>
			<td>RayBiotech</td>
			<td>CoV-Pos-S-500</td>
			<td>500 microliters of PCR+ serum</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>&#160;A412-1</td>
			<td>1 Liter</td>
		</tr>
		<tr>
			<td>Microwell-integrad microchips</td>
			<td>Protochips, Inc.</td>
			<td>EPB-42A1-10</td>
			<td>10x10-mm window arrays</td>
		</tr>
		<tr>
			<td>TEMWindows microchips</td>
			<td>Simpore Inc.</td>
			<td>SN100-A10Q33B</td>
			<td>9 large windows, 10-nn thick</td>
		</tr>
		<tr>
			<td>TEMWindows microchips</td>
			<td>Simpore, Inc.&#160;</td>
			<td>SN100-A05Q33A</td>
			<td>9 small windows, 5-nm thick</td>
		</tr>
		<tr>
			<td>Top microchips</td>
			<td>Protochips, Inc.</td>
			<td>EPT-50W</td>
			<td>500 mm x 100 mm window</td>
		</tr>
		<tr>
			<td>Whatman #1 filter paper</td>
			<td>Whatman</td>
			<td>1001 090</td>
			<td>100 pieces, 90 mm</td>
		</tr>
		<tr>
			<td>Equipment&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DirectView direct electron detector</td>
			<td>Direct Electron</td>
			<td></td>
			<td>6-micron pixel spacing</td>
		</tr>
		<tr>
			<td>Falcon 3 EC direct electron detector</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>14-micron pixel spacing</td>
		</tr>
		<tr>
			<td>Gatan 655 Dry pump station</td>
			<td>Gatan, Inc.</td>
			<td></td>
			<td>&#160;Pump holder tip to 10-6 range</td>
		</tr>
		<tr>
			<td>Mark IV Vitrobot</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>state-of-the-art specimen preparation unit&#160;</td>
		</tr>
		<tr>
			<td>PELCO easiGlow, glow discharge unit</td>
			<td>Ted Pella, Inc.</td>
			<td></td>
			<td>&#160;Negative polarity mode</td>
		</tr>
		<tr>
			<td>Poseidon Select specimen holder</td>
			<td>Protochips, Inc.</td>
			<td></td>
			<td>&#160;FEI compatible;specimen holder</td>
		</tr>
		<tr>
			<td>Talos F200C TEM</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>200 kV; Liquid-TEM</td>
		</tr>
		<tr>
			<td>Titan Krios G3</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>300 kV; Cryo-TEM</td>
		</tr>
		<tr>
			<td>Freely available software</td>
			<td>Website link</td>
			<td></td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>cryoSPARC</td>
			<td>https://cryosparc.com/</td>
			<td></td>
			<td>other image processing software</td>
		</tr>
		<tr>
			<td>CTFFIND4</td>
			<td>https://grigoriefflab.umassmed.edu/ctffind4</td>
			<td></td>
			<td>CTF finding program</td>
		</tr>
		<tr>
			<td>MotionCorr2</td>
			<td>https://emcore.ucsf.edu/ucsf-software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RELION</td>
			<td>https://www3.mrc-lmb.cam.ac.uk/relion/index.php?title=Main_Page</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SerialEM</td>
			<td>https://bio3d.colorado.edu/SerialEM/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UCSF Chimera</td>
			<td>https://www.cgl.ucsf.edu/chimera/</td>
			<td></td>
			<td>molecular structure analysis software package</td>
		</tr>
	</tbody>
</table>

advancing high-resolution imaging of virus assemblies in liquid and ice

近年来，人们对液电子显微镜（liquid-EM）的兴趣激增，因为科学家现在可以观察纳米级的实时过程。将高分辨率冷冻电镜信息与动态观察相结合是非常理想的，因为许多事件发生在快速的时间尺度上 - 在毫秒范围内或更快。提高对柔性结构的了解也有助于设计新型试剂来对抗新出现的病原体，例如SARS-CoV-2。更重要的是，在流体环境中观察生物材料可以对它们在人体中的表现进行独特的一瞥。这里介绍的是新开发的方法，用于研究液体和玻璃冰中病毒组装的纳米级特性。为了实现这一目标，使用定义明确的样本作为模型系统。并排比较了样品制备方法和代表性结构信息。显示了在~3.5-Å-10 Å范围内解析的结构的亚纳米特征。支持这一互补框架的其他最新结果包括候选疫苗和液体成像的基于抗体的疗法的动态见解。总体而言，这些相关应用提高了我们可视化分子动力学的能力，为它们在人类健康和疾病的使用提供了独特的背景。

所有液电镜成像实验均使用工作在200 kV的液电镜，工作电压为300 kV的冷冻电镜用于所有冷冻电镜数据收集。展示了多种病毒的代表性图像和结构，以证明这些方法在各种测试对象中的实用性。这些包括重组腺相关病毒亚型 3 （AAV）、源自患者血清的 SARS-CoV-2 亚病毒组件以及猿轮状病毒双层颗粒 （DLP）、SA11 菌株。首先，演示在液体缓冲溶液（50 mM HEPES，pH 7.5;150 mM NaCl;10 mM MgCl2;10 mM CaCl2<...

Watch this Scientific Journal Video about Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice at JoVE.com

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

这里描述了使用透射电子显微镜制备适用于纳米级液体电镜和冷冻电镜分析的病毒组件的协议。

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 ...

近年来，人们对液体电子显微镜的兴趣激增，因为我们现在可以在纳米级实时过程中进行可视化。对这些柔性结构的了解的提高可以帮助我们开发新型试剂来对抗SARS-CoV-2等新兴病原体。在流体环境中观察蛋白质有助于模仿生物系统，并让我们有机会以更动态的方式观察蛋白质。这个实验将向您展示如何在玻璃冰和液体环境中使用和可视化蛋白质的新技术。最近的结果包括候选疫苗在液体成像的基于抗体的疗法中的动态见解。相关的液体和冷冻电镜应用提高了我们可视化分子动力学的能力，为人类健康和疾病提供了独特的背景。采用传统TEM或冷冻电镜技术的读者可以考虑实施液体电镜工作流程，以补充其当前策略的方式为其样品提供新的动态观察。市售的液态电镜系统可以提供流动、混合、电化学刺激和温度控制，这对于许多实时成像应用至关重要。这里介绍的微芯片夹心方法描述了一种简单的入门级方法，在跳跃到更复杂的商业系统进行原位实验之前，首先查看液体中的样品。首先，通过将每个芯片在150毫升丙酮中孵育两分钟，然后在150毫升甲醇中孵育两分钟来清洁氮化硅微芯片。让芯片在层流气流中干燥。使用辉光放电仪器使用氩气在标准条件下运行 45 秒的等离子体清洁干燥的芯片。接下来，将干燥的基础微芯片装入样品支架的尖端，并将大约 0.2 微升的样品添加到基础芯片中。孵育一到两分钟后，将顶部芯片放在含有样品的湿基芯片上。将组件放在一起，形成一个密封的外壳，由三个黄铜螺钉机械地固定到位。使用涡轮泵送干泵站将尖端泵送到 10 至负 6 托。支架现在已准备好插入 TEM。等离子体使用辉光放电仪器清洁微芯片和碳网格45秒。并将大约两微升样品添加到放置在凝胶包上的发光放电微芯片中。使用滤纸或移液管取出多余的溶液，孵育一到两分钟。然后将发光放电的碳网格添加到包含样品的湿微芯片中。在室温下使用单个倾斜试样支架将组件种植在一起，以形成密封的外壳。或者，使用自动加载网格夹并将三明治组件放在底部 C 夹上。将顶部夹放在组件的顶部，并使用标准夹紧工具将组件密封在一起。试样现在已准备好插入TEM。检查在低温条件下或室温下放置在自动进样器中的样品。对于液电镜成像，将样品支架装入配备场发射枪的 TEM 中，并在 200 千伏下运行。打开喷枪，使用摆动功能调整显微镜载物台相对于标本的共心高度，将样品在柱中从零下 15 度倾斜到正 15 度。此过程在Z方向上调整载物台以适应样品厚度，并有助于确保图像记录期间的准确放大倍率。使用串行数据采集软件包将图像记录为长帧短片或单个图像，实现自动成像程序。在低剂量条件下以 28， 000X 到 92， 000X 和每秒 40 帧的放大倍率采集图像。调整曝光时间以尽量减少光束对标本的损坏，并在指定放大倍率下使用负一到四微米的散焦范围。如果遇到厚溶液，请使用更高的散焦值或选择不同的感兴趣区域。通过将电子束聚焦在不用于数据收集的牺牲区域上，直到形成气泡，确保溶液在整个成像过程中存在于样品中。使用 RELION-3.0.8 程序或任何其他图像处理软件分析电影中的 SARS-CoV-2 颗粒。使用 MotionCor2 程序执行运动校正。更正后，使用程序软件包中的自动拾取工具提取颗粒。液体标本的典型框尺寸为 330 像素，冰标本的典型框尺寸为 350 像素。使用程序的 3D 初始模型例程和数据处理软件包中的从头模型选项使用 C1 对称性计算初始重建。然后在数据处理软件中执行细化协议。使用分子结构分析软件包检查结果，同时评估动态变化。此处显示了腺相关病毒亚型 3 或 AAV 中液体电镜和冷冻电镜结构的比较。代表性图像显示了AAV在溶液和冰中的结构。此处显示了从液体和冰结构中提取的AAV VP1亚基的旋转视图。这些图像表示使用分子结构分析软件中的形态图函数生成的液体结构中的动态值。来自多个病毒组件的平均结构显示出构象变化，使用EM数据测量了近5%的直径变化。此处显示了从 COVID-19 患者的血清组分中分离出的 SARS-CoV-2 亚病毒组件的图像。这些白色气泡表示样品中存在液体。这里显示了这些亚病毒组件的EM重建，通过地图显示了五纳米切片的彩色径向密度。代表性图像描述了使用微芯片夹心技术在玻璃体冰中制备的轮状病毒双层颗粒的分析。液体电镜成像应尽量减少或避免使用去垢剂、甘油、聚乙烯、乙二醇和高浓度糖。这些试剂可能会引入伪影，产生过多的鼓泡、水解产物和束损伤引起的自由基。这些协议的使用将使科学家能够研究原子细节的动态过程。这跨越了多个科学领域，包括医学、生命科学和材料研究。这里介绍的协议描述了最先进的工具如何提供一种令人兴奋的方法，通过新的眼睛可视化生物大分子。液体电子显微镜领域可能会提升我们研究这些对人类健康构成威胁的新型病毒的方式，甚至可能有助于我们的大流行防范措施。

Research

JoVE Journal

Biochemistry

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

植物冰结合蛋白通过冰再结晶抑制和隔离的评估使用冰亲和力纯化鉴定

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

科研

生物化学

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

固体核磁共振波谱法研究高分子组件的原子尺度结构

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

通过低温BN-MS分析进行高分辨率复杂分析

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

肽聚糖的液相色谱质谱和生物信息学半定量分析

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

呼吸系统同步病毒病毒特定核胶囊的生成和组装

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

FÃƒÆ’Ã‚Â¶rster Resonance Energy Transfer Measurements in Living Plant Cells

活植物细胞中的Förster共振能量转移测量

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...