我们感谢杰夫·丹尼尔斯的技术援助。这项工作是由R01 AI067946到JSO

1。大衣盖玻片与抗体<ol><li> Prewarm（37℃）30毫升的RPMI 10％FCS培养基和1ml BD Cytofix / Cytoperm的。 </li><li>制备的磷酸盐缓冲盐水（PBS）5微克/纯化的抗体的溶液中。对于激活NK92细胞系，利用反CD18和抗NKp30的推荐。 <ol><li>商标申请一个约角钱大小的圆圈为每个条件上使用PAP笔在＃1.5盖玻片。对于双色彩实验，应该有四个条件:未染色，双染，和两个单染条件。免除200微升的抗体溶液在每个区域中，孵育在37℃下30分钟。 </li><li>在室温下轻轻地浸渍在50ml锥形管中的每个在50 1毫升的PBS洗涤盖玻片。洗涤应在加入的细胞和应小心以避免在盖玻片抗体烘干立即发生。 </li></ol></li></ol> 2。启动封面上​​的自然杀伤细胞单;修复和通透<ol><li>每个隔离条件5×10 5 NK92细胞。离心，倒出上清液。用10ml预热的媒体从步骤1.1洗一次。离心，倒出上清液。 <ol><li>重悬的细胞在从步骤1.1预热介质以2.5×10 6 / ml的浓度。 </li><li>轻轻倒出200μl到1.2.1节中创建的区域的中心。在37℃下进行20分钟，在5％CO 2。 （注:这个时间可以延长或减少取决于利益的生物学功能对于NK细胞颗粒极化，20分钟就足够了。）。 </li></ol></li><li>下列细胞的孵育后，轻轻地通过在50ml锥形管中浸渍各50毫升室温的PBS洗涤盖玻片。 </li><li>加入1μl的Triton X-100的1毫升彻底从步骤1.1和涡流预热的修复/烫发液。固定和通透加入200微升的修复/彼尔姆缓冲（步骤2.3）的细胞。孵育10米在黑暗中在室温下进行。 </li></ol> 3。细胞染色<ol><li>制备染色缓冲液:磷酸盐缓冲盐水（PBS），1％BSA，0.1％皂素。 <ol><li>制备第一抗体的溶液中 200微升 染色缓冲液（见步骤3.1）。 （注:抗体应在使用前进行滴定）。避免使用时引发在用于涂覆的盖玻片（步骤1.2）的同种第一抗体。此外，还要避免Strepatividin-生物素的联系为STED成像。 </li><li>下节2.3.1，轻轻在50毫升染色缓冲洗盖玻片。轻拍PAP笔区域的边缘用棉签去除多余的缓冲区。适用于3.1.1节创建的抗体溶液。孵育30分钟，在黑暗中在室温下进行。 （建议:在孵育箱滑动盖玻片用潮湿的纸巾以保持湿度）。 </li></ol></li><li>制备第二抗体溶液中 200微升 染色法缓冲区。推荐荧光团的Alexa Fluor 488，太平洋橙，和V500。一般来说，一个1:200稀释，适合STED成像。 <ol><li>在50毫升染色缓冲液轻轻冲洗盖玻片。轻拍PAP笔区域的边缘用棉签去除多余的缓冲区。适用于二次抗体溶液。孵育30分钟，在黑暗中在室温下进行。 </li></ol></li><li>重复洗涤和染色的其他蛋白。如果检测到的F-肌动蛋白的鬼笔环肽用，这可以包括与二次抗体，一般在1:200稀释。 </li></ol> 4。摩盖玻片上的幻灯片<ol><li>准备安装介质。注:时延或延长黄金是可取的。 VECTASHIELD必须避免，因为它不是用受激发射损耗兼容。必须避免2,2 - thiodioethanol如鬼笔环肽被使用。 MOWIOL是可以接受的。 </li><li>将约10-20微升幻灯片上的安装介质。反转盖玻片（细胞面朝下），并安装盖玻片轻轻地，小心地避免引入气泡。幻灯片孵育24小时（盖玻片上）前成像。 </li><li>密封盖玻片的边缘用指甲油。 </li></ol> 5。实验装置<ol><li>启动所需的激光器和软件。启动STED枯竭激光以100％功率。对齐STED激光，这在商业系统的情况下通常是一个自动化的过程。 </li><li>重点样本，单染控制开始，使用目镜显微镜。 </li></ol> 6。设置的优化<ol><li>扫描第一信道和优化激光功率，激发光束位置和检测器的范围。如果可能的话，避免&gt; 100的增益。捕获图像中的共聚焦到优化设置。线和/或帧平均会提高分辨率。检查像素的饱和度。注意:某些饱和度是在共聚焦接受的应用受激发射损耗将减少排放的强度。对于受激发射损耗，最佳的像素尺寸将是30nm以下如何以往更好的分辨率将与更小的像素尺寸来获得。被成像的感兴趣区域的大小将决定像素大小的下限。更小的像素尺寸可能会增加漂白。 <ol><li>应用STED光束枯竭和捕捉图像，从50％枯竭激光功率。如果在分辨率的改善可以看到的，更耗尽激光功率可被应用。在此阶段，可能有必要调整激发激光功率，平均线，和/或增益。 </li><li>申请时间选通，以降低背景（最低0.3纳秒）。调整设置，直到过共聚焦分辨率的提高可以看出。分辨率可以通过估计半峰全宽（FWHM）来近似。这代表在通过在感兴趣的结构画线配置文件中创建一个高斯峰的半最大强度的距离，估计是分辨率的一种广泛使用的方法。 </li><li>一旦第一个通道是令人满意的，启动第二序列为SEquential扫描。在一般情况下，最好是扫描较长波长的荧光团第一。在第二个通道重复步骤6.1。 </li></ol></li><li>由成像单染控制与两个扫描序列确认缺乏光谱重叠的。温和的光谱重叠可以使用软件中的频谱未混合的特点予以纠正，但应尽可能避免。 </li></ol> 7。图像采集<ol><li>获取图像。对于定量成像，建议以得到至少20张/条件。确切的数字，但是，应根据实验的问题在演唱会与统计方法，如样本量的计算定义。保存实验。 </li></ol> 8。解卷积<ol><li>用反卷积软件或批量处理器打开文件。检查参数使用软件每个通道。确认每个通道的激发和发射光谱，受激发射损耗消耗排放，成像方向化（上升或下降，如果该图像是3维）尤其如此。 </li><li>使用去卷积反卷积向导。默认设置是通常足够，但是信噪比（SNTR）会有所不同荧光团的荧光团，以及将需要对每个通道和每个单独的实验来确定。 </li></ol>

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作者宣称自己有没有竞争的经济利益。这篇文章的部分出版物成本拨款支付徕卡Microsystems公司

在过共聚焦分辨率的提高将会有所取决于其无法控制的因素。这些因素包括在盖玻片厚度和安装介质不一致轻微畸变。它保留在成像室尽可能一致的温度和湿度是重要的，并且受激发射损耗光束应被拉直，大约每60分钟。正如上文中的程序，必须避免使用VECTASHIELD安装介质，因为这是不符合STED兼容。除了这些，每个人都应该使用＃1.5盖玻片，可能的话，使用那些已被证实为一个特定的厚度。 这里描述的方法的一个改进是在共聚焦图像附加信道，使用能发出在更长的波长比STED光束的荧光团。以这种方式，人们可以像多达四个通道（两个在共焦，2在STED）。如果采用这种方法，但是，通道荧光Ëmitting的受激发射损耗耗尽激光上面将需要被第一成像，如应用程序中的受激发射损耗光束将耗尽的光子在这些信道。一个优点是该技术是时间选通的应用，这也将通过从光子寿命短34消除排放改善在共聚焦分辨率。特别地，使用时间选通，发射检测器的时序，以脉冲激发受激发射损耗相对应，将成像接近它时，降低背景荧光从反射离开玻璃盖玻片。即使是在一个实验不适于受激发射损耗，如果使用脉冲声源，选通时间可以是用于提高分辨率的共焦的有用工具。 有可以被利用来改善分辨率在STED各种修改。之一是降低从标准1艾里单元针孔的大小，虽然这也将降低光到达样品的量。这可以通过增加拉斯来补偿呃功率或增益。另一个是提高平均线，这将增加收集的信息对每个光子的数量，提高分辨率。再次，然而，这可能是在样品的光漂白的成本，所以平衡需要的分辨率和漂白之间取得平衡。同样，利用荧光蛋白如GFP都将需要仔细优化，以避免脱色。这可以通过受激发射损耗减小激光功率，如果有必要来完成。更长的时间点也将允许更大的光子恢复，减少漂白。校正漂白应占现场分析时，受激发射损耗的。 当然，成像在3维空间中受激发射损耗也是可能的，并且也将得到改进，常规共焦成像。这是如果它是在与去卷积组合完成，尤其如此，虽然应注意，以校正期间在z轴成像的多个平面中发生的漂移。如果使用惠更斯软件去卷积，使用"图像稳定"功能获得此修正。使用这种方法，在沿z轴的分辨率将得到改善。这是一个很大的改善优于传统共焦成像，具有较差的轴向分辨率，甚至超过刚刚STED本身，它也有比较差的z轴的分辨率。而在STED获取多个堆栈，必须小心，以避免样品的漂白，并在必要时可以减少为了这样做行平均或激光功率强度。再次，应当注意的是，如果成像其它荧光团是不适合STED，在第一顺序扫描应用的耗尽束将防止从这些频道发射。因此，一个混合的受激发射损耗/共聚焦的方法（在在发射波长大于592纳米的渠道使用共焦扫描时），将不幸不适合3D。 概括地说，我们已经选择STED作为一种方法，由于其相对容易的应用程序的的复制和改善超过标准的共聚焦成像分辨率。成像免疫突触，它已被证明是有效的和有价值的技术，让我们看到了超过200纳米的分辨率在F-肌动蛋白的架构不可能的详细信息。虽然许多这些细节看似细微，他们可以对NK细胞功能产生深远的影响。因此，我们采用最新的纳米级成像技术，推导是维持人体健康的重要信息。

免疫突触的信号转导蛋白和细胞骨架元素的复杂环境。溶细胞突触最初被描述为具有"公牛眼"状结构与围绕中心分泌域1-4肌动蛋白和黏附分子的环。但是，我们现在知道，它是由活跃的信令微观领域需要连续动态细胞骨架重组，功能5-11。很多我们现在掌握的信息对突触是来自于显微镜和免疫学家一直尖端成像技术的早期采用者。 一个这样的新技术是超高分辨率显微镜。常规的光学显微镜是由光的衍射屏障，它设置分辨率的下限为所有荧光显微镜，共聚焦包括，在大约200纳米空间的限制。近年来，一些技术已经下发展d表示允许分辨率小于衍射障碍。这些措施包括刺激发射损耗显微镜（STED），结构照明显微镜（SIM卡），随机解决显微镜（STORM），和光活化的光学显微镜（PALM）。在详细介绍这些技术已经在别处审查12-15，但现简介如下。 Subdiffraction分辨率有限，在每个系统以独特的方式产生。超分辨率技术的选择，因此，应通过感兴趣的实验和实验系统所决定的。 STED超分辨率是使用高强度的托洛伊德尔耗尽束周围的每个感兴趣的荧光团，其选择性地"沉默"荧光激发后，产生subdiffraction有限荧光显微镜16-18实现。受激发射损耗的一个优点是，图像采集速度快，并且需要相对少的后处理。而染料的选择是由规格决定耗尽光束，这在商业上可用的系统位于592纳米的TRAL位置，一些市售的染料可用，使两个荧光团的组合成为可能。此外，常用的荧光报告如GFP可以成像，使得活细胞实验未能19,20。 我们先前使用受激发射损耗，以确定和量化由NK细胞被用于脱粒21,22使得F-肌动蛋白的低密度区域。我们建议STED是用于成像的免疫突触，由于其相对柔性的荧光基团和在xy轴的分辨率优于改进的可用的良好选择。此外，用于这些实验的市售STED系统上，使用一个高速（12000赫兹）共振扫描仪允许快速采集与对样品的损伤最小的图像。在染料的选择有限的灵活性被认为是受激发射损耗12的缺点，然而双色受激发射损耗是相对简单的几个市售荧光。受激发射损耗与激光扫描共聚焦显微镜的集成还允许在受激发射损耗与附加组合共聚焦成像，因此而受激发射损耗限制为两个通道，额外的结构可以在成像的共焦约200纳米（E.锤，未发表意见的决议）。尽管我们描述了使用受激发射损耗的用于成像的免疫细胞，该技术被应用到多种细胞类型，包括神经细胞，以及用于可视化的各种细胞结构23-26。 SIM卡采用了不同的方法来产生subdiffraction有限的图像。通过可视化的已知周期激励模式，信息然后可以对未知结构如下数学变换27正在研究中获得的。这产生以〜100nm的横向28,29增加分辨率。 SIM的优点是，它我s的所有标准的共聚焦染料和探针兼容的，但缺点是，它是慢得多的获取图像和这些需要长时间的后加工12。这也限制了它的使用活细胞成像。 最后，超分辨率图像可以由随机的光开关的荧光基团的产生。这种方法是利用在光活化定位显微镜（PALM）和随机光学重建显微镜（STORM）。通过扫描多个摄像头帧和本地化随机激活的分子，是"开"和"关"随着时间的推移，从累计帧数30-32产生20-30 nm分辨率的图像。权衡就本决议案而言是获取图像所需的时间。 在这里，我们表明，在细节，协议准备和成像双色样本中受激发射损耗。在这个系统中，激励是用脉冲，可调谐的，白色的光的激光。由于自然脉冲激发光束，检测的时间门控成为可能，并进一步提高了分辨率。此外，该系统配备了钆混合（HYD）检测器，它比常规的光电倍增管更敏感，从而允许较低的激光功率的要求。耗尽光束为STED被连续地施加和被调谐到592纳米，这将决定提供两种色STED染料的选择。常用的染料组合，一般包括一种可激发由488纳米（如的Alexa Fluor 488，俄勒冈绿，DyLights绿色，或做Chromeo 488）和一个兴奋由458纳米（如太平洋橙色或地平线V500）。因此，尽管检测的两种染料的将在一个类似的范围（又都是由耗尽激光访问），激励会发生不同波长。用可调白光激光器和可调探测器，同时消除了光谱重叠最大化的信号是由相当容易。因此，我们曾与COMBINAT良好的成功市售的染料离子，如太平洋橙色和Alexa Fluor 488（此处使用）。我们的协议是朝着量身打造，并介绍人类NK细胞的评价作为代表我们实验室的历史焦点。我们具体地，利用本例中的NK92细胞系，因为这是1，我们已经在我们的实验工作21,33经常应用。

<table><tbody><tr><td>#1.5 cover slips</td><td>VWR</td><td>48393-172</td><td></td></tr><tr><td>BD Cytofix/Cytoperm</td><td>BD Biosciences</td><td>554722</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>Cotton tipped applicator</td><td>Fisher Scientific</td><td>S450941</td><td></td></tr><tr><td>Falcon centrifuge tubes (50 ml)</td><td>VWR</td><td>352070</td><td></td></tr><tr><td>Fetal calf serum (FCS) (500 ml)</td><td>Atlantic Biologicals</td><td>S11050</td><td></td></tr><tr><td>Goat anti-rabbit Pacific Orange</td><td>Life Technologies</td><td>P31584</td><td></td></tr><tr><td>Laboratory tissue wipers</td><td>VWR</td><td>82003-820</td><td></td></tr><tr><td>Nail polish</td><td>VWR</td><td>100491-940</td><td></td></tr><tr><td>NK-92 cells</td><td>ATCC</td><td>CRL-2407</td><td></td></tr><tr><td>Phalloidin Alexa Fluor 488</td><td>Life Technologies</td><td>A12379</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Life Technologies</td><td>14190250</td><td></td></tr><tr><td>Prolong anti-fade reagent</td><td>Life Technologies</td><td>P7481</td><td></td></tr><tr><td>Purified anti-CD18</td><td>Biolegend</td><td>301202</td><td></td></tr><tr><td>Purified anti-NKp30</td><td>Biolegend</td><td>325202</td><td></td></tr><tr><td>Purified anti-perforin</td><td>Biolegend</td><td>308102</td><td></td></tr><tr><td>RPMI 1640 medium (500 ml)</td><td>Life Technologies</td><td>11875-093</td><td></td></tr><tr><td>Saponin from Quillaja bark</td><td>Sigma</td><td>S4521</td><td></td></tr><tr><td>Super PAP pen</td><td>Life Technologies</td><td>008899</td><td></td></tr><tr><td>Triton X-100</td><td>Electron Microscopy Sciences</td><td>22142</td><td></td></tr><tr><td>Huygens deconvolution software</td><td>SVI</td><td>Contact company</td><td></td></tr><tr><td>Leica SP8 TCS STED microscope</td><td>Leica Microsystems</td><td>contact company</td><td></td></tr></tbody></table>

visualization of the immunological synapse by dual color time-gated stimulated emission depletion (sted) nanoscopy

自然杀伤细胞形成严格的监管，微调免疫突触（IS）以裂解病毒感染或肿瘤发生细胞。动态肌动蛋白重组，是NK细胞的功能和IS的形成是至关重要的。 F-肌动蛋白在突触成像历来利用激光共聚焦显微镜，然而光的衍射极限的限制荧光显微镜的分辨率，包括共焦，约200纳米。在成像技术的最新进展已经启用的subdiffraction有限的超分辨率成像的发展。为了形象化的F-肌动蛋白架构的就是我们坚持的NK细胞在玻璃上激活受体概括NK细胞的细胞毒性突触。然后，我们的兴趣采用双色受激发射损耗显微镜（STED）图像的蛋白质。这将导致在&lt;80 nm的分辨率在突触。在这里，我们描述的样品制备和收购采用双山坳图像的步骤或受激发射损耗纳米显微可视化的F-肌动蛋白在NK IS。我们也使用莱卡SP8软件和时间选通STED说明样品采集的优化。最后，我们利用惠更斯软件对图像的后处理反褶积。

显然，超分辨率成像的主要目标将是一个改进常规共焦显微镜。不过，也有可能导致次优解决一些常见的陷阱。这些要求在每个实验中被单独优化。在我们的代表性的实验，我们在成像的F-肌动蛋白网络中的NK细胞通过抗体结合到玻璃激活。是常见原因（和更正）缺乏受激发射损耗过共聚焦提高分辨率如下: <ol><li> 欠采样（图1a），这可能导致颗粒性和像素信息的损失，如图F-肌动蛋白丝的分辨率差。增加行或帧平均经常可以纠正这一点。 </li><li> 漂白和/或过取样（图1b），这可能是由于长的像素停留时间，因为过度的行平均的结果所造成的。或者，它可能是之前采集过扫描的图像，包括过度使用而引起的耗尽激光。这通常会导致朦胧或模糊的图像。这可以通过扫描感兴趣的字段仅最低限度地获取之前，或者，如果可能的话，增加激光的扫描速度进行校正。如果问题仍然存在，耗尽激光功率可以降低。 </li></ol>通过实现的像素停留时间的正确平衡，激发激光功率和损耗激光功率，具有更高的分辨率和足够信息的图像可以产生（ 图1c）。分辨率可以通过使用去卷积（ 图1d）的进一步改进。当收购进行了优化，将反褶积提高分辨率定性和定量和亚100纳米的分辨率应常规实现的。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51100/51100fig1highres.jpg" src="/files/ftp_upload/51100/51100fig1.jpg" /> 图1。 优化采集和STED成像的常见缺陷。NK92细胞活化的抗CD18和-NKp30的镀膜玻璃20分钟，然后固定，透化和染色的F-肌动蛋白的鬼笔环肽用的Alexa Fluor 488。的一个）的一个例子图像信息的损失，由于欠采样。b）决议的损失是由于漂白/过采样C）条件下的优化四）优化条件导致更大的改善与反褶积分辨率的例子。比例尺= 5微米。

Watch this Scientific Journal Video about Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy at JoVE.com

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

在这里，我们示出了协议，用于成像由两色STED纳米显微NK细胞扼要重述在玻璃上的细胞毒免疫突触。使用这种方法，我们获得的突触蛋白和细胞骨架的亚100纳米分辨率。

免疫突触由双色时间选通受激发射损耗（STED）纳米显微可视化

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

visualization-immunological-synapse-dual-color-time-gated-stimulated

Research

JoVE Journal

Immunology and Infection

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

78.0K 次观看.  Texas Children's Hospital and Baylor College of Medicine. 在这里，我们示出了协议，用于成像由两色STED纳米显微NK细胞扼要重述在玻璃上的细胞毒免疫突触。使用这种方法，我们获得的突触蛋白和细胞骨架的亚100纳米分辨率。 

视频: Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

科研

免疫与感染

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

内吞体动力学在生活神经末梢与四维荧光成像可视化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

神经科学

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

成像免疫突触动力学内皮平面细胞模型

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

利用受刺激的排放损耗 (STED) 显微镜可视化肌动蛋白和微管骨架在 B 细胞免疫突触中的应用

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

成像人类免疫学突触

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

超分辨率成像研究原发神经元中蛋白质和突触标记的共同定位

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. ...

Immunological Synapse Formation Between Helper T Cells and Antigen-Presenting Cells

Source: Bello-Gamboa, A. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/60312">Imaging the Human Immunological Synapse.</a>&#160;J. Vis. Exp.&#160;(2019) This video demonstrates the in vitro study of immunological synapse formation between an antigen-presenting cell and helper T lymphocytes using a fluorescence microscope. T cell receptors of helper T lymphocytes interact with an antigen-MHC-II complex in antigen-presenting cells and form immunological synapses, followed by secretory vesicle movements toward the synapse, which is visualized by fluorescent microscopy.

辅助性 T 细胞和抗原呈递细胞之间的免疫突触形成

Source: Bello-Gamboa, A. et al., Imaging the Human Immunological Synapse.&#160;J. Vis. Exp.&#160;(2019) This video demonstrates the in vitro study of ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Assays and Functional Profiling

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Source: Zheng, P., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52502">Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer.</a> J. Vis. Exp. (2015).
This video demonstrates a technique for visualizing the formation of the immunological synapse in natural killer (NK) cells on a supported lipid bilayer (SLB). The SLB is first labeled with a fluorophore-tagged NK cell activating receptor ligand, followed by incubation with NK cells. Subsequently, the cells are stained for polymerized actin and perforin-positive lytic granules. This approach enables the visualization of synapse formation using stimulated emission depletion (STED) microscopy.