Name: live-cell fluorescence microscopy to investigate subcellular protein localization and cell morphology changes in bacteria
Uploaded: 2019-11-23T14:00:00
Description: Watch this Scientific Journal Video about Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria at JoVE.com

我们感谢我们的实验室成员对本文的评论。这项工作由南佛罗里达大学（PE）的启动赠款资助。

1. 一般增长条件
<ol><li>用抗生素（必要时）补充2 mL的适当生长介质，并结合要成像的菌株单一菌群。在摇动的培养箱中，在22°C中孵育这些种子培养物过夜。 注：本文中使用的特定细菌生长条件在代表性结果部分下提供。</li>
	<li>在125 mL瓶中稀释新鲜介质中的过夜培养物1：20，补充抗生素（如果需要）。</li>
	<li>在摇动的培养箱中生长37°C的培养基，直到中对数阶段（OD600 = 0.5）。 ?...

<ol>
	<li>Coltharp, C., Xiao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superresolution+microscopy+for+microbiology.">Superresolution microscopy for microbiology.</a> Cellular Microbiology. 14 (12), 1808-1818 (2012).</li><li>Holden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+mechanistic+principles+of+bacterial+cell+division+with+super-resolution+microscopy.">Probing the mechanistic principles of bacterial cell division with super-resolution microscopy.</a> Current Opinion in Microbiology. 43, 84-91 (2018).</li><li>Hsu, Y. 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=Full+color+palette+of+fluorescent+d-amino+acids+for+in+situ+labeling+of+bacterial+cell+walls.">Full color palette of fluorescent d-amino acids for in situ labeling of bacterial cell walls.</a> Chemical Science. 8 (9), 6313-6321 (2017).</li><li>Nonejuie, P., Burkart, M., Pogliano, K., Pogliano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+cytological+profiling+rapidly+identifies+the+cellular+pathways+targeted+by+antibacterial+molecules.">Bacterial cytological profiling rapidly identifies the cellular pathways targeted by antibacterial molecules.</a> Proceedings of the National Academy of Sciences of the USA. 110 (40), 16169-16174 (2013).</li><li>Rueden, C. T., 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=ImageJ2:+ImageJ+for+the+next+generation+of+scientific+image+data.">ImageJ2: ImageJ for the next generation of scientific image data.</a> BioMed Central Bioinformatics. 18 (1), 529 (2017).</li><li>Colville, K., Tompkins, N., Rutenberg, A. D., Jericho, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+poly(L-lysine)+substrates+on+attached+Escherichia+coli+bacteria.">Effects of poly(L-lysine) substrates on attached Escherichia coli bacteria.</a> Langmuir. 26 (4), 2639-2644 (2010).</li><li>McNally, J. G., Karpova, T., Cooper, J., Conchello, J. A. <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+imaging+by+deconvolution+microscopy.">Three-dimensional imaging by deconvolution microscopy.</a> Methods. 19 (3), 373-385 (1999).</li><li>Wallace, W., Schaefer, L. H., Swedlow, 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=A+workingperson's+guide+to+deconvolution+in+light+microscopy.">A workingperson's guide to deconvolution in light microscopy.</a> Biotechniques. 31 (5), 1076-1082 (2001).</li><li>Eswara, P. 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=An+essential+Staphylococcus+aureus+cell+division+protein+directly+regulates+FtsZ+dynamics.">An essential Staphylococcus aureus cell division protein directly regulates FtsZ dynamics.</a> Elife. 7, (2018).</li><li>Haeusser, D. P., Margolin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splitsville:+structural+and+functional+insights+into+the+dynamic+bacterial+Z+ring.">Splitsville: structural and functional insights into the dynamic bacterial Z ring.</a> Nature Reviews Microbiology. 14 (5), 305-319 (2016).</li><li>Du, S., Lutkenhaus, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=At+the+Heart+of+Bacterial+Cytokinesis:+The+Z+Ring.">At the Heart of Bacterial Cytokinesis: The Z Ring.</a> Trends in microbiology. , (2019).</li><li>Haranahalli, K., Tong, S., Ojima, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+discovery+and+development+of+antibacterial+agents+targeting+the+cell-division+protein+FtsZ.">Recent advances in the discovery and development of antibacterial agents targeting the cell-division protein FtsZ.</a> Bioorganic and Medicinal Chemistry. 24 (24), 6354-6369 (2016).</li><li>Brzozowski, R. 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=Deciphering+the+Role+of+a+SLOG+Superfamily+Protein+YpsA+in+Gram-Positive+Bacteria.">Deciphering the Role of a SLOG Superfamily Protein YpsA in Gram-Positive Bacteria.</a> Frontiers in Microbiology. 10, 623 (2019).</li><li>Elsen, N. 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=Mechanism+of+action+of+the+cell-division+inhibitor+PC190723:+modulation+of+FtsZ+assembly+cooperativity.">Mechanism of action of the cell-division inhibitor PC190723: modulation of FtsZ assembly cooperativity.</a> Journal of the American Chemical Society. 134 (30), 12342-12345 (2012).</li><li>Haydon, D. 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=An+inhibitor+of+FtsZ+with+potent+and+selective+anti-staphylococcal+activity.">An inhibitor of FtsZ with potent and selective anti-staphylococcal activity.</a> Science. 321 (5896), 1673-1675 (2008).</li><li>Pilhofer, M., Ladinsky, M. S., McDowall, A. 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=Bacterial+TEM:+new+insights+from+cryo-microscopy.">Bacterial TEM: new insights from cryo-microscopy.</a> Methods in Cell Biology. 96, 21-45 (2010).</li><li>Ni, Q., Mehta, S., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+of+cell+signaling+using+genetically+encoded+fluorescent+reporters.">Live-cell imaging of cell signaling using genetically encoded fluorescent reporters.</a> Federation of European Biochemical Societies Journal. 285 (2), 203-219 (2018).</li><li>Weiss, C. A., Hoberg, J. A., Liu, K., Tu, B. P., Winkler, W. C. <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+Microscopy+Reveals+That+Levels+of+Cyclic+di-GMP+Vary+among+Bacillus+subtilis+Subpopulations.">Single-Cell Microscopy Reveals That Levels of Cyclic di-GMP Vary among Bacillus subtilis Subpopulations.</a> Journal of Bacteriology. 201 (16), (2019).</li><li>Schneider, J. P., Basler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shedding+light+on+biology+of+bacterial+cells.">Shedding light on biology of bacterial cells.</a> Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 371 (1707), (2016).</li><li>Yao, Z., Carballido-Lopez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+imaging+for+bacterial+cell+biology:+from+localization+to+dynamics,+from+ensembles+to+single+molecules.">Fluorescence imaging for bacterial cell biology: from localization to dynamics, from ensembles to single molecules.</a> Annual review of microbiology. 68, 459-476 (2014).</li><li>Ettinger, A., Wittmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+live+cell+imaging.">Fluorescence live cell imaging.</a> Methods in Cell Biology. 123, 77-94 (2014).</li><li>Fredborg, 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+image+analysis+for+quantification+of+filamentous+bacteria.">Automated image analysis for quantification of filamentous bacteria.</a> BioMed Central Microbiology. 15, 255 (2015).</li><li>Ursell, T., 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=precise+quantification+of+bacterial+cellular+dimensions+across+a+genomic-scale+knockout+library.">precise quantification of bacterial cellular dimensions across a genomic-scale knockout library.</a> BioMed Central Biology. 15 (1), 17 (2017).</li></ol>

显微镜一直是有关微生物的研究的支柱。鉴于其微米级细胞大小，单细胞水平研究传统上依赖于电子显微镜（EM）。虽然EM近年来已经成为一种非常强大的技术，但它除了有限的用户访问16之外，还有它自己的内在限制。荧光显微镜技术的改进和不同荧光探针（如FDAA3）的开发为微生物细胞生物学家提供了大量工具，用于研究活细胞中的各种细胞过程。研究人员?...

最近显微镜技术1，2的进步，大大加强了细菌细胞生物学领域。在其他仪器中，能够进行延时荧光显微镜实验的显微镜仍然是一种宝贵的工具。研究者可以使用荧光蛋白实时监测各种生理事件，如绿色荧光蛋白（GFP）基转录和翻译报告器融合、荧光D-氨基酸（FDAA）3，或者使用其他污渍标记细胞壁、膜和DNA。因此，荧光显微镜在微生物细胞生物学家中仍然很受欢迎也就不足为奇了。除了简单地显示末端表型，提供有关观察到的表型如何产生的信息使用延时显微镜可以增加重大价值的发现，并可能提供线索，什么细胞过程是潜在的药物候选者的目标4。
本文提供了使用全电动、倒置广域荧光显微镜进行高分辨率成像的协议（参见材料表）。这些方案可以适应其他荧光显微镜的需求，这些显微镜能够进行延时显微镜检查。虽然这里讨论的软件对应于材料表中所示的特定制造商提供的软件，但其他显微镜制造商通常提供的软件或免费提供的 ImageJ5具有用于分析显微镜数据的等效工具。对于时间推移不利于的条件，可以进行时间过程实验，如本文所述。此处描述的协议为研究两种不同细菌物种的树型变化提供了详细的指南：B. subtilis和S. aureus。有关使用的菌株，请参阅表 1。

<table>
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Fisher BioReagents</td>
			<td>BP160-100</td>
			<td>Molecular Biology Grade - Low EEO</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>FM4-64</td>
			<td>Invitrogen</td>
			<td>T3166</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Fisher BioReagents</td>
			<td>BP1755-10</td>
			<td>Dioxane-free</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>GE</td>
			<td>DeltaVision Elite</td>
			<td>Customized Olympus IX-71 Inverted Microscope Stand; Custom Illumination Tower and Transmitted Light Illuminator Module. Objectives: PLAPON 60X (N.A. 1.42, WD 0.15 mm); OLY 100X OIL (N.A. 1.4, WD 0.12 mm); DIC Prism Nomarski for 100X Objective; CoolSnap HQ2 camera; SSI Assembly 7-color; Environmental control chamber - opaque.</td>
		</tr>
		<tr>
			<td>PC190723</td>
			<td>MilliporeSigma</td>
			<td>3445805MG</td>
			<td>FtsZ inhibitor</td>
		</tr>
		<tr>
			<td>SoftWorx</td>
			<td>GE</td>
			<td></td>
			<td>Manufacturer-supplied imaging software</td>
		</tr>
	</tbody>
</table>

live-cell fluorescence microscopy to investigate subcellular protein localization and cell morphology changes in bacteria

对影响细菌细胞分裂和细胞形状的因素的调查通常与高分辨率荧光显微镜一起进行，因为在总体水平上进行的观察可能无法真正反映单个细胞水平上发生的情况。活细胞延时显微镜使研究人员能够监测细胞分裂或细胞形态的变化，从而提供有关蛋白质亚细胞定位和基因表达时间的宝贵见解，从而有可能有助于回答重要的生物学问题。在这里，我们描述了使用高分辨率去卷积显微镜监测亚基利杆菌和金黄色葡萄球菌的形态变化的协议。本报告的目的是提供一个简单明了的协议，可以由有兴趣进行荧光显微镜实验的其他研究人员采用，以研究细菌和其他生物体的不同生物过程。

GpsB 表型 以前我们已经表明，Sa-GpsB是一种必需的蛋白质，因为使用反义RNA消耗GpsB会导致细胞解毒9。在这里，我们描述了如何使用本文中描述的延时显微镜协议来捕获各种细胞分裂表型的出现和蛋白质定位的变化。为此，S. aureus菌株 RB143 [SH1000 怀有 pEPSA5（空载体）] 和 GGS8 [SH1000 窝藏 pGG59（Pxyl-gpsB反义布猫） ] 之前报告的<...

Watch this Scientific Journal Video about Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria at JoVE.com

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

本文提供了一个分步指南，以研究蛋白质亚细胞定位动力学，并使用高分辨率荧光显微镜监测葡萄球菌和金黄色葡萄球菌的形态变化。

活细胞荧光显微镜研究细菌的亚细胞蛋白定位和细胞形态变化

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

该协议有助于在单细胞水平上直接观察各种表型变化，并允许持续监测蛋白质定位和基因表达时间。荧光显微镜的主要优点是，它是一种直接的方法，用于监测各种生物过程，如细胞分裂和活细胞的细胞形态变化。请务必特别注意样品制备协议，并探索显微镜软件，以便能够找到该协议中列出的设置和选项。此协议的可视化表示至关重要，因为它允许用户在设置自己的实验时观看和学习技术的重要步骤。首先，用适当的膜染色染料标记感兴趣的细胞培养物的5至50微升等同物，并在35毫升玻璃底部培养皿底部的盖滑上添加5微升染色细菌细胞。将直径为 11 毫米的 agarose 板放在样品上，轻轻敲击以确保板与盖滑平。然后在盖滑周围加入大约五微升的水，以防止红糖垫干燥，并允许培养皿与高分辨率去卷积显微镜的孵化室内的温度平衡15至20分钟。要对样品进行成像，请使用粗调整焦点旋钮，以确保在显微镜软件中初始化显微镜之前，目标完全降低。将1.517折射率油滴入100x油浸油目标，将含有样品的玻璃底盘转移到金属外壳棺材中。轻轻地将盘子滑入舞台夹，并使用粗糙的调整旋钮提升目标，直到油与盘子的玻璃底部接触。使用目镜和微调旋钮将样品对焦，然后切换到相机模式。在"解决 3D"窗口中的映像软件中，选择"设计/运行实验"图标。将出现一个标题为"设计/运行实验"的新对话框。打开对话框中的"设计和剖面"选项卡，以设置 Z 堆栈的数量和样本厚度。要测量样本中单元格的厚度，请使用"解决 3D"对话框中的向上和向下箭头进行增量调整 Z 平面，使单元格超出焦点作为图像采集的上限和下限。在"解决 3D"对话框中调整"光强度的"传输百分比"和"单个选定通道的曝光持续时间"后，单击"点列表"以打开点列表。在"设计和时间推移"选项卡中，选择"时间推移"复选框并输入时间推移参数。选择"访问点"列表选项，并在文本框中输入要成像的点，用逗号或连字符分隔点，以完成序列。然后在"运行"选项卡中编辑文件名和文件位置，并在"开始实验"对话框中选择"播放"以开始实验。在分析结束时，在适当的去卷积程序中打开感兴趣的 RAW 图像文件，然后选择"过程"选项卡和去卷。在将图像保存为 TIFF 文件之前，请根据需要在任何或所有波长通道中执行任何手动背景减噪和亮度对比度调整。要进行单元长度量化，请打开制造商提供的软件或自由成像软件（如 ImageJ）中的去扩算图像文件，然后打开"工具"选项卡。然后选择"测量距离"，然后左键单击所需图像文件上的开始点和终点，以测量单元格长度。对于荧光信号量化，选择数据检查器。绘制一个框，将"列/行"选项设置为特定感兴趣的维度，然后选择要量化信号的区域。添加木糖诱导GGS 8菌株会导致六细胞表型。而空向量控件看起来类似于在没有诱导剂的情况下生长的对照细胞。金黄色葡萄球菌 gpsB 的超生产会破坏 B.子的细胞分裂，通过时间推移显微镜和细胞长度定量进行测量。此外，FtsZ或与此蛋白质相关的蛋白质的定位模式可用于记者通过时间推移成像和细胞长度定量来研究和/或识别新的抗菌化合物。请务必使用具有适当折射率的机油，因为折射率可能会根据成像使用的温度而变化。如果用户希望消除焦点外噪声，可以执行去卷积，还可以执行涉及荧光信号量化和细胞长度和宽度测量的数据分析。

Research

JoVE Journal

Immunology and Infection

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

科研

免疫与感染

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

活细胞视频显微镜病病原菌的吞噬作用

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

顺行运输Alphaherpes病毒传播的活细胞成像

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

荧光<em&gt;原位</em&gt;杂交（FISH）技术对病毒和植物和昆虫组织内共生细菌的本土化

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

蛋白质中 Plasmodesmal 定位序列的识别在底中

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...