由美国国立卫生研究院授予U01 GM06230 qFSM发展。

第1部分：获取荧光标记的肌动蛋白为密克罗尼西亚所需材料：纯化的肌动蛋白，荧光（Alexa的，X -罗丹明建议）。 G缓冲区，超速离心机<ol><li>取得良好的密克罗尼西亚电影的一个重要组成部分，需要肌动蛋白与您所选择的荧光团适当的标记（或其他细胞骨架蛋白）。 我们建议使用Alexa的荧光团（488，568波长）和X -罗丹明琥珀酰亚胺酯目标暴露在表面的肌动蛋白赖氨酸残基的衍生物。 </li><li>当荧光团和适当波长的选择，确保您的显微镜设置相应的过滤器（激发和发射）和照明灯（汞弧灯和/或激光），以满足您的荧光标记。我们建议，选择橙红色光波的波长（560-630纳米），以尽量减少自体荧光和光损伤的影响，并确保其兼容性与GFP -共轭。 </li><li>纯肌动蛋白可从老鼠或鸡的肌肉组织中提取，但是，需要一个漫长的肌肉丙酮粉提取的过程。肌肉丙酮粉可购自Invitrogen。印制标签的程序是在本议定书中没有讨论，但可以在这里找到[1] </li><li>或者，您可以购买包括骨架公司和Invitrogen在内的几家厂商标记的肌动蛋白。 Invitrogen公司也可以通过自定义标签服务。纯净，非标记的肌动蛋白（肌肉和非肌肉肌动蛋白），就可以买到从骨架公司</li><li>无论您决定购买或编写您自己的标记的肌动蛋白，你想，以确保标签的比例是大约0.3％至0.7％的肌动蛋白单体染料。标签比率过高可能影响肌动蛋白丝的营业额动力学（和相关蛋白的结合），而标签的比例过低将导​​致穷人寻找斑点（低信噪比），并可能导致显微注射的问题（见下文）。这是一个得到良好的散斑图像的关键因素。 </li><li>长期存储之前，标记的肌动蛋白溶液应澄清纺纱75000 - 80000 20分钟，在4摄氏度的XG。标记的肌动蛋白应存放在G -缓冲液，并保持-80摄氏度。强烈建议准备2 UL等份，以减少反复冻融导致不溶性聚合的不利影响。避免从标记的肌动蛋白溶液暴露在直射光线下（将漂白的荧光基团）。我们建议长期贮存使用不透明的离心管。 </li></ol>第2部分：标记的肌动蛋白细胞和显微注射的制备所需材料：显微注射针，microsyringe，microloaders，预热细胞媒体，注入缓冲，冰桶，显微镜（相环，相衬的目标40X，transjector，针拔取器，显微） <ol><li>许多类型的细胞适合密克罗尼西亚，但得到良好的散斑图像的显微注射过程中的成功直接依赖。当选择密克罗尼西亚细胞，细胞要大（直径大于50微米），应分散镀时，很自然地附着在基板（或塑料组织培养皿）。它还有助于细胞的细胞质中占有面积大于它的原子核。上述标准，确保适合以显微注射的细胞。成功的候选人细胞系，包括（但不仅限于）PtK1，蝾螈肺细胞[2,3] [4]，和海兔的神经元[3]。 </li><li>应镀平方米的盖玻片（22 × 22毫米; 1-1/2）已酸洗细胞。可应用于标准基材涂布盖玻片，并不会影响显微注射。 </li><li>显微注射针的正确选择是依赖于尖端张开的大小。通常情况下，孔尺寸大小从0.5微米到3微米一样大。不过，我们建议，肌动蛋白的注射需要0.5到1微米之间的最佳开口尺寸。如果遇到堵塞问题的一致性，可容纳较大的孔尺寸为单体交付以及有害的聚合的微丝。显微注射针，可通过购买几家厂商，包括Eppendorf公司的系统，使femtotip显微注射针头。或者，如果一个商业针拔取器是可用的，玻璃显微注射管可以从沙特购买，并拉来规范。对于初学者，我们建议手头上有10-20拉针才开始注射过程。 </li><li>注射理想的“浓度”的肌动蛋白（肌动蛋白终浓度载入针）的基础上的染料40％的标签肌动蛋白单体的比例为0​​.5至1.0微克/ UL。肌动蛋白的标签比下可以补偿在较高浓度（1-3微克/ UL）注射。然而，你的针堵塞的频率会增加。用针开口更大，这可以部分缓解。可稀释，加入冰冷的注射缓冲库存的肌动蛋白的浓度。冰冷ddH2O也可用于快速注射稀释。在这一点上，所有的工作方案应保持在冰上。 </li><li>负载0.5至1.0 UL您的肌动蛋白使用microsyringe（窄轨 - 汉密尔顿）的解决方案，或者使用microloader枪头。 </li><li>仔细免除的肌动蛋白的解决方案，轻轻释放的解决方案。应避免泡沫，但占用多余的液体，周围的泡沫，可以很容易地删除。 </li><li>确保肌动蛋白的解决方案是在针尖完全休息。多余的溶液可以破坏任何地方沿毛细管针注射流。 </li><li>现在，认真重视针，避免与针尖接触，喷油器的持有人。持有人的位置，使针头一个35-50度角显微镜阶段的基本。下角是首选。 </li><li>下针尖，直到它几乎打破了媒体沐浴你的细胞的表面。 </li><li>现在的位置针尖（不降低），以便它直接由上述中心的目标。如果针尖与客观相一致，你应该通过目镜看到一个耀眼光环。这是针尖结束。缓慢移动的针一侧到另一侧（使用的操盘）可能有助于更好地找到的光环。 </li><li>调整显微镜的Z -焦点，直到你的细胞是明确的说法。现在，慢慢的提示，直到光环逐渐收缩，直到最后到针尖的汇合 - 你会同时需要控制的重点（Z -位置）。寻找另外一个特点是轴针，这将投线性阴影。降低针头，将逐步界定针轴。当完成正确的针和你的细胞，都应该在明确的说法。相环的小幅调整可能需要提高对比度。 </li><li>航行时，确保远高于你的细胞，以防止打破针尖针尖。 </li><li>当选择注入的细胞，细胞与自由边应面临的针尖。这将确保细胞的斜率下降（从厚点，细胞核所在的领先优势，最薄处），将满足针尖，而不是平行。 </li><li>应设置在0.3至0.8磅针压力。不断施加压力，将导致在源源不断的肌动蛋白溶液。 </li><li>决定一个细胞注入后，定位针，使针尖旁核（核周区域，最厚的部分细胞的细胞核是不是直接在下面）。 </li><li>缓慢降低，并调整针的位置，直到提示按下膜，现在逐步降低，直到针尖刺穿膜。如果针完全划破膜，细胞会显得更亮，只要你看到这种差别，提高针，直到它不再是接触的细胞。实际穿刺和注射过程将需要0.5到2秒。 </li><li>做得正确时，会出现细胞形态保持不变，而过多注射会导致细胞边缘的快速回缩，并在某些情况下，你会真正看到的细胞爆炸。 </li><li>如果你没有看到在细胞中的变化时，显微注射（不变亮），针尖可能被关闭。您可以按&#39;干净&#39;的按钮，如果您transjector的测试。如果开放，这将显着增加的压力恒定流，可以通过目镜看到的 - 它会出现波纹和附近杂物可以看出分散。 </li><li>如果针尖是封闭的，你可以选择插入一个新的显微注射针或试图打破轻轻地降低，直到对玻璃盖玻片针印刷机的尖端针针尖，从而造成尖端的打破。破损面积不应该比针点（针，开始收敛到一个点的部分）的面积的1 / 5日。 </li><li>继续注射，花费不超过20 - 45分钟，每个显微注射会议（将取决于细胞类型）。作为一般规则，建议缩短会期，以尽量减少对细胞的压力。 </li><li>的显微注射完成后，改由在培养皿的细胞介质，加入新鲜预热媒体。然后使细胞恢复为30分钟前成像的孵化器。这也使肌动蛋白融入现有的骨架网络。 </li></ol>第3部分：组装影像厅所需材料：玻璃罩，双面胶带，Q -技巧，镊子，valap，金擦拭，oxyrase，影像媒体，刀片，P200 PIpette，纯乙醇<ol><li>由prewarming成像介质和解冻15 Oxyrase UL等分的开始。确保免受光线的感光oxyrase。 oxyrase此外，将大大降低漂白的影响。 </li><li> Prewarm valap，通过设置热板温度低。不要过热/燃烧的valap，它会失去其作为密封剂的疗效。 </li><li>浇上纯乙醇清洁和清除小碎片，一个kimwipe擦拭玻璃罩。 </li><li>堆栈彼此顶部的双面胶带两个同等大小的长度（2.5厘米长），作为一个平台，使用一个干净平坦的表面。按任何气泡，以创建一个完全平坦的平面。 </li><li>使用刀片，切两条直线条，沿长轴地带的措施，使每个宽0.5厘米。 </li><li>使用镊子，对您的玻璃罩的长边各放置地带，使磁带上的两个带创建一个在您的玻璃罩中心的差距。在磁带上轻轻按下一个很好的密封玻璃。磁带的带将作为玻璃罩和盖玻片之间的间隔。 </li><li>确保已完全液化，然后再开始下一步的valap。 </li><li>找回您的预热成像介质，加入15 oxyrase UL 500 uL的每一种媒体。 </li><li>折叠成三角形kimwipes创建3个硬边。这将用来创建一个磁带表面干燥，要坚持。 </li><li>从孵化器中检索你的细胞。使用镊子，抢盖玻片的一个角落。快速放置盖玻片上kimwipe（细胞的一面朝上，不接触的组织），并使用折叠kimwipe擦拭盖玻片的两个对立的双方，创造一个半干燥的表面（表面上的细胞休息）。 </li><li>使用镊子，抢一个角落盖玻片，放置在您的玻璃罩，使磁带两个条是一致的，干燥的表面。请注意，现在你有两个封​​闭两侧和两个开放式开衩。慢慢地吸液管200 UL成像媒体oxyrase解决方案接近一开口。更重要的是，无气泡，应引入。该解决方案应该被吸成新组装的毛细力的商会，从而充分浸泡在溶液中的细胞。 </li><li>使用棉条民建联四个角你融化valap迅速加盖盖玻片和稳定的盖玻片。你会发现，valap干燥瞬间（在室温秒内）。 </li><li>现在开放两岸（非磁带边）开始，打下一个valap使用棉条和模仿刷牙中风薄带钢。重复封闭的两侧。慢慢建立valap大衣，从而强化了密封重复涂层步骤。应限于该valap的边缘，使盖玻片中心明确。 </li><li>轻轻擦拭，使用棉条与ddH2O以除去残留的媒体，不被铭记粉碎细胞的盖玻片的中心部分。重复用纯酒精清洗步骤。 </li><li>现在，您将有一个完全封闭的散斑成像成像室进行了优化。拥有一个完全密封的外壳，防止快速漂白的荧光团。 </li></ol>第4部分：成像细胞所需材料：广角倒置显微镜，mecury灯，适当的过滤器，冷却6.7微米像素，高数值孔径复消色差油浸计划目标（60X，100X，相位或DIC）的CCD摄像头。振动台。成像软件。 <ol><li> Prewarm显微镜阶段到37度。广场新落成的成像室，并等待10-15分钟的温度稳定之前成像。 </li><li>开始集中在明场细胞，并切换正确荧光设置，然后找到你注射的细胞。尽量减少荧光曝光时间，减少漂白。 </li><li>寻找注入的细胞，具有健康弯曲前缘。在注射细胞（爆炸细胞）将缩回，锯齿状边缘的特点，类似许多丝状伪足 - 避免这些细胞的成像。 </li><li>采集设置，将取决于细胞类型，标记的肌动蛋白的质量，和显微镜组件。然而，曝光设置不应大于2秒，5至10秒和帧之间的时间间隔应设置。典型的时间序列长度（每成像单元）可以相差10至30分钟之间的任何地方，并且是由漂白效果有限。可以打开相机的增益设置，提高信噪比。 </li><li>理想的斑点特点，应该有每个斑点间隔1〜2未来邻近斑点斑点直径。这将保证最佳的肌动蛋白结构的空间和时间采样。上皮细胞，斑点肌动蛋白网络将出现同质化，均匀分开的斑点。细胞株，功能密集的应力纤维或lamellipodia将区别，但将有一条虚线/斑点的外观。应调整曝光设置，以获得最佳的斑点图像。 </li><li>在显微注射细胞将有密密麻麻的斑点，不会出现离散的。应力纤维和lamellipodia将失去它的点缀外观。 </li><li>在显微注射细胞会显得非常暗淡，通过目镜，含有相距甚远（&gt; 10斑点除了），并出现随机分布的斑点。应力纤维，并在这些细胞中的lamellipodia将没有什么区别。 </li><li>获得“好”的关键斑点电影是维护的重点。涉及散斑测量流量和营业额的分析后，这一点尤为重要。因此，定量分析的质量将取决于重点锁定延时时间。如果成像平面是比较薄的，这可以是特别具有挑战性的。显微镜系统的稳定，尤其是舞台住房和成像室，将取决于其他因素。如果图像的时间间隔的长度足以容纳此功能，可用于基于对比度的重点。其他选项包括购买一个基于近红外对焦系统，提供可靠的实时集中调整。从尼康，奥林巴斯和ASI，你可以找到这些产品。 </li></ol>第5部分：qFSM分析本节中详细讨论，但分析软件上的信息可以在这里找到[5]。软件下载请求，可对我们的网站（lccb.scripps.edu）。 内容包括： <ul><li>噪音校准</li><li>散斑检测</li><li>跟踪使用一个基于图像相关的粗运动跟踪混合方法去斑领域和单粒子追踪个别斑点的详细议案。 </li><li>从强度波动过程中聚合物的组装和拆卸率计算斑点的外观和失踪事件。 </li></ul>

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	<li>Waterman-Storer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+speckle+microscopy+(FSM)+of+microtubules+and+actin+in+living+cells.">Fluorescent speckle microscopy (FSM) of microtubules and actin in living cells.</a> Curr Protoc Cell Biol. Chapter 4, Unit 4.10-Unit 4.10 (2002).</li><li>Ponti, A., Machacek, M., Gupton, S. L., Waterman-Storer, C. M., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+actin+networks+drive+the+protrusion+of+migrating+cells.">Two distinct actin networks drive the protrusion of migrating cells.</a> Science. 305, 1782-1786 (2004).</li><li>Zhang, X. F., Schaefer, A. W., Burnette, D. T., Schoonderwoert, V. T., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-dependent+contractile+responses+in+the+neuronal+growth+cone+are+independent+of+classical+peripheral+retrograde+actin+flow.">Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow.</a> Neuron. 40, 931-944 (2003).</li><li>Salmon, W. C., Adams, M. C., Waterman-Storer, 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=Dual-wavelength+fluorescent+speckle+microscopy+reveals+coupling+of+microtubule+and+actin+movements+in+migrating+cells.">Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells.</a> J Cell Biol. 158, 31-37 (2002).</li><li>Danuser, G., Waterman-Storer, 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=Quantitative+fluorescent+speckle+microscopy+of+cytoskeleton+dynamics.">Quantitative fluorescent speckle microscopy of cytoskeleton dynamics.</a> Annu Rev Biophys Biomol Struct. 35, 361-387 (2006).</li></ol>

The authors have nothing to disclose.

几个关键因素是至关重要的，成功获得散斑图像，但是良好的斑点，开始和结束与荧光标记的肌动蛋白的质量。一个标签的比例高，肌动蛋白单体，约0.4至0.7的染料将确保斑点会显得明亮和离散，标记的蛋白质本身，而应可溶性聚集。同样重要的是显微注射过程。为了保证正常细胞动态平衡和生存，细胞需要被注入一个低流量的压力，引入低浓度的标记蛋白。这需要一定的技术专业知识/经验的显...

注射缓冲器（IB）， 50毫米钾谷氨酸 0.5 M的氯化镁（ 附录2A） 在-20 ° C储存长达2年 &nbsp; G缓冲区 2毫米的Tris⋅氯，pH值8.0（ 附录2A） 0.2毫米氯化钙（ 附录2A） 只是在使用前添加： 0.2毫米ATP（见100毫米配方） 0.5毫米2 - 巯基乙醇店1天4 ° C

live cell imaging of f-actin dynamics via fluorescent speckle microscopy (fsm)

在这个协议中，我们描述了使用荧光斑点显微镜（FSM）来捕获PtK1细胞的肌动蛋白动力学的高清晰度图像。密克罗尼西亚的一个独特的优势是它能够捕捉到活细胞内的流动和周转动力学的F -肌动蛋白网络（组装/拆卸）。这种技术是有用的，特别是在衍生F -肌动蛋白动力学的定量测量与计算机视觉软件（qFSM）配对。我们描述了选择，显微注射法和荧光的肌动蛋白探针在活细胞中的可视化。更重要的是，类似的程序，适用于可视化等macomolecular集会。密克罗尼西亚已经证明微管，中间丝和粘附复合物。

Watch this Scientific Journal Video about Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM at JoVE.com

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

的选择，显微注射，并成像荧光标记的F -肌动蛋白，通过荧光斑点显微镜（密克罗尼西亚）。

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

live-cell-imaging-f-actin-dynamics-via-fluorescent-speckle-microscopy

Research

JoVE Journal

Biology

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

15.8K 次观看.  Scripps Institute. 的选择，显微注射，并成像荧光标记的F -肌动蛋白，通过荧光斑点显微镜（密克罗尼西亚）。

视频: Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

三维超F-微丝高分辨率显微镜通过干涉光敏定位显微镜（iPALM）

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

活细胞成像，以评估在斜接主轴扰动后元相定时和细胞命运的动态

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

通过总内部反射荧光显微镜与数量和亮度分析相结合，活细胞中细胞表面受体的显微化动力学

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...