Name: cortical actin flow in t cells quantified by spatio-temporal image correlation spectroscopy of structured illumination microscopy data
Uploaded: 2015-12-17T15:00:00.000+00:00
Duration: 9 min 9 s
Description: Watch this Scientific Journal Video about Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data at JoVE.com

D.M.O. acknowledges European Research Council grant No. 337187 and the Marie-Curie Career Integration Grant No. 334303. P.W.W. acknowledges the Natural Sciences and Engineering Research Council of Canada's Discovery Grant program.

注:阶段成像的前一天1和2来进行;确保前或上成像的天平衡到所使用的所有媒体和补充剂。平衡是通过媒体和补充变暖在培养箱设定为5％的CO 2来实现至37℃。所有细胞培养物的工作和盖玻片电镀步骤在无菌条件下在层流罩。 1.细胞转染<ol><li>转染T细胞进行成像24小时的实验用质粒编码荧光融合构建的F-肌动蛋白GFP标记和成像之前。注:细胞应分裂24小时，在转染前（48小时之前，成像），以确保细胞在最佳的健康，并在其对数生长期。 <ol><li>放置5毫升的RPMI补充剂（RPMI 1640 + 10％FBS和1％青霉素 - 链霉素（可选））在6孔板的单个孔进入孵化器中平衡。 </li><li>沉淀2×10 7个T细胞崇拜使用离心机管中于327×g离心5分钟，置的，在37℃5％的CO 2在RPMI补充和洗涤在预热的电介质的等体积。注:电穿孔介质是含有缓冲区和补充（见消耗品）降低血清培养基。 </li><li>后重新制粒在327×g离心5分钟，重悬细胞于500μl电介质，慢慢添加至试管中，添加5微克质粒的PCR级的dh 2 O用于标记F-肌动蛋白。 </li><li>轻轻拍打引擎盖上的工作区的反应杯，以确保去除任何气泡在媒体上，这可能会导致电力的电弧和向媒体内均匀地分布在细胞。 </li><li>打开电穿孔装置和设置电压300 V和电容290Ω。使用指数衰减为20毫秒电穿孔样品。注:可能需要不同的细胞和不同的质粒构建优化。 </li><li>一旦转染协议完成后，存储单元冰上继续之前1分钟。 </li><li>小心从试管转移细胞到从步骤1.1.1含有平衡的RPMI补充的5毫升的6孔板中。 </li><li>孵育细胞O / N在37℃+ 5％CO 2。 </li></ol></li></ol> 2.外套盖玻片和预暖成像补充<ol><li>外套盖玻片与抗体诱导T细胞刺激。 <ol><li>涂层的8孔盖玻片用1微克/毫升αCD3和αCD28在200μlPBS中的各腔室。 </li><li>储存在4°CO / N。可替代地，涂层盖玻片2小时之前，成像和保持在培养箱中在37℃至通过最大限度地减少温度变化到盖玻片在成像过程中减少漂移。 </li></ol></li><li>创建具有0.1μm的子衍射荧光微球盖玻片测试样品。 <ol><li>珠粒涡旋原液，以避免团块。 </li><li>在th的一种新的，干净的盖玻片e型中的那些相同步骤2.1.1使用，吸管5微升荧光微球原液到表面上（根据制造商的说明）。 </li><li>分布在与枪头盖玻片微球的解决方案。 </li><li>晾干，然后申请200微升安装介质如PBS。 注意:安装介质应与用于成像的细胞样品。 </li></ol></li><li>平衡的HBSS（+ 20mM的HEPES，以下称为"补充的HBSS&#39;）在一个离心管放置在培养箱中预暖O / N在37℃+ 5％CO 2。 注:加HEPES的是，如果使用的是孵化室的二氧化碳输入跳过此补充缓冲CO 2。 </li></ol> 3.显微镜建立<ol><li>打开TIRF-SIM显微镜和地方足够的蒸馏水的在显微镜的贮存阶段顶培养箱完全覆盖加热元件的基座。允许水以电子邮件quilibrate至37℃。加热套环添加到物镜，也设定为37℃。 注:由于在设置了灵敏度，这些步骤是以前做过的晚上，所以光学组件处于稳定的温度之前，成像。 </li><li>放置TIRF 488兼容光栅挡在显微镜的光路，并固定到位。 <ol><li>切换通道模式和光纤电缆连接到激光床和显微镜的阶段，"单"的位置分别从事的TIRF-SIM模式，正确的光路。注意:跳过此步骤会导致软件窗口内的错误（图1A）。 </li></ol></li><li>打开所有的显微镜和计算机部件，打开采集控制软件。 <ol><li>打开超频板，钛垫，N-SIM卡垫和ND采集窗口（ 图1B）。在"超频面板"中选择选项"TIRF 488&#39;（ 图2B，黄色箭头）。 </li&gt; <li>在N-SIM板窗口中，选择"TIRF-SIM"和"移动" 设置 （图2C，黄色箭头）。选择设置按钮 （图1C，黄色方框），然后选择"全内反射荧光488（用于100X TIRF 488nm的）&#39;从下拉菜单中，点击"应用"和"确定"。 </li><li>帧速率50毫秒，读出模式，EM增益3 MHz的14位和EM增益倍数300为1的转换增益（ 图1C，绿框）内的"DU897设置"来设置相机设置。 注:请参阅帧速率的考虑代表性的成果。 </li><li>通过选择100×1.49 NA CFI复消色差透镜的TIRF目的，对"N-SIM垫&#39;校准为TIRF-SIM显微镜的照明转动488纳米的激光功率为10％。 注:原始图像的像素大小为60纳米，与美景的512×512像素的领域。 </li><li>清洁使用镜头纸，以去除灰尘和油物镜。 </li></ol></li&gt; <li>找到TIRF-SIM焦平面。 <ol><li>开始与在尽可能低的z平面的目标。放置透镜油对目标的下降，并从步骤2.2到显微镜阶段添加荧光微球样品。 </li><li>选择位于显微镜的身体完美对焦系统（PFS）的功能。 </li><li>通过Z平面缓慢移动的目标，直到PFS按钮停止闪烁，以确认焦平面已经达到。 </li><li>切换到控制软件和图像的微球通过使用PFS微机械手轮做最后的调整"现场"视图。 </li></ol></li><li>观察均匀照明与离焦的无荧光以上75nm的渐逝波发射。 注意:确认结构照明通过观察从荧光微球的样品波动的照明模式实现。 </li><li>测量荧光微球的样品和量化改进成像resolut离子通过观察全宽的强度分布的半最大值（FWHM）。 <ol><li>打开分析软件（v4.20.01），安装了NIS-A N-SIM分析模块，并选择"应用程序" - &gt;"显示为N-SIM窗口"。 </li><li>与照明调制对比度（IMC）和高分辨率的噪声抑制（HRNS）设置为1的TIRF-SIM数据集（图2），重建微球体样本图像生成的1024×1024个像素的重建图像以30像素尺寸纳米。 </li><li>与1个像素（ 图3A中，黄色盒）的宽度使用线轮廓工具，通过一个单一的微球体的中心画线。 </li><li>选择将FWHM 按钮 （图3B，黄色盒），并点击在高斯分布的强度最大值，随后其强度最小量。 </li><li>确认微球体的FWHM是在100nm（图3C）的范围内。注:分辨率阿赫ieved成像当生物样本将取决于信噪比从标记效率和背景荧光变化。 </li></ol></li></ol> 4.样品制备成像<ol><li>盖玻片准备。 <ol><li>从冰箱或保温箱步2.1检索αCD涂层的盖玻片，并从每个孔取出PBS含αCD3和αCD28。 </li><li>从步骤2.3加入200μl预热HBSS补充到盖玻片的每个孔中。洗净并除去，以确保最小αCD3和αCD28留在溶液中。添加另一个200微升HBSS补充盖玻片井。 </li><li>清洁盖玻片的下侧与镜头纸以除去油和颗粒。 </li></ol></li><li>在显微镜台位置盖玻片并通过使用PFS z平面找到盖玻片TIRF焦平面如在步骤3.4中找到。 </li><li>细胞的准备。 <ol><li>吸取200微升媒体CON的泰宁转染的细胞从步骤1.1.8成微离心管中。 </li><li>沉淀细胞在微离心机中以268×g离心20秒并除去细胞介质。 </li><li>悬浮细胞在200μl来自步骤2.3预先温热的HBSS补充。 </li></ol></li></ol> 5.成像细胞<ol><li>取含细胞的HBSS补充到显微镜，轻轻移液加入200μl至孔中的一个。 </li><li>而在盖玻片（在PFS模式）留在TIRF焦平面，用显微镜目镜落射荧光照明跟踪荧光细胞接近盖玻片。 </li><li>一旦细胞土地上的盖玻片，切换到"现场"模式，在N-SIM垫带来的图像上的显示器，并检查细胞处于焦点的电脑屏幕上。 </li><li>要记录时间的课程，打开ND采集窗口，选择"时间"选项卡上，单击"添加"，并设置"间隔"为无延迟，"长"至10分钟，"环路"设置为1。 注意:持续时间可以根据所关注的方法来改变;当以"无延迟"捕获的STICS软件可以提取与≈10帧图像堆栈定量结果。 </li><li>以相同的采集设置到胎圈样品，首先在ND采集窗口中选择"立即运行"记录数据。 </li></ol> 6.重建SIM图片<ol><li>成像完成后，打开分析软件（v4.20.01），安装了NIS-A N-SIM分析模块，并选择"应用程序" - &gt;"显示为N-SIM窗口"。 </li><li>与照明调制对比度（IMC）和高分辨率的噪声抑制（HRNS）设置为1的TIRF-SIM数据集（图2），重建一个（或多个使用批量工具）原始SIM文件，以产生重构的SIM图像。 </li><li>确认重构文件具有30nm的像素大小，在所指示的图像状态栏。 注意:像素大小不影响STICS分析，但它应该是在尺寸比在PSF空间分辨率的至少2倍小，以确保像素之间的空间相关性。 </li><li>导出数据为.tiff文件，准备STICS分析。 </li></ol> 7. STICS分析<ol><li>下载并解压缩STIC（C）S Matlab软件包（怀斯曼实验室;可作为补充信息）。脚本stics_vectormapping.m是单通道STICS分析的核心和第一30行的代码被用作在手册的第2.1节中定义来定义的输入参数。打开这个脚本，并定义的输入参数TIRF-SIM卡的数据分析。建议也与子文件夹添加STIC（C）S文件夹复制到本地机器Matlab的路径，该手册的附录中详细说明。 <ol><li>通过编辑代码的相应行定义的输入参数的数据进行分析</li><li>要执行全日空的TIRF-SIM数据的裂解，以产生1向量地图，在第31行设置瞬时参数到颞分区域的帧的最大数量，并在管线32到1的时移为获得时间序列的矢量地图改变这些参数。 </li><li>对于不动去除过滤器，设置参数&#39;过滤&#39;（23行），以"移动平均"，并设置为21&#39;MoveAverage"参数（第24行）。 注意:该设置已被显示能用于移动较慢的和大的荧光信号，因为它是基于减去帧的串行数据帧被分析。 </li><li>更改线路33-38来设置输出文件和输出矢量地图的标题名称，以及输出目录路径，并确定以何种格式的矢量地图的图像应保存。 </li></ol></li></ol><table><tbody><tr><td>像素尺寸（微米） </td><td> 0.03微米</td><td>第19行</td></tr><tr><td>帧速率（秒） </td><td> 1秒</td><td>第20行</td></tr><tr><td>最大延迟时间（帧） </td><td> 20帧</td><td>第21行</td></tr><tr><td>分区域的大小（像素） </td><td> 8个像素</td><td>第29行</td></tr><tr><td>分区域的间距（像素） </td><td> 1个像素</td><td>第30行</td></tr></tbody></table><ol start="2"><li>打开在Matlab编辑器窗口中的脚本run1ChannelSTICS。通过运行线这个脚本的1-23加载图像序列。 注意:此脚本可用于加载和预处理图像系列，绘出矢量地图和速度直方图和运行批处理处理多个文件。如果需要的话，通过使用这个脚本的线24-26剪切图像的感兴趣的区域。 <ol><li>运行线路run1ChannelSTICS 29-35启动STICS分析。提示时，选择一个多边形来表示的感兴趣区域（ROI）的特定区域，如果需要的话。确保将ROI不与小区边缘重叠，边界伪影出现时STICS分析这些重gions。 </li><li>运行线路38-52，显示矢量地图。要绘制速度幅度直方图，运行线路53-72。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

该技术将允许研究人员的形象和以前无法解决的量化细节细胞表达荧光。在我们的结果，我们表明，F-肌动蛋白以对称方式从向小区中心中的T细胞是细胞周流动。这些发现与来自1D线轮廓kymograph数据13之前的结果一致，并延伸分析的能力，以二维和超分辨率数据。 所提出的技术是从kymograph成像它仅限于通过单个1D线随时间绘制荧光强度波动进展。在比较当前技术允许全...

该实验的目标是图像，并为了在免疫突触（IS）的形成建立流动方向和幅度表征filamentous-（F - ）的肌动蛋白的分子流在活的T细胞，超出衍射极限。这种技术将进行使用结构照明显微镜（SIM）中的全内反射荧光（TIRF）模式，成像的Jurkat T细胞上形成激活盖玻片包被有抗CD3-和抗CD28抗体的人工突触。 T细胞和目标之间的IS形式;抗原呈递细胞（APC）在T细胞受体（TCR）活化1,2。由于这是在确定是否适应性免疫系统产生要感染的免疫应答的第一步，不正确的响应性对刺激的后果可能导致许多疾病。的T细胞的APC相互作用的重要性是有据可查的，然而，初始TCR SI后成熟的作用（S）为gnal内化是尚未得到充分的理解。 作为细胞骨架被认为有助于两个链接到TCR活化（分子在质膜的移动和信号分子依赖于肌动蛋白骨架3,4的控制），了解如何细胞骨架重排的空间过程和时间上会阐明的步骤在突触形成的分子重组。 F-肌动蛋白逆行流动被认为是托起TCR集群对免疫突触的中心，这种易位被认为是信号停止和回收至关重要;辅助T细胞活化5的良好的平衡。 而标准荧光显微镜是一种灵活的工具，继续提供对多生物学事件，包括细胞 - 细胞相互作用的洞察力，它是由该系统的准确地解决多个荧光团位于比DIFFR接近能力的限制对方的行动限制（≈200纳米）。当细胞内的许多分子事件涉及的分子密集的人口，并表现出无论是在静止状态或在特定的刺激动态重配置，常规光学显微镜不​​能提供完整的图片。 采用超分辨率成像已经使研究人员能够绕过使用多种技术的衍射极限。单分子定位显微镜（SMLM）如PALM和STORM 6,7-必须解决分子的位置最多约20纳米的精度的能力，然而，这些技术依赖于长采集时间，建立一个图像在许多帧。通常这要求样品是固定的或对结构表现出低移动性，以便单一荧光团不误解为多个点。而受激发射损耗（STED）成像允许超分辨率通过非常有选择性的共焦激发8，所需的扫描方法可以是在视全细胞字段慢。受激发射损耗还演示显著光毒性更高的分辨率增加所致在耗尽光束的功率。 结构照明显微镜（SIM 9）提供了一个替代这些方法，能够在标准的荧光显微镜的分辨率增加一倍，并且是与活细胞成像比目前SMLM技术更兼容。它采用较低的激光功率，在一个宽视场系统的视图全细胞领域;提供更高的采集速度，并与标准荧光标记兼容。 SIM的宽视场的性质也允许全内反射荧光（TIRF），用于高选择性激励的利用率，以在&lt;100纳米的轴向尺寸的穿透深度。 图像相关光谱（ICS）是荧光强度相关的波动的方法。 ICS的演变;时空IM年龄相关光谱（STICS 10）相关的时间和空间内的荧光信号。通过关联像素强度与经由相关函数围绕滞后帧中的像素，将信息获得关于流的速度和方向。为了确保静态对象不与STICS分析干扰，不动的对象滤波器实现的，它的工作原理是减去像素强度的移动平均值。 这里提出的技术被认为是图像相关光谱的被施加到超分辨率显微镜数据，量化活细胞11的分子流的第一个示范。此方法对经由STICS分析12 F-肌动蛋白流衍射限制成像改进并会适合研究者谁希望确定二维分子流在活细胞中，从在超出光的衍射极限的空间分辨率获得的数据。

<table><tbody><tr><td>&lt;strong&gt;Consumables:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Jurkat E6.1 cells</td><td>APCC</td><td>ATTC TIB-152</td><td></td></tr><tr><td>RPMI</td><td>Life Technologies</td><td>21875-091</td><td></td></tr><tr><td>FBS</td><td>Sigma</td><td>F7524-500ML</td><td></td></tr><tr><td>Penicillin-Strepromycin&amp;nbsp;</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>HBSS</td><td>&amp;nbsp;Life Technologies</td><td>14025-050</td><td></td></tr><tr><td>HEPES</td><td>Life Technologies</td><td>15630-056</td><td></td></tr><tr><td>#1.5 8-well Labtek coverslips</td><td>NUNC, Labtek</td><td>155409</td><td></td></tr><tr><td>LifeAct GFP construct</td><td>Ibidi</td><td>60101</td><td></td></tr><tr><td>OptiMem</td><td>Life Technologies</td><td>51985-042</td><td></td></tr><tr><td>anti-CD3</td><td>Cambridge Bioscience</td><td>317315</td><td></td></tr><tr><td>anti-CD28</td><td>BD Bioscience / Insight Biotechnology</td><td>555725</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>20012-019</td><td></td></tr><tr><td>Lens oil</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gene Pulsar Xcell System</td><td>BioRad</td><td>165-2660</td><td></td></tr><tr><td>Cuvette (0.4cm)</td><td>BioRad</td><td>165-2088</td><td></td></tr><tr><td>Centrifuge (5810R)</td><td>Eppendorf&amp;nbsp;</td><td>5805 000.327</td><td></td></tr><tr><td>Centrifuge (Heraeus)</td><td>Biofuge pico</td><td>75003235</td><td></td></tr><tr><td>6 well plate</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>Stage-top incubator</td><td>Tokai Hit</td><td>INUH-TIZSH</td><td></td></tr><tr><td>Nikon N-SIM microscope</td><td>Nikon</td><td>Contact company</td><td></td></tr><tr><td>Nikon Analyse software</td><td>Nikon</td><td>Contact company</td><td></td></tr><tr><td>Incubator (190D)</td><td>LEEC</td><td>Contact company</td><td></td></tr></tbody></table>

cortical actin flow in t cells quantified by spatio-temporal image correlation spectroscopy of structured illumination microscopy data

丝状肌动蛋白起着在大多数细胞过程，包括运动，并在免疫细胞，被称为免疫突触的关键细胞间相互作用的形成至关重要的作用。 F-肌动蛋白还可以推测，以在细胞中包括子膜囊泡动力学和蛋白质聚类的膜调节分子分布发挥作用。虽然标准的光学显微镜技术，允许进行广义和衍射极限的观察，许多细胞和分子事件，包括集群和分子流发生在人群的长度尺度远远低于标准光学显微镜的分辨能力。通过全内反射荧光结合超分辨率成像方法结构照明显微镜，记录F-肌动蛋白中的T细胞的免疫突触二维分子流。时空图像相关光谱（STICS）随后被施加，其产生量化resulTS在速度直方图和代表流动方向性和大小的矢量地图的形式。这个协议描述的超分辨率成像和STICS技术相结合，以产生在细节的子衍射水平流矢量。这个技术被用于确认一​​个肌动蛋白的流动是对称逆行和向心力整个T细胞的周后突触的形成。

显微镜设置在成功地实现所有步骤研究者将具有F-肌动蛋白的流动是T细胞中突触的完成结构照明（SIM）成像（视频1），并量化这个分子流通过时空图像相关光谱（图4 11）。记录图像之前，确保样本照明是在9个原始图像均匀且在莫尔条纹在样品中观察到的形式，结构照明（图5），这些都是指?...

Watch this Scientific Journal Video about Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data at JoVE.com

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

cortical-actin-flow-t-cells-quantified-spatio-temporal-image

Research

JoVE Journal

Immunology and Infection

9.6K 次观看.  King's College London. To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy. 

视频: Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

科研

生物工程

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 ...

免疫与感染

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

生物化学

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 ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

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 ...

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

Photoactivatable 激动剂对 T 细胞活化的时空控制

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

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 ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

生物学

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate multiple functions, including adhesion, proliferation, migration, and differentiation. Mechanical forces can be transmitted from the cell via the adhesion receptor integrin to ligands in the ECM. The amount and spatial organization of these cell-generated forces can be modulated by growth factor receptors, including epidermal growth factor receptor (EGFR). The tools currently available to quantify crosstalk-mediated changes in cell mechanics and relate them to&#160;focal adhesions, cellular morphology, and signaling are limited. DNA-based molecular force sensors known as tension gauge tethers (TGTs) have been employed to quantify these changes. TGT probes are unique in their ability to both modulate the underlying force threshold and report piconewton scale receptor forces across the entire adherent cell surface at diffraction-limited spatial resolution. The TGT probes used here rely on the irreversible dissociation of a DNA duplex by receptor-ligand forces that generate a fluorescent signal. This allows quantification of the cumulative integrin tension (force history) of the cell. This article describes a protocol employing TGTs to study the impact of EGFR on integrin mechanics and adhesion formation. The assembly of&#160;the TGT mechanical sensing platform is systematically detailed and the procedure to image forces, focal adhesions, and cell spreading is outlined. Overall, the ability to modulate the underlying force threshold of the probe, the adhesion ligand, and the type and concentration of growth factor employed for stimulation make this a robust platform for studying the interplay of diverse membrane receptors in regulating integrin-mediated forces.

TGT surface is an innovative platform to study growth factor-integrin crosstalk. The flexible probe design, specificity of the adhesion ligand, and precise modulation of stimulation conditions allow robust quantitative assessments of EGFR-integrin interplay. The results highlight EGFR as a 'mechano-organizer' tuning integrin mechanics, influencing focal adhesion assembly and cell spreading.

用于量化生长因子介导的整合素力学和附着力的张力计系绳探头

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate ...

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

摘要

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此视频中的章节

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

T细胞实时成像实时信号复合物的形成

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

成像免疫突触动力学内皮平面细胞模型

通过全内反射荧光（TIRF）显微镜在体外可视化肌动蛋白和微管耦合动力学

影像性流式细胞仪对人体免疫突触的定性和定量分析

Photoactivatable 激动剂对 T 细胞活化的时空控制

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

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

用于量化生长因子介导的整合素力学和附着力的张力计系绳探头

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

摘要

探索更多视频

此视频中的章节

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

T细胞实时成像实时信号复合物的形成

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

成像免疫突触动力学内皮平面细胞模型

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

影像性流式细胞仪对人体免疫突触的定性和定量分析

Photoactivatable 激动剂对 T 细胞活化的时空控制

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

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

用于量化生长因子介导的整合素力学和附着力的张力计系绳探头

通过全内反射荧光（TIRF）显微镜在体外可视化肌动蛋白和微管耦合动力学