在体内成像设施国子监的由MRC资助。

1。小鼠准备<ol><li>购买或滋生足够18-20克小鼠研究所需。 </li><li>如果老鼠从外部供应商购买，运输设施后，习惯于老鼠1周蒸压食物和水稳定肠道菌群。 </li><li>称重，耳切迹，并分离出所需数量的小鼠每笼。 </li><li>感染前一天，之前执行任何麻醉，检查是否有足够的氧气和异氟醚的程序的时间。如有必要，添加更多isofluorane或更换氧气瓶。随后，检查麻醉拾荒者的重量，如果他们已经获得了超过50克，因为他们第一次使用，丢弃它们更换新鲜的拾荒者。 </li><li> Anaesthetise老鼠，用3％isofluorane XGI-8麻醉系统和脱毛使用VEET为7分钟。 </li><li> 附注： <ul><li>彻底脱毛是必不可少的，尤其是在黑老鼠的毛发中黑色素显着衰减的发光信号3,15。 </li><li>皮草之前开始再生，老鼠的毛发通常持续8-10天。要执行一段时间后，4D多模态成像脱毛老鼠时必需的，以便感兴趣的领域是裸体成像会议。 </li><li>小鼠被安置在一二级生物安全（BSL-2）遏制设施。 </li></ul></li></ol> 2。细菌细胞的制备<ol><li>准备足够的卢里亚Burtani肉汤和琼脂平板上卡那霉素[50微克毫升-1]补充所需的研究和储存在4°C，直到需要。 </li><li>感染前一天解冻冻存管C. rodentium应变ICC180，立即加入一个cryobead至15毫升LB培养基辅以卡那霉素[50微克毫升-1]，在220 rpm培养过夜，37℃为中等污染控制，准备一个额外的未接种15毫升LB培养基辅以卡那霉素[50微克毫升-1]和文化在220转，37°C。 </li><li>第二天早上（约16小时），检查介质控制浊度细菌生长的标志。如果控制是明确的，转让ICC180文化猎鹰管，并在4000转离心。随后，冲洗细菌沉淀在PBS。然后重悬在1.5毫升PBS（辅以卡那霉素的LB原体积的1/10 日 ），调匀创建细菌接种。 </li><li>为了验证ICC180已正确培养和生物发光，图像在IVIS频谱CT的使用BLI生活图像4.3.1向导和自动设置如前所述1接种感染小鼠前。 注意事项： <ul><li>应变ICC180是一个发光衍生野生型C. rodentium ICC169 2。这种细菌的菌株具有的勒克斯 CDABE操纵包括一个Kanamycin抗性基因插入xylE假基因在染色体上的2。 </li><li>开展所有的细菌培养，使用标准的无菌技术，如果需要购买无菌耗材/试剂或高压灭菌，使用前。 </li><li>在步骤2.4中，生物发光读数（对/秒/厘米2 / SR）可以用来估计小鼠感染前细菌数。 </li></ul></li></ol> 3。与生物发光C小鼠感染的rodentium和细菌定植的评估<ol><li>感染前，检查是否有足够的氧和异氟醚的持续时间的程序。如有必要，添加更多isofluorane或更换氧气瓶。随后，检查麻醉拾荒者的重量，如果他们已经获得了超过50克，因为他们第一次使用，丢弃它们更换新鲜的拾荒者。 </li><li> anaesthetise小鼠用3％isofluorane使用XGI-8 AnaestheSIA系统提供人性化的克制。 </li><li>彻底混合细菌接种注射器吸取0.2毫升（约5×10 9 CFU ICC180）。 </li><li>删除小鼠从麻醉系统在时间和颈背他们用拇指和食指牢牢把握脖子后面的皮肤。 </li><li>灌胃针推往嘴里的屋顶，在舌头，食道和胃注​​入细菌接种到。 </li><li>小鼠灌胃，监视他们的步态和呼吸窘迫的迹象，这可能是一个迹象，细菌接种到肺部已交付。安乐死已接种的任何小鼠在肺部。 </li><li>管饲法用200μlPBS中的车辆控制的未感染的小鼠，中所描述的步骤是3.1-3.6。 </li><li>要确认正确，进行灌胃，图像的感染和模拟感染小鼠在IVIS频谱CT的使用BLI和生活中自动设置图像4.3.1向导如前所述，1。 </li><li>通过在PBS中连续稀释，斑点到添加有卡那霉素的LB琼脂上，一式三份，确定在接种物中的活菌数[50微克毫升-1]。 </li><li> ICC180 CFU /毫升计算通过寻找稀释大约有5-50菌落计数菌落数，并记录每个点，稀释稀释，将菌落计数。随后，计算菌落的平均数（三个点），这个值乘以稀释因子50（每个点是原来的1毫升样品20微升），将菌落计数，给人CFU /毫升的稀释。 </li><li>定植小鼠每日监控到预先称重的Eppendorf管中（称重良好的平衡），从每只小鼠中收集粪便，并根据样品的重量（0.1克毫升-1）稀释在PBS中的1/10。 </li><li>确定活菌数在PBS连续稀释的粪便和点状出血，一式三份到LB琼脂辅以卡那霉素[50微克毫升-1]。 </li><li>通过以下的步骤（3.9），计算每克粪便的存活细菌的数量，再乘以数个菌落形成单位/毫升在PBS中的粪便稀释因子（步骤3.10），得到0.1克·毫升-1个菌落形成单位/克。 </li><li>要确定用纯ICC180培养小鼠接种和所有殖民地的生物发光。从步骤3.8以琼脂平板上，用眼观察菌落形态评估或挑选有代表性的殖民地，从板块和通过光学显微镜进行分析。如果有必要，细菌鉴定可以执行的特定菌株的菌落PCR，以评估培养的纯度。随后，图像板块在IVIS频谱CT的使用BLI所描述的步骤3.7和检查，在平板上的菌落是100％的生物发光。 附注： <ul><li>步骤3.4，当灌胃针插入如果感觉到有阻力不要强行灌胃针，因为这会导致接种进入肺部。相反，重新插入针头，轻轻地再次尝试。 </li><li>第3.1步是可选的，取决于机构的动物福利政策无需麻醉，可口服灌胃小鼠。 </li><li>必须镀凳，他们收集了一天。C. rodentium将增长，即使留在4°C过夜在PBS，将导致量化菌落/克粪便是不正确的。 </li></ul></li></ol> 4。每日综合3D漫射光成像断层扫描成像μCT（DLITμCT）感染小鼠 动物福利的考虑：DLITμCT涉及DLIT的光学成像结合一个快速，低辐射剂量μCT扫描（约23 MGY两个鼠标的各种扫描，〜53 MGY为一个单一的鼠标扫描）的固定化的动物。这个剂量积累与每个成像会议，目的是保持剂量为卤味w为可能（且始终远低于LD 50/30），同时还完成了研究。在某些情况下，如果没有在传统的BLI扫描小鼠感染的迹象，μCT扫描可以被避免的，以进一步减少剂量。即使剂量保持尽可能低，在长期的研究中，如果有任何辐射暴露的担忧，老鼠将被剔除不利症状或在年底μCT成像期间的第一个迹象。 <ol><li>在成像之前，初始化IVIS频谱CT和X射线安全联锁装置的工作和激烈的阶段是在正确的温度（37°C）成像检查。随后，检查水平的isoflourane麻醉系统和麻醉剂的清除剂的重量。如有必要，添加更多isofluorane和，如果拾荒者已经获得了超过50克的，因为他们第一次使用，将它们丢弃，更换新鲜拾荒者。 </li><li>设置，使摄像参数，频谱CT运行自动曝光功能在生活图像4.3.1优化成像细菌荧光素酶。选择编辑&gt;首选项&gt;收购和自动曝光窗口。然后选择范围值&gt;地契。时间（秒）和设置&gt;最大。 300S。最后，选择&gt;目标计数（最小）&gt;发光，并设置为10,000计数。 </li><li>将鼠标成像平台到频谱CT和下探到麻醉。 </li><li>上的X-射线安全联锁开关，并初始化IVIS。 </li><li>一旦IVIS频谱CT的已初始化，使用XGI-8的麻醉系统来提供人性化的克制与3％isofluorane，麻醉小鼠。 </li><li>要确定是否有必要进行一个的μCTDLIT扫描图像前使用BLI小鼠如前所述，一个鼠标成像平台。如果获得一个强大的信号，鼠标是适合成像DLIT。麻醉鼠标离开鼠标成像platfor米的BLI扫描后准备为DLITμCT。 </li><li>使用生活图像软件4.3.1设置的μCTDLIT的扫描成像向导。成像向导会自动确定的视野，曝光时间，F-停止和分级的DLIT和视野，曝光时间，过滤网套和分级μCT成像提供最好的信噪比。当影像向导的提示，包括560，580，600，620 nm发射过滤器，并选择一个鼠标μCT扫描。然后点击&gt;收购开始成像。 </li><li>返回麻醉鼠标成像后的笼子里，取出一个鼠标成像平台从频谱CT和消毒，用1％三基因。如果需要，更换海绵垫，鼠标定位到小鼠之间的交叉污染的风险降到最低。 </li><li>图片小鼠每天最多8天感染后（PI）使用DLITμCT，中所描述的步骤是4.1-4.8，生成的成像数据的长度须作出4D莫感染争夺。 注意事项： <ul><li>在第4.7步中，我们建议使用560-620 nm发射滤波器的细菌生物发光图像。虽然细菌的生物发光的峰值大约是485纳米，由于组织的光学元件（蓝色/绿色的光子的小鼠组织的显着衰减），重要的是要使用用于三维重建的橙色/红色光子，因为它们便于信号深度的计算。 </li><li> &#39;自动&#39;的曝光设置在住图片4.3.1为DLIT需要进行调整，以优化捕获的光子量和用于每个过滤器设置的最大成像时间。 </li><li>它仅需要执行的BLI预成像描述时，在步骤4.6中，研究者不能确定时，如果有一个可检测的生物发光信号，例如使用一个新的感染模型。 </li></ul></li></ol> 5。三维重建DLITμCT成像数据<ol><li>开放的生活图像软件4.3.1和选择&gt;浏览和打开文件夹包含μCTDLIT的文件。 </li><li>在的生活图像浏览器，例如C.打开的μCTDLIT的文件rodentium日1 PI通过点击加载。 </li><li> Tool Palette中选择表面地形&gt;裸鼠，调整阈值，然后单击“&gt;”生成曲面。如果表面重建是成功的表面轮廓将显示在3D视图窗口。 </li><li>要在3D视图“窗口中查看呈现的μCT扫描，隐藏的表面轮廓（点击&gt; 3D光学工具，取消选择”显示曲面对象），或降低表面不透明（点击&gt; 3D光学工具“&gt;”调整不透明度滑块）。 </li><li>提供详细的解剖定位的的3D DLIT重建有必要改变的三维空间数据（μCT扫描），以便只有鼠标的骨架是可见的，它具有最佳的对比度。点击&gt; 3D多模工具和选择&gt;对数直方图，Gradient照明，质量和降噪。最后，调整滑块向右直方图和观看的三维立体数据的变化对比。不断调整数据，直到只剩下骨头都清晰可见。如果需要，更改颜色直方图的传递函数点。 </li><li>下一页选择&gt; DLIT三维重建中的工具调色板，并确保只有560，580，600和620纳米过滤套，所必需的成像细菌体内荧光素酶，已被选定。点击“&gt;”开始“。数据现在会出现预览窗口“菜单中显示鼠标在2D成像光谱过滤BL信号。这些信号被自动阈值由软件实现，但也可以手动调整，如果需要使用上面的菜单底部的选项卡。阈值确定，包括作为在重建的数据的最小数据的强度（以百分比表示的图像中的最大信号）。 </li><li>接下来选择&gt; DLIT Tool Palette中的三维重建，并检查用于DLIT重建的光学特性是正确的。选择“&gt;”属性“选项卡，并确保组织属性的设置被设置为鼠标组织，设置源频谱细菌。 </li><li>点击&gt;改造执行的DLIT重建。 </li><li>要生成的μCTDLIT的3D电影点击&gt;工具&gt; 3D动画和选择&gt;预设动画&gt; CCW Y-轴和总时间10秒（每秒25帧）。一旦3D动画的设置是正确的按&gt;记录和保存的μCTDLIT的三维重建。avi文件在一个单独的文件夹中，实验组和时间点标记。 注意事项： <ul><li>这是很重要的μCTDLIT数据进行三维重建时，生活图像4.3.1和服务包1安装和使用PC电脑有一个显卡适合运行多模态成像软件。钍软件可以下载，GPU规格都包含在发行说明（ <a href="http://www.caliperls.com/support/software-downloads.htm" target="_blank">http://www.caliperls.com/support/software-downloads.htm</a> ）。 </li><li>如果出现向后的μCTDLIT的三维重建被救活后，您的计算机运行的是老版本的显卡驱动程序，这可以从制造商的网站下载更新纠正。 </li><li>在步骤5中，我们提出我们的首选设置用于生成3DμCTDLIT的重建。但是，也有可以进行修改，在此协议中未讨论的参数的过程中三维重建的多个点。 4.3.1生活图像三维重建对于一个全面的指南，请参阅制造商的说明书。 </li><li>重要的是要创建的三维重建，使用相同的设置在多模态成像工具，所以它们具有相近。除了，当创建的μCTDLIT的三维重建，这是重要的，以使用相同数目的帧每秒和总持续时间（长度），否则4D视频定时是不是同质视频。 </li></ul></li></ol> 6。 C.生成一个4D电影rodentium感染<ol><li>清楚标示的μCTDLIT的三维重建正确的实验，时间点和组在一个易于访问的文件夹。 </li><li> Windows Live影音制作中，并创建一个新的文件，按时间顺序从第1天PI从文件夹中使用“工具”选项卡上的步骤6.1编制插入的μCTDLIT的重建。 </li><li>描述的时间点开始的每一个视频中添加字幕，例如1天PI，通过选择适当的μCTDLIT的视频，然后选择首页&gt;添加标题。一个文本框出现在屏幕上，添加适当的传说。调整字体大小，样式，颜色和DES的理由IRED使用相同的标签微软Word软件中的工具栏。最后，将左上角的视频标题。 </li><li>重复步骤7.3所有的μCTDLIT的视频。 </li><li>添加标题页，例如C。天rodentium感染1-8通过点击首页&gt;标题。适当的标题被添加到一个空白幻灯片在屏幕上显示一个文本框。调整字体大小，样式，颜色，随意使用相同的标签微软Word软件中的工具栏和理由。 </li><li>将项目保存为一个Movie Maker项目* MMP在适当的文件夹的标题电影。这一步是必不可少的，如果你想修改的影片。 </li><li>保存电影。wmv文件。点击影片制作菜单&gt;电影&gt;计算机。 </li><li>转换电影制作使用的文件转换器。avi或其他合适的文件类型与您的计算机兼容的文件。 附注： <ul><li>这是建议的4D电影生成的PC上安装的Windows Live Movie Maker中（ <a href="http://windows.microsoft.com/en-GB/windows7/products/features/movie-maker" target="_blank">http://windows.microsoft.com/en-GB/windows7/products/features/movie-maker</a> ）和良好的多媒体程序可以播放WMV。 avi文件类型。 </li></ul></li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+antituberculosis+drug+activity+in+vitro+and+in+macrophages+using+bioluminescence.">Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence.</a> J. Antimicrob. Chemother. 67, 404-414 (2012).</li><li>Kuo, C., Coquoz, O., Troy, T. L., Xu, H., Rice, B. W. <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+reconstruction+of+in+vivo+bioluminescent+sources+based+on+multispectral+imaging.">Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging.</a> J. Biomed. Opt. 12, 024007 (2007).</li><li>Cronin, 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+in+vivo+bioluminescent+imaging+for+the+study+of+bacterial+tumour+targeting.">High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.</a> PLoS One. 7, e30940 (2012).</li><li>Mundy, R., MacDonald, T. T., Dougan, G., Frankel, G., Wiles, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrobacter+rodentium+of+mice+and+man.">Citrobacter rodentium of mice and man.</a> Cell Microbiol. 7, 1697-1706 (2005).</li><li>Curtis, A., Calabro, K., Galarneau, J. R., Bigio, I. J., Krucker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+variations+of+skin+pigmentation+in+C57BL/6+mice+affect+optical+bioluminescence+quantitation.">Temporal variations of skin pigmentation in C57BL/6 mice affect optical bioluminescence quantitation.</a> Mol. Imaging Biol. 13, 1114-1123 (2011).</li><li>Wiles, S., Crepin, V. F., Childs, G., Frankel, G., Kerton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+biophotonic+imaging+as+a+training+aid+for+administration+of+substances+in+laboratory+rodents.">Use of biophotonic imaging as a training aid for administration of substances in laboratory rodents.</a> Lab Anim. 41, 321-328 (2007).</li><li>Crepin, V. 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=Dissecting+the+role+of+the+Tir:Nck+and+Tir:IRTKS/IRSp53+signalling+pathways+in+vivo.">Dissecting the role of the Tir:Nck and Tir:IRTKS/IRSp53 signalling pathways in vivo.</a> Mol. Microbiol. 75, 308-323 (2010).</li><li>Willekens, I., 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=Evaluation+of+the+radiation+dose+in+micro-CT+with+optimization+of+the+scan+protocol.">Evaluation of the radiation dose in micro-CT with optimization of the scan protocol.</a> ContrastMedia Mol. Imaging. 5, 201-207 (2010).</li><li>Boone, J. M., Velazquez, O., Cherry, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-animal+X-ray+dose+from+micro-CT.">Small-animal X-ray dose from micro-CT.</a> Mol Imaging. 3, 149-158 (2004).</li><li>Sato, F., Sasaki, S., Kawashima, N., Chino, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+effects+of+whole+or+partial+body+x-irradiation+on+mice:+life+shortening.">Late effects of whole or partial body x-irradiation on mice: life shortening.</a> Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 39, 607-615 (1981).</li><li>Kohn, H. I., Kallman, R. 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+influence+of+strain+on+acute+x-ray+lethality+in+the+mouse.+II.+Recovery+rate+studies.">The influence of strain on acute x-ray lethality in the mouse. II. Recovery rate studies.</a> Radiat. Res. 6, 329-338 (1957).</li></ol>

生产和自由地进入本文珀金埃尔默赞助。 作者凯文·P，弗朗西斯，杰夫Meganck和Chaincy郭卡尺珀金埃尔默公司的雇员。 根据动物科学程序法1986进行了所有的动物实验，并分别由当地的伦理审查委员会的批准。

4D电影细菌感染提供了一个有用的工具，可视化和解释大量的多模态成像数据快速，轻松地。这种技术有助于详细分析了如何通过单个鼠标感染扩散，并可以用来研究如何删除主机或细菌基因或特定的干预策略效果细菌负荷，分销和本地化在一项纵向研究，7。这些影片也提供了有用的教具和向公众传播信息的一种手段。 有几个关键步骤，在这个协议中，可能会影响获得的数据从DLIT-μCT成像和能够编译感染的4​​D视频质量。在此协议中最重要的部分是成功的和均匀的感染与C小鼠， rodentium。至关重要的是，用于研究小鼠在18-20克之间，并且bacte里亚尔接种新鲜烹制的约5×10 9 CFU，如前所述2,3。小鼠感染之前，重要的是要检查，使用频谱C​​T，一旦已接种物制备接种物的生物发光，它必须不断地均质化之前，每只小鼠灌胃，以确保小鼠获得类似的感染剂量。老鼠的的μCTDLIT成像进行了优化，使自动曝光功能的生活图像4.3.1软件自动确定优化成像参数的信号要远高于噪声。然而，在自动曝光功能依赖于用户定义的设置和参数的程序中所描述的，这需要进行修改。如果不这样做会导致图像不佳，收集的光子数量较少，不会导致感染的一个明显的进展，作为频谱CT的出厂设置为自动曝光编程肿瘤成像表达萤火虫荧光素酶。重组使用560-620毫微米，得到最佳的模拟和测量数据之间的协议，因此，更可靠的数据包括在重建进行。 μCTDLIT使用的一个限制μCT扫描，电离辐射会导致亚致死辐射伤害是累积一项纵向研究，18。亚致死辐射暴露可能减弱免疫反应，引起DNA损伤，凋亡的内脏19。最终，累积亚致死辐射损伤可导致死亡的LD 50/30，如果超过电离辐射，这是在5至7之间戈瑞取决于小鼠品系和年龄的小鼠用18,20,21。虽然一些分子电离辐射损伤可以治愈，因为总体的原则是保守估计剂量，这是通常不占研究规划。相反，其目的是尽量留BEL嗷嗷这些限制可能，同时还完成了研究目标。这一点尤为重要，因为正常的免疫反应的感染在本研究中，可能会更容易电离辐射成像的频率，而事实上，转基因，免疫组成的，或严重感染的动物。 规划时，在实验过程中产生感染的4D电影时，重要的是要考虑实验的长度，在此期间要求μCT扫描的数量和LD 50/30的电离辐射所使用的小鼠品系。另一个潜在的限制到DLITμCT内的细菌菌株正在使用的记者表达的是实力，因为这会影响细菌的检出限和成像时间。强烈建议，研究人员使用经过验证是完全致命的细菌菌株，但最大的lux操纵子的表达进行了优化，证明以前BLI2,3。 当前设计的四维成像的一个需要注意的是，每部电影是由个别μCTDLIT的扫描有不同的光子缩放。这可以使图像难以解释的本地化的BL灶，其强度的变化是微妙的，如果有一个强烈的BL焦点周围由多个弱灶。因此，对于纵向的可视化，重要的是保持一致的时间点的彩条。 可以应用到任何适当标记的细菌病原体感染的4D电影的概念。这项技术的未来发展将致力于使用荧光成像断层扫描（FLIT）的以及DLIT的方便使用相结合的生物发光细菌病原体和荧光近红外探头调查感染的宿主反应注射感染的宿主反应的调查。除了这个，在这个协议中，我们只描述了使用生物发光的细菌感染创建4D电影。然而，在某些情况下，可能会需要使用荧光标记的细菌，例如用IRFP标签，所以可以用于调查在感染过程中的主机遗传学，生物发光记者。重要的是，使用的多模态成像结合DLIT /熔接μCT将让我们的非侵入性调查多个参数过程中的细菌感染，这将有助于显着减少，改进和更换使用动物在科研中列出的NC3R主动（ <a href="http://www.nc3rs.org.uk/" target="_blank">http://www.nc3rs.org.uk/</a> ）。

小动物模型，特别是那些利用小鼠，常规用于研究细菌致病或测试感染，如抗生素，益生菌，益生元和疫苗1-7干预策略。主要实验小动物感染的病原体负荷读数，空间和时间的本地化的感染，受感染的机体的免疫反应， 在体内光学成像传染病研究的一个有价值的工具，可以用来监视多个实验通过使用读出的报告基因（荧光素酶，荧光蛋白，β-内酰胺酶，等），荧光染料，纳米颗粒或化学发光探针对象的蛋白质，生物过程或微生物6。 生物发光成像（BLI）是一种光学成像方式用于监视的小动物，如小鼠和大鼠的定植，致病bacteRIA 3,6,8,9。小鼠感染的重组细菌表达一个荧光素酶，如从发光光勒克斯 CDABE操纵子。这些细菌可以然后可以通过它们的光的生产，使用基于在体内成像系统3,6,9的CCD检测。重要的是，唯一的代谢活性的微生物是生物发光（BL），意思是唯一可行的被检测到的细菌细胞通过这种方法10,11。使用2D BLI，BL源的位置是推断，从动物的表面，其中的信号被发射8。确切的解剖定位在体内的BL灶，必须确定通过体外分析器官3,6,9相反，复合3D漫射光成像断层扫描（DLIT）可用于编译的定量三维重建的BL来源12。通过收集BL拍摄的图像进行DLIT使用定义的窄带通光学滤光器和随后输入到弥漫光断层扫描三维重建算法1,7,12,13。 目前，多模态成像是唯一的方法，可用于体外分析，而不需要得到真正的非侵入性的解剖定位在体内的生物发光灶。最近，我们使用的组合DLIT共同注册μCT成像评估枸橼酸杆菌rodentium（C. rodentium）定植动态预防性治疗益生菌7。C. rodentium是鼠科的特定的肠道病原体用于模型人类感染肠致病性和埃希氏 enterhemorrhagic的大肠杆菌14。C. rodentium感染引起的肠炎，通常伴有轻度减肥，腹泻，极化的Th1免疫反应和不同的病理变化，包括结肠隐窝增生和附加谦虚病变formati14。此外，C. rodentium发病已经彻底使用BLI和定植动态C57BL/6J小鼠是有据可查的，这种细菌的理想模型使用的多模态成像3,4,7微生物研究。 该协议是第一μCTDLIT集成使用一个多模态成像平台，IVIS频谱CT，和新一代的4D电影的真正动力的非侵入性感染的细菌感染成像勾勒出一个方法。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Bioluminescent C. rodentium</td>
			<td>Frankel lab</td>
			<td>ICC180</td>
			<td>Wiles et al., 2004</td>
		</tr>
		<tr>
			<td>Veet</td>
			<td>Boots</td>
			<td>&nbsp;</td>
			<td>Optimal depilation time is 7 min. Depilation works better if the cream is rubbed in well.</td>
		</tr>
		<tr>
			<td>Isofluorane (100% v/v)</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Medical Oxygen</td>
			<td>BOC Medical</td>
			<td>&nbsp;</td>
			<td>Size F Cylinder. Note: an appropriate regulator is required.</td>
		</tr>
		<tr>
			<td>Luria Bertani broth</td>
			<td>Merck</td>
			<td>1.10285.0500</td>
			<td>25 g in 1L Demineralised water.</td>
		</tr>
		<tr>
			<td>Luria Bertani agar</td>
			<td>Merck</td>
			<td>1.10283.0500</td>
			<td>37 g in 1L Demineralised water.</td>
		</tr>
		<tr>
			<td>Kanamycin sulphate</td>
			<td>Sigma (Fluka)</td>
			<td>60615</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml Polypropylene conical Falcon tubes</td>
			<td>BD (Falcon)</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Universals</td>
			<td>Corning (Gosselin)</td>
			<td>E5633-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD (Plastipak)</td>
			<td>300013</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oral dosing needle (16G x 75 mm) curved</td>
			<td>Vet Tech</td>
			<td>DE005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microbanks (Cryovial)</td>
			<td>Pro-Lab Diagnostics</td>
			<td>PL.170/Y</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IVIS Spectrum CT</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>133577 Rev A/ Spectrum CT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>6kVA UPS</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>XGI-8 anesthesia system</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>118918</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>XAF-8 Anaesthesia filter charcoal</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>118999/00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Living Image v4.3.1 SP1</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Benchtop shaking incubator</td>
			<td>New Brunswick Scientific</td>
			<td>Innova 44</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

4d multimodality imaging of citrobacter rodentium infections in mice

此协议概述纵向监测的生物发光细菌感染，使用复合3D漫射光成像断层扫描集成μCT（μCTDLIT）和随后利用这些数据来生成一个四维（4D）电影感染周期所需的步骤。要发展感染4D电影和验证的μCTDLIT的使用IVIS频谱CT成像细菌感染的研究中，我们使用生物发光C.感染rodentium，这会导致自限性的小鼠结肠炎。在这个协议中，我们勾勒出感染小鼠生物发光C. rodentium和日常的μCTDLIT成像列举8天从粪便和细菌定植的非侵入性的监测。 的IVIS频谱CT的使用，有利于无缝配准的光学和μCT扫描，使用一个单一的成像平台。低剂量μCT方式使老鼠的摄像在感染过程中的多个时间点，提供详细的解剖定位，而不会造成文物的累积辐射生物发光细菌灶3D。更重要的是，受感染的小鼠的4D电影提供了一个功能强大的分析工具来监测体内的细菌定植动态。

感染的C57BL/6J小鼠用5×10 9个菌落形成单位C. rodentium是一个很好的描述的细菌感染模型和一种自限性的胃肠道感染的结果，峰之间的第6-8天感染后持续3〜4周2,14之间。这种感染是局限于肠腔通过小鼠的免疫系统，并作为一个结果，细菌在粪便中不断脱落。 C.小鼠定植rodentium可以监视非侵入性细菌枚举直接从粪便，或生物发光成像2,3。 这份手稿概述了一种优化的协议执行3D和4D C.多模态成像监控细菌的感染过程中的负荷和本地化小鼠rodentium内。 图1中的结果表明控制所需要的成功的4D成像。小鼠感染前，它是必不可少的，以防止我所使用的细菌接种物是生物发光（ 图1A），与生物发光C鼠标灌胃后rodentium，该信号中可以观察到的动物的胃，而不是在肺中（ 图1B）16。 除了 ​​监测细菌定植，利用生物发光成像，它是很好的实践量化粪便中的细菌数量是胃肠黏膜的细菌定植的间接测量使用。 图2展示的典型细菌负荷粪便中C57BL6 / J 8 〜5×10 9 CFU ICC180已经感染了8天PI定植增加6-7天，直到第2天PI监控小鼠感染峰〜5×10 10个/ g， 这是在以前的报告符合2,3,17。 要强调的重要性的评价方法婷在一个单一的鼠标， 图1b和3以及视频1感染的传播已经产生相同的鼠标C. rodentium感染，如上面所述。每日DLITμCT被用来评估在此鼠标的发光细菌的空间分布，使用骨骼解剖参考图3演示了ICC180在感染3个关键的时间点的PI于的μCTDLIT重建的C57BL/6J小鼠3天PI小，生物发光病灶可以观察到结肠，生物发光强度第5天PI与空间分布变化不大，表现出温和增长。在第7天的PI中，我们观察到了显着性增加，生物发光和生物发光灶遍布整个结肠。 视频1是一个汇编的3DμCTDLIT的1-8天PI ICC180重建，并说明C.rodentium 1-2天PI之间蔓延到天3 PI 4-6天PI在结肠中的细菌数量，直到在第7天达到高峰PI细菌病灶覆盖整个结肠扩张结肠近端胃肠道内。在8天的PI只有两种截然不同的细菌灶中存在的近端和远端结肠。在每个时间点按照时间顺序查看完整的三维重建是一个功能强大的工具来分析宿主病原体相互作用的能力，很容易解释，比3D仍是相同的数据集。 <img alt="图1" src="/files/ftp_upload/50450/50450fig1.jpg" /> 图1。二维生物发光成像的A）生物发光C. rodentium ICC180接种和B）的小鼠灌胃后用200μl的接种物。箭头（&gt;）说明生物发光C. rodentium </<html> em&gt;的胃。 <img alt="图2" src="/files/ftp_upload/50450/50450fig2.jpg" /> 图2 C。 rodentium定植动态量化C.从粪便rodentium菌落形成单位8天PI <img alt="图3" fo:content-width="3in" fo:src="/files/ftp_upload/50450/50450fig3highres.jpg" src="/files/ftp_upload/50450/50450fig3.jpg" /> 图3。漫射光断层扫描成像的生物发光C-μCT扫描从一个鼠标rodentium感染监测3天，5和7 PI箭头（&gt;）说明了结肠的定植。 <a href="https://www.jove.com/files/ftp_upload/50450/50450fig3large.jpg" target="_blank">点击这里查看大图</a> 。 视频1。p :/ / www.jove.com/files/ftp_upload/50450/LAB_MEDIA_50450_Frankel_Video1.wmv“的目标=”_blank“&gt;点击此处查看视频。4D电影从一个鼠标C. rodentium感染监测1-8天PI

Watch this Scientific Journal Video about 4D Multimodality Imaging of Citrobacter rodentium Infections in Mice at JoVE.com

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

多模态成像是一种有价值的方法，在小动物模型研究细菌定植。此协议概述感染小鼠生物发光<em&gt;柠檬酸rodentium</em和纵向监测细菌定植复合3D弥漫的光成像断层扫描成像μCT创建一个4D电影<em&gt; C。 rodentium</em&gt;感染。

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

4d-multimodality-imaging-of-citrobacter-rodentium-infections-in-mice

Research

JoVE Journal

Immunology and Infection

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

12.9K 次观看.  Imperial College London. 多模态成像是一种有价值的方法，在小动物模型研究细菌定植。此协议概述感染小鼠生物发光柠檬酸rodentium

视频: 4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

“<em&gt;枸橼酸rodentium</em&gt;鼠标产品型号：研究感染性结肠炎的病原体和宿主的贡献

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

在抗菌蓝光治疗的体内研究的多药耐药鲍曼不动杆菌感染烧伤利用生物发光成像

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

成像 Ca2 +在志贺菌感染上皮细胞期间的响应

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...