LWE和RK支持由美国国立卫生赠款R37 NS033506-16和R01 NS060699-03。 MPT支持美国癌症协会的博士后研究奖学金（PF-10-057-01-MPC）。马修·莱曼博士和奥伦Kobiler博士的咨询和知识的的显微镜配置和活细胞成像方法设计。我们也感谢尼尔·巴洛和Brian T.凯因尼康仪器的购置，安装和性能的显微镜技术援助。

第1节 - 环境控制条件为活细胞荧光显微镜<ol><li>任何成像实验前30分钟连接，并开始升温，在显微镜阶段顶部孵化。 <ol><li>打开显微镜，透射和落射荧光光源，和自动化的硬件控制。打开显微镜控制软件，NIS元素，连接到显微镜，并开始冷却EM-CCD相机。 </li><li>附加镜头温暖适当的目标（60X或100X油浸） 注：根据实验类型选择物镜。 100X微分干涉对比（DIC）是最好的60X阶段目标跟踪顺运送病毒颗粒的装配，同时适用于可视化顺传播。较低的放大倍率，非油浸目标不需要镜头回暖的，因为他们不直接与样品接触。 </li><li>附加阶段顶部孵化到机动阶段英里<html> croscope。确保样品在适当的插入，将举行。将加湿后的空气从孵化器控制线的阶段顶部孵化器的输入端口。 </li><li>打开孵化器和镜头温暖的控制和调整温度设定具体的目标和成像样品（见讨论）先前验证条件。 </li><li>打开控制阀上的5％CO 2/95％的大气中的CO 2补充空气罐提供至台顶部的培养箱。为流动相，流速应为每分钟在60〜80毫升的气体。更快的速度，将导致大量的样品脱水尽管加湿室。 </li></ol></li><li>添加油物镜镜头，并立即放置到舞台顶部孵化器的文化，将映像。 </li><li>允许在培养的温度达到平衡至少10分钟。一小时是优选的，特别是对于第3节中所描述的成像过夜。 </li></ol>ve_title“&gt;第2部分 - 实时成像的顺行性病毒粒子运输活动协议<ol><li>成像前六至八个小时，感染了35毫米盖玻片底菜成熟，游离SCG神经元与1×10 6病毒兴趣斑块形成单位（例如病毒，请参阅表1）使用Taylor 等描述的方法2012a的人 11。如果测试一个以上的病毒，关闭设定的接种由30-60分钟，以允许在相似时间顺序样本成像感染后。 </li><li>将菜进入阶段顶部孵化器运行任何实验前10分钟最低。同时将温度达到平衡，焦平面转移和将产生负面影响的任何成像实验。 </li><li>使用透射光显微镜目镜，找到一个神经元的细胞体，具有明确孤立的轴突延长。另外，使用适当的荧光照明找到一个表达探测器荧光蛋白的表达。重要的是限制暴露的样品荧光灯照明系统，在该步骤和后续步骤中，由于光诱导的细胞损伤和漂白的荧光蛋白。 </li><li>衰减荧光照明，以减少激励光的强度的细胞暴露。从事光衰减荧光照明光路中的中性密度（ND）滤镜。对于持续时间超过1分钟，ND4最少的电影必须使用，以防止光漂白的荧光蛋白和广泛的光损伤的神经轴突。高荧光的结构与ND8过滤器后，通常可以被可视化。 </li><li>使用显微镜的软件（ 图2和图补充电影1A），EM-CCD相机的光路切换。 </li><li>确定最佳成像时间和条件，每个荧光通道。 <ol><li>启动实时图像窗口中使用“播放”按钮。&lt;/ LI&gt; <li>确定每个荧光蛋白的最佳相机的曝光设置。不要超过300毫秒，每通道的运动像差的快速移动的病毒粒子结构会发生。利用EM增益的摄像头设置300（制造商建议的最大增益），以尽量减少暴露时间，提高检测昏暗的信号。正确的曝光时间应该产生的图像，背景低强度和高比信号。 </li></ol></li><li>设置后的风险，并找到一个明确的区域轴突，该软件必须被配置为执行自动图像采集。停止实时成像窗口，防止样品漂白。 <ol><li> NIS Elements软件，选择6D -定义从“应用程序”下拉菜单/运行实验应用。 </li><li>在ND收购“窗口中，单击时间选项卡，这将决定频率的图像采集。设置“无延迟”，持续时间为5分钟的时间间隔。钍E软件将获取图像帧尽可能在最高频率，持续时间为5分钟。 </li><li>选择“拉姆达”选项卡，可以选择预先设定的用于多个荧光图像采集的硬件配置。点击第一个方块，并在其后的下拉菜单中选择一个合适的硬件配置。重复所有的荧光蛋白进行成像。 </li><li>确保“XY位置”，“Z系列”和“大图片”的选项卡选择。 </li><li>以上选项卡，选择“栏内填上”保存到文件“在收购过程中自动将图像保存到硬盘。配置实验输出文件的位置和文件名。正如顺序进行实验分析，该软件将在所指定的文件名的末尾添加一个序列号。 </li><li>一旦所有的标签都被选中。点击“运行”按钮，开始实验。 </li><li>通过曝光设置优化的图像采集率和IMAGE尺寸。使用一个小区域的成像区域，往往会加快帧采集速率。使用ROI工具在NIS-元素，选择图像总面积的25％和50％之间的区域定义区域的利息。或者，更短的曝光时间将增加帧采集速率，但会降低信号的质量，（见讨论）。 </li></ol></li></ol>第3节 - 隔夜时间推移成像顺行传播活动<ol><li>四小时前成像，感染在35毫米μ-菜文化构建一个条块分割的神经。的神经元细胞体，接种1×10 6噬斑形成单位的病毒还使用Taylor 等人所描述的方法（例如病毒，请参阅表1）。2012B 12。 </li><li>轻轻放在培养皿中到舞台顶部培养箱。确定的目标的位置被设置为侧烹调。慢慢地使目标成为关注的焦点，确保ing的目标不推到样品中。一旦已确定的重点领域，慢慢移动目标进入中心的菜，附近的轴突车厢内部侧内部屏障demarking。在运动过程中，确保维持聚焦深度，避免推高到样品。客观的塑料表面的偏转将损坏的轴突和塑料。 </li><li>添加约2毫升的温水（〜37°C）磷酸盐缓冲盐水的培养皿以外的特氟隆环。这周围的神经元文化与环PBS，减少样品脱水，并提供保温。 </li><li>允许至少1小时的温度达到平衡之前，启动自动图像采集（3.8）。在这段时间内，可以配置步骤3.5至3.7。 </li><li>光照强度限制到可能的最低水平，让所有中性密度滤光片。这将允许频繁图像采集的爱ŗ长的时间周期，没有过多的光损伤。减少照明将导致低信号，需要更长时间的曝光（见讨论）。 </li><li>使用年前的曝光设置或之前的实验设置生成目标荧光蛋白的表达，以建立的曝光设置，第2.5节中所描述的一个平行的盘感染的上皮细胞。 </li><li>设置曝光后，进行必要的渠道，它是时间配置自动图像采集软件。停止实时成像窗口，以减少样本的漂白。 <ol><li>在Elements软件（ 图2和补充电影2），选择6D - “应用程序”下拉菜单，弹出ND采集窗口定义“/”运行试验申请。 </li><li> ND采集窗口，单击“时间”选项卡。设置Interval“5分​​”。设置持续时间为20小时。循环字段的数目重复的程序，自动计算m将执行。 </li><li>选择“拉姆达”的标签。点击第一个方块，并在其后的下拉菜单中选择适当的lambda配置相衬。重复后续位置，选择合适的配置，每个荧光蛋白成像。 </li><li>选择“大图象”选项卡，它决定了参数的多个图像被组合成一个单一的大视场。一个很好的大小的区域由5×2使用7％的拼接重叠的图像。这将允许每个位置的单元格的最大数量的图像采集的频率，而不会影响成像。此外，取消选择“关闭主动式快门在第一阶段的运动”的复选框。此选项提高采集速度，不关闭图像之间的百叶窗。 </li><li>选择“XY位置”选项卡，通宵电影的过程中，将依次并行成像识别多个职位。选择定义宝中心的位置int对于每一个大的图像区域。选择6-8个样品中的图像位置。一个良好的形象区将有小簇细胞紧密并列明确界定的神经轴突。细胞不应该被彼此和轴突的同装堆的顶部，应尽量减少。对焦点的细胞的细胞核。的目标定位，使得一个明确定义的核膜被看见的大部分细胞。 </li><li>确保“Z系列”选项卡，取消选择。 </li><li>配置如上所述2.6.5节中的自动保存功能。 </li><li>测试曝光和点击“1时间循环”位置设置。评估图像透射光照明，重点和框架。如果焦点已经转移，焦点重置为每个位置的XY选项卡下。等待10分钟，然后重复此步骤，直到重点是稳定的。 </li><li>已验证和测试的所有设置后，单击“立即运行”按钮，启动自动实验。 </li></ol></lI&gt; </ol>第4节 - 图像处理与出口数据导出<ol><li>运输或传播事件在活细胞成像获得的数据集应该从NIS Elements软件出口到常用的文件类型，在演讲和出版物（补充电影3）供以后使用。要导出电影打开相应的原料。ND2文件NIS元素，并选择“拆分组件”可视化所需的荧光通道。播放的文件在一个合适的速度，每秒5张的可视化快速交通是一个很好的起点。 </li><li>包括一个时间戳代表在电影播放过程中的实时时钟。右键单击图像并选择添加ND信息。数字计数器将出现在左上角。 <ol><li>编辑时间标记来显示信息。在柜台上按一下滑鼠右键，选择“编辑”ND信息。在随后弹出的窗口中，编辑文本，反映了想象力ng的参数是重要的。编辑字体，颜色和大小的清晰度。 </li></ol></li><li>进入“编辑”菜单，找到“创建视图快照”，或按“X”键。这个命令打开“视图快照”窗口。选择“适用于所有的帧，以产生一个8位的RGB，出口准备的文件中含有的荧光从整个电影的频道。 </li><li>除作为跨平台兼容性。avi文件。设置在200毫秒每帧的播放速率。与其他转换软件。由NIS元素生成的AVI文件可以被进一步压缩成所需的文件类型。 </li></ol>

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpesviruses+use+bidirectional+fast-axonal+transport+to+spread+in+sensory+neurons.">Herpesviruses use bidirectional fast-axonal transport to spread in sensory neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (6), 3466 (2001).</li><li>Kramer, T., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alphaherpesvirus+Infection+Disrupts+Mitochondrial+Transport+in+Neurons.">Alphaherpesvirus Infection Disrupts Mitochondrial Transport in Neurons.</a> Cell Host &amp; Microbe. 11 (5), 504-514 (2012).</li><li>Swedlow, J. R., Platani, M. <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+using+wide-field+microscopy+and+deconvolution.">Live cell imaging using wide-field microscopy and deconvolution.</a> Cell Structure and Function. 27 (5), 335-341 (2002).</li><li>Kaech, S., Huang, C. F., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-Term+High-Resolution+Imaging+of+Developing+Hippocampal+Neurons+in+Culture.">Short-Term High-Resolution Imaging of Developing Hippocampal Neurons in Culture.</a> Cold Spring Harbor Protocols. 2012 (3), (2012).</li><li>Frigault, M. M., Lacoste, J., Swift, J. L., Brown, C. M. <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+microscopy+-+tips+and+tools.">Live-cell microscopy - tips and tools.</a> Journal of Cell Science. 122 (6), 753-767 (2009).</li></ol>

我们什么都没有透露。

活细胞成像的目的是观察生物过程，因为他们没有发生显着改变的行为观察。实现此目标，通过优化三个变量：环境控制，成像速度和荧光照明。必须平衡这些相互依存的变量，以实现可行的成像条件。协议利用特定的条件下产生的有代表性的结果。我们将简要讨论环境控制和观测损坏，然后详细介绍了具体的方法。 在显微镜上建立和维护相关的生物条件下的细胞事件监测?...

外周神经系统（PNS）alphaherpes病毒如单纯疱疹病毒（HSV）-1，-2，伪狂犬病病毒（PRV）感染涉及到一些复杂和高度调节的步骤，整个病毒的生命周期。 PNS的神经元内的交通运输是至关重要的两个主要病毒感染和后续主机间传播的事件期间。调节的分子机制，病毒的生命周期的两个组成部分;定向运输的轴突（顺运输）和后续传输的病毒粒子易感细胞（顺价差）从细胞组织内的病毒组件是重要的是要了解疱疹的发病机制。 病毒颗粒神经元的运输和出口的依赖于一个成熟的传染性病毒粒子1,2组装。此前固定的检测，包括免疫荧光（IF）和电子显微镜（EM），分别用于研究粒子组装状态和PROT运输和传播的病毒粒子3-6艾因相互作用。然而，运送病毒颗粒和实验文物化学固定推出的动态性质混淆固定图像7,8的解释。最近，已经描述了一些病毒荧光融合蛋白，单纯疱疹病毒，伪狂犬病毒，可以忽略不计的蛋白质的功能上的影响。绿色荧光蛋白（GFP）和红色荧光蛋白（mCherry或MRFP）的荧光基团往往是成对两个成熟的病毒粒子的衣壳，表膜，和糖蛋白9-11的三个结构部件，以允许成像。活细胞成像的顺交通使用双标记病毒的病毒颗粒的装配状态可视化，在运输过程中。类似的荧光基团，表示病毒株用于可视化传播12,13后的病毒基因组的数目和多样性。荧光蛋白的属性，（评论于14,15）极大地影响病毒或细胞的组件可视化的能力。时，应考虑的内在特性，荧光蛋白，包括自我相互作用和稳定性，设计和测试新的蛋白质融合的野生型功能的保存。 结合荧光蛋白融合，两个特征在体外细胞培养系统的就业和条块分割疱疹病毒感染活细胞成像：游离16 17（ 图1A）大鼠颈上神经节（SCG）神经元。在这两种系统中，胚胎SCG的解剖，解离为单细胞体，并在体外培养18镀。 SCG神经是植物神经系统的一部分，并可以很容易地通过神经生长因子（NGF）成为一个成熟的偏振态体外培养及诱导分化。游离SCG神经元的轴突形成扩展网​​络，使f或可视化的病毒颗粒，因为它们经过顺行运输6。条块分割的的SCG文化提供流体隔离神经细胞体（S舱）和远端轴突末端（N舱）19。先前已经公布的一些条块分割的神经元，使用原来的20改性Campenot室17的建设和使用的详细协议。允许选择性地感染神经元细胞体和子代病毒检测后交通隔离轴突末端流体隔离。电镀上皮细胞感染前末端提供了受体细胞病毒感染的传播。 有多种人体必需的元素，重要的是所有活细胞成像实验，但最相关的我们的协议是：自动化图像采集，荧光灯照明系统，成像速度和环境控制。对于所有成像实验中，我们使用一个倒置的，自动化的，落射荧光照明显微镜（ 图1B）。该显微镜是围绕尼康Eclipse钛基地迅速重新配置在自动显微镜图像采集，并采用了微机控制的电动系统。对于荧光照明，我们使用一种广谱汞弧灯，可以中性密度滤光片照明路径衰减。激发滤光片荧光灯照明系统的频谱限制，并搭配多通可视化特异性荧光化合物的分色镜和荧光发射滤波器。被安装在独立的，快速的切换滤光器轮的激发和发射滤波器，使不同的荧光通道的快速顺序采集。的图像采集速度得到进一步增强，有灵敏和快速的EM-CCD摄像头，用于检测低强度的信号和短的图像读取时间。环境控制上力显微镜OPE是实现与一个阶段顶部加热孵化器和物镜加热器，以保持饱和湿度和CO 2富集的气氛是通过在升高的温度下，同时放入培养箱样品。显微镜是保持在一个黑暗的房间里，环境温度保持接近25°C和外界光线，遮光窗帘上的所有窗口最小化。以下协议描述了使用该系统的顺运输和蔓延检测。 存在一些替代品来控制活细胞成像的变量。显微镜设置的控制可以通过手动进行，或者依赖于专有软件的自动化系统。荧光照明，可以实现用卤素，LED或激光源。修改成像的速度由速度的滤波器切换可视化成对的检测系统中的信号的时间。环境控制，可以实现与专用硬件英里croscope阶段，外壳加热和加湿的框中，然后在显微镜的全部或部分，或通过将执行显微镜的房间的升温。这些替代品中的每一个都涉及到成本和性能都有优点和缺点。 在随后的协议，我们详细使用活细胞成像研究通过使用重组病毒株的快速顺运输顺alphaherpes病毒蔓延。活细胞成像实时可视化衣壳，皮层和/或糖蛋白共定位11轴突内进行运输的动态结构。条块分割的神经文化隔夜慢速成像可视化轴突病毒粒子出口和感染易感细胞12。这里介绍的协议已经使用我们独特的成像系统进行了优化，但在广义上讲，相对于活细胞成像的四大要素。在我们的讨论将进一步详细一些优化成功的实验是必要的。

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>35 mm glass bottom culture dish</td>
 <td>MatTek Corporation; Ashland, MA </td>
 <td>P35G-1.5-20-C</td>
 </tr>
 <tr>
 <td>35 mm &mu;-Dish </td>
 <td>Ibidi USA LLC, Verona, WI </td>
 <td>81156</td>
 </tr>
 <tr>
 <td>Microscope body </td>
 <td>Nikon Instruments Inc. </td>
 <td>Nikon Eclipse Ti</td>
 </tr>
 <tr>
 <td>Motorized microscope X-Y stage</td>
 <td>Prior Scientific; Rockland, MA </td>
 <td>H117 ProScan Flat Top</td>
 </tr>
 <tr>
 <td>Motorized filter wheels</td>
 <td>Prior Scientific</td>
 <td>HF110 10 position filter wheel</td>
 </tr>
 <tr>
 <td>Fluorescent illumination source </td>
 <td>Lumen Dynamics; Mississauga, Ontario Canada </td>
 <td>X-Cite 120Q </td>
 </tr>
 <tr>
 <td>Multi-band Fluorescence filter sets</td>
 <td>Chroma Technology Corp.; Bellows Falls, VT</td>
 <td>89000 Sedat Quad - ET 89006 ECFP/EYFP/mCherry - ET</td>
 </tr>
 <tr>
 <td>EM-CCD camera</td>
 <td>Andor Technology USA; South Windsor, CT</td>
 <td>iXon3 897 </td>
 </tr>
 <tr>
 <td>Chamlide stage top environmental incubator</td>
 <td>Live Cell Instruments; Seoul, South Korea</td>
 <td>TC-L-10</td>
 </tr>
 <tr>
 <td>Objective lens heater</td>
 <td>Bioptecs; Butler, PA</td>
 <td>150803 Controller 150819-12-08 Heater</td>
 </tr>
 <tr>
 <td>Analysis software</td>
 <td>Nikon Instruments Inc.; Melville, NY</td>
 <td>NIS Elements </td>
 </tr>
 </tbody>
</table>

live cell imaging of alphaherpes virus anterograde transport and spread

活细胞荧光显微技术，以及建设反映荧光融合蛋白的重组病毒株的进展已使能运输和扩散的alphaherpes病毒感染的神经元的实时可视化。本实用新型荧光融合蛋白病毒膜，皮层和衣壳，结合活细胞成像，确定病毒颗粒组件轴突内进行运输。类似的工具已经成功地采用了细胞 - 细胞间传播的病毒颗粒定量细胞间传送的病毒粒子的数量和多样性分析。更重要的是，顺行运输和传播的活细胞成像技术产生财富包括粒子输运速度，颗粒的分布，时间和分析蛋白质的本地化的信息。除了经典的病毒的基因技术，这些方法提供了重要的见解进入重要的机械问题。在这篇文章中，我们详细描述的成像方法，回答基本问题的alphaherpes病毒运输和蔓延。

顺行交通此协议的应用与PRV 348游离SCG文化的感染，表达GFP-US9和GM-的mCherry膜融合蛋白的重组伪狂犬病毒株，促进病毒颗粒（ 图3和图4）补充电影顺行运输的可视化。这些融合蛋白掺入到病毒颗粒输送泪点检测结果，上述的成像条件尽量减少每个滤波器切换过程中在一个移动的粒子的荧光基团的偏移量。在一个三分钟的成像窗口，众多运输泪点通常观察?...

Watch this Scientific Journal Video about Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread at JoVE.com

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

活细胞成像的alphaherpes病毒感染使动态的定向运输和细胞间传播的事件分析。在这里，我们提出的方法，利用重组病毒株表达荧光融合蛋白，以方便在感染病毒组件可视化的初级神经元。

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 ...

live-cell-imaging-alphaherpes-virus-anterograde-transport

Research

JoVE Journal

Immunology and Infection

11.0K 次观看.  Montana State University. 活细胞成像的alphaherpes病毒感染使动态的定向运输和细胞间传播的事件分析。在这里，我们提出的方法，利用重组病毒株表达荧光融合蛋白，以方便在感染病毒组件可视化的初级神经元。

视频: Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

科研

免疫与感染

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

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

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

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

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 ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

使用真空稳定成像系统的实验性急性肺损伤中肺微循环的活体内宽视场荧光显微镜

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

小鼠下颌下腺再生的实时实时成像

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

作者聚焦：研究损伤后唾液腺再生中巨噬细胞-上皮细胞的相互作用 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

通过离体活细胞成像询问唾液腺中的细胞间相互作用

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...