Name: visualizing single molecular complexes in vivo using advanced fluorescence microscopy
Uploaded: 2009-09-08T16:30:00
Description: Watch this Scientific Journal Video about Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy at JoVE.com

我们承认，从教授朱迪思阿米蒂奇（英国牛津大学）和康拉德Mullineaux教授（英国伦敦大学玛丽皇后学院）组菌株的实物捐赠。 IMD是共同出资的生物化学部（牛津大学）和OCISB; AR是由工程和物理科学研究理事会（EPSRC）的DTC的助学金的资助; ND是从生物技术和生物科学研究理事会（BBSRC）资助; MCL由英国皇家学会大学研究奖学金。

<ol><li>要开始此过程中，50μL荧光蛋白表达大肠杆菌细菌细胞冷冻库存首次解冻和成长翻天覆地的5毫升LB生长介质一夜之间在37度C.在早上有氧，提取50μL这个饱和文化和子成最小的M63葡萄糖培养基，在30度4至6小时的彗星孵化，培养。在这里，我们展示了使用两种不同的细胞株，其中一个表示电子传输的细胞色素融合到绿色荧光蛋白，表示有关的蛋白质融合，以绿色荧光蛋白在细菌鞭毛电机的其他。 </li><li>细?...

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	<li>Leake, M. C., Chandler, J. H., Wadhams, G. H., Bai, F., Berry, R. M., Armitage, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+and+turnover+in+single,+functioning+membrane+protein+complexes.">Stoichiometry and turnover in single, functioning membrane protein complexes.</a> Nature. 443, 355-358 (2006).</li><li>Leake, M. C., Greene, N. P., Godun, R. M., Granjon, T., Buchanan, G., Chen, S., Berry, R. M., Palmer, T., Berks, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+stoichiometry+of+the+TatA+component+of+the+twin-arginine+protein+transport+system+observed+by+in+vivo+single-molecule+imaging.">Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.</a> Proc Natl Acad Sci U S A. 105, 15376-15381 (2008).</li><li>Leake, M. C., Wilson, D., Gautel, M., Simmons, R. M., M, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+single+titin+molecules+using+a+two-bead+optical+tweezers+assay.">The elasticity of single titin molecules using a two-bead optical tweezers assay.</a> Biophys. J. 87, 1112-1135 (2004).</li><li>Leake, M. C., Wilson, D., Bullard, B., Simmons, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+single+kettin+molecules+using+a+two-bead+laser-tweezers+assay.">The elasticity of single kettin molecules using a two-bead laser-tweezers assay.</a> FEBS Lett. , 535-555 (2003).</li><li>Lenn, T., Leake, M. C., Mullineaux, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+clustering+and+dynamics+of+cytochrome+bd+complexes+in+the+Escherichia+coli+plasma+membrane.">In vivo clustering and dynamics of cytochrome bd complexes in the Escherichia coli plasma membrane.</a> Mol. Microbiol. 70, 1397-1407 (2008).</li><li>Lenn, T., Leake, M. C., Mullineaux, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+Escherichia+coli+OXPHOS+complexes+concentrated+in+specialised+zones+within+the+plasma+membrane.">Are Escherichia coli OXPHOS complexes concentrated in specialised zones within the plasma membrane.</a> Biochem. Soc. Trans. 36, 1032-1036 (2008).</li><li>Lo, C. J., Leake, M. C., Pilizota, T., Berry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+measurements+of+Membrane+Potential,+Sodium-Motive+Force+and+Flagellar+Motor+Speed+in+Escherichia+coli.">Single-cell measurements of Membrane Potential, Sodium-Motive Force and Flagellar Motor Speed in Escherichia coli.</a> Biophys. 93, 294-302 (2007).</li><li>Lo, C. J., Leake, M. C., Berry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+measurement+of+intracellular+sodium+concentration+in+single+Escherichia+coli+cells.">Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells.</a> Biophys. J. 90, 357-3565 (2006).</li></ol>

必须小心不要“剪切”拴细菌细胞，因为这可能会损害鞭毛马达的功能。重要的是使用细胞的显微镜玻片上一次更长的时间不到一个小时，因为他们可能成为氧气耗尽。可能需要相当大的优化，正在调查中找到最好的显微镜成像条件满足您特定的生物系统。这可能是明智的做法是企图用纯化GFP的单独确定正确的强度激光激发您的特殊的显微镜系统所需的成像。

visualizing single molecular complexes in vivo using advanced fluorescence microscopy

全面洞察到活细胞的机制，可以实现只有通过调查的关键工序引出并直接在细胞水平上的事件。迄今为止的生物系统的剪切复杂性造成了精确的单分子实验过于苛刻，而不是单一系统使用相对粗糙的散装合奏平均测量的研究重点。然而，许多重要的过程发生在活细胞只是一个水平或几个分子;合奏测量一般掩盖这些事件的随机性和异质性。在这里，使用先进的光学显微镜和分析图像分析工具，我们演示了如何在一个单一的生活细菌细胞的单个分子的精确度，以及如何运作的生物机器，我们可以观察到分子复合物内的动态监测的蛋白质。该技术直接相关的生理。他们微创微扰和非侵入性正在研究的生物样品，并完全合拍，在物质生活的调查功能，没有现成的其他单分子生物物理的方法。此外，生物标本研究所有在未修改的细胞株（“基因组编码”），产生显着更多的蛋白质比自然发生的，而不是比较常见的，但不太理想的方法几乎相同的水平产生荧光标记的蛋白质（“质粒表达”）。因此，将调查的实际生物样品中有显着接近自然的有机体，因此观测真正的生理过程有关。

Watch this Scientific Journal Video about Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy at JoVE.com

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

在这里，我们展示了单分子荧光显微镜，对生活的细菌细胞，使功能分子复合物进行检测，跟踪和量化的执行协议。

可视化单分子络合物在体内使用高级荧光显微

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Research

JoVE Journal

Biology

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

科研

生物学

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有丝分裂的siRNA转染后的时间推移成像

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使用荧光显微镜可视化中的细菌线虫

 
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采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

可视化细胞质流动过程中单细胞伤口愈合<em&gt;蓝斑宏声</em

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

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快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

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可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

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基于3D 荧光显微镜的病毒转录工厂功能成像

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

使用前 Vivo 共聚焦显微镜可视化淋巴节点结构和细胞定位

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

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荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...