笔者想承认UNIVERSITA德利阿布鲁Studi住宅米兰向埃利安娜迪卡伊拉诺（博士后奖学金）和斯特凡莫雷蒂（博士奖学金）的支持。这项工作是由大学研究计划PUR到CP支持我们要感谢教授杰里米·M.亨利，生物化学学院，布里斯托，英国的大学，为pHluorin建设和Dotti弗朗西斯博士在数据分析的援助，和Silvia Marsicano技术援助。

1.细胞培养和转染<ol><li> SH-SY5Y细胞培养 注意：使用人神经母细胞瘤SH-SY5Y（ATCC＃CRL-2266）15实验已经完成。 SH-SY5Y细胞成长为浮动集群和贴壁细胞的混合物。按照报告的协议（细胞密度，分光比等 ），以有生长牢固地附着在玻璃盖，其用于TIRFM关键细胞说明书。 <ol><li>在开始之前，层流生物安全柜下，使无菌磷酸盐缓冲生理盐水（PBS）和培养基的时机音量。 <ol><li>制成50毫升的PBS以150 mM氯化钠，24 mM磷酸盐缓冲液，pH 7.4中的浓度。过滤该溶液。 </li><li>使从Dulbecco改进的Eagle具有高葡萄糖，10％热灭活的小牛血清（FBS），青霉素（100单位/毫升），链霉素（100微克/毫升），L-谷氨酰胺（培养基（DMEM）50 ml的细胞培养基2毫摩尔），和丙酮酸钠（1毫米）。过滤该溶液。 </li></ol></li><li>除去完全生长培养基，并用3毫升的PBS洗涤细胞。 </li><li>孵育细胞用2ml的0.05％胰蛋白酶-乙二胺四乙酸（EDTA）（对于6cm培养皿中）中5分钟，在37℃，5％的CO 2，并使用移液管分离的细胞。 </li><li>灭活胰蛋白酶通过加入2ml DMEM中，并通过离心收集细胞，在300×g离心5分钟。 </li><li>除去上清液，加入1 ml DMEM中，以沉淀和吸取的溶液上下充分分散细胞分化成单细胞悬浮液。 </li><li>他们分成1：4在一个新的直径6厘米的Petri装有3毫升完全培养基菜。在一个5％CO 2培养箱保持培养中的细胞在直径6cm的培养皿，在37℃下。子培养，每周一次，或当它们已经覆盖80 - 90％的表面区域。 </li></ol></li><li> SH-SY5Y细胞接种于摄像 <ol><li>对于TIRFM实验，板细胞在玻璃盖。采用玻璃覆盖与0.17±0.005毫米的厚度和1.5255±0.00015折射率。开始前，准备盖玻片如下： <ol><li>干净的玻璃覆盖90％的乙醇，O / N。 </li><li>在蒸馏水（蒸馏水三个转变）彻底冲洗。干燥时的玻璃覆盖在干燥炉中。 </li><li>地方覆盖在玻璃培养皿和消毒在预热烘箱中在200℃下进行3小时。 </li></ol></li><li>在转染前一天，将每个盖玻片在3.5公分培养皿，加入1 ml培养基并在5％CO 2的培养箱中孵育在37℃。 </li><li>如在步骤1.1.3描述Trypsinize细胞 - 1.1.5，暂停将细胞沉淀在1ml完全培养基和计数。计算细胞悬浮液的正确体积添加至每个培养皿中，得到3×10 5细胞/孔。此密度是必需的以获得最佳的细胞生长和有效转染。孵育在37℃下在一个5％CO 2培养箱O / N。 </li></ol></li><li> SH-SY5Ytransfection由聚乙烯亚胺（PEI） 注意：为了形象化突触小泡动力学，已使用含突触-pHluorin pCB6载体。所述突触-pHluorin已在绿色荧光蛋白（GFP）16和小泡膜蛋白突触2.构建体已被广泛用于研究神经元9内突触小泡特性的PH敏感的变体的框内融合生成由。 <ol><li>在开始转染前，打10毫升以下的解决方案。保持解决方案，最大为1个月。 <ol><li>做一个150毫米的NaCl溶液。调节至pH 5.5 0.01N HCl中。 </li><li>使在10％的聚乙烯亚胺（PEI; 25kDa的线性）一个PEI溶液在150mM的NaCl溶液。溶液的pH升高到8.8。调节pH至7.8用0.01N HCl中。 </li></ol></li><li>铺板后24小时，去除培养基，并用1.5ml的刷新完全培养基。保持所述细胞在37℃，在5％CO 2的培养箱中培养。 </li><li>下的层流生物安全柜，在1.5ml微量离心管中，加入3微克质粒DNA到25微升150 mM氯化钠溶液和100微升每3.5厘米培养皿的PEI溶液。 </li><li>涡旋10秒，然后孵育该DNA / PEI混合物进行30分钟，在RT。 </li><li>小心将DNA / PEI混合物添加到含有盖玻片与细胞培养皿并轻轻摇动，以平均分配，试剂中的培养皿。 </li><li> 4小时后，改变培养基，并在5％CO 2的培养箱中孵育细胞O / N在37℃。转染后48小时 - 进行成像试验24。 </li></ol></li></ol>通过全内反射荧光显微镜2.细胞成像（TIRFM） <ol><li> 成像设置 <ol><li>与图1中描述的设置执行的TIRF成像，它包括一个机动倒置显微镜（ 图1，小图a），激光源（ 图1，小图B）和TIRF滑块（ 图1，小图C）。通过高数值孔径（NA 1.45阿尔法普兰-Fluar）100X油，浸泡的目的达到TIRFM照明。 </li><li>对于TIRFM照明，采用多线（458/488/514纳米）100毫瓦氩离子激光。使用单模式光纤，引入线偏振激光光进入光束路径，经由TIRF滑块。插入的TIRF滑块成反射光束路径的发光场光阑平面。 <ol><li>对宽视场照明，显微镜连接到常规汞短弧灯HBO白光。在滑块的偏振波保持双棱镜可以确保的TIRF照明和白光的同时的联合。 </li></ol></li><li>过滤该激光光用激发滤波器（频带宽度十分之四百八十八纳米）安装在滤光轮中，被引入激光路径。聘用高速，软件控制，快门，以便快速控制激光照射。对于pHluorin分析，安装一个带通五十分之五百二十五nm发射过滤器。在快速冷却的CCD摄像头，图像PROPLUS软件捕捉数字影像（512×512像素）。 </li></ol></li><li> 实现的TIRF照明（图2） <ol><li>打开激光，计算机，照相机，滤光轮，并且快门控制器;然后，等待20分钟开始实验的激光器需预热和稳定之前。 </li><li>影像之前，请通过以下方法解决的时机音量。 <ol><li>制成50毫升克雷布斯（KRH）溶液在125 mM氯化钠，5mM的氯化钾，1.2mM的MgSO 4干燥，1.2毫KH 2 PO 4，25mM的4-（2-羟乙基）哌嗪-1-乙磺酸（HEPES）（缓冲至pH为7.4），2mM的氯化钙 ，以及6毫米的葡萄糖。 </li><li>使溶于10ml氯化钾-KRH溶液（pH 7.4）中，在80毫米的NaCl，50mM的氯化钾，1.2mM的MgSO 4干燥，1.2毫磷酸二氢钾，25毫米的HEPES（缓冲到pH 7.4），2mM的氯化钙 ，以及6毫米的葡萄糖。 </li></ol></li><li>与转染细胞中取出玻璃盖，并在适当的成像室插入。装配该室，并在玻璃的中心加入500微升的KRH溶液。 </li><li>加油过的目标。将成像室在显微镜的阶段，并在该玻璃盖玻片定位目标。定位安全盖在样本。 </li><li>在落射荧光模式，专注于盖玻片（上面），然后选择转染细胞放置在室内中心。选择单元格，其荧光信号可以用低于80​​毫秒的曝光时间可以清楚地记录下来。 </li><li>在软件控制下，切换到TIRF照明在实时模式。 </li><li>要设置的TIRF配置，检查浮现出的目标，在样品上覆盖（ 图2B）的光束的位置。当光束被定位在吨的中心他物镜（ 图2A，左），一个光点是在TIRF样品盖的中心可见（ 图2B，左）和该信元被成像在落射荧光模式（几个聚焦平面，高背景荧光; 图2C，左） 。 </li><li>使用上的TIRF滑块（ 图1C）的角度调整螺钉;达到临界角，将聚焦光点在Y方向（ 图2B中，中心向前或向后）。当光束以一个角度大于临界角大（ 图2A，右）会聚在样品平面，光点消失，一个直的，薄的，聚焦线是显而易见的，在样品盖的中部（ 图2B，右）。 </li><li>要微调的TIRF角度使用细胞样本（ 图2C）。观看的视频的荧光图像，在这个阶段中，落射荧光状图象仍是可见的。轻轻地，将螺丝，直到TIRF CONDITION实现：仅一个光电池的平面是在聚焦（ 即，在与盖滑动接触质膜），这导致在一个平面图像具有高对比度（ 图2C，右）。 </li></ol></li><li> 样品成像 <ol><li>设置单声道的时间推移的实验。为了最大限度地减少漂白，使用低曝光时间和高增益捕获图像。适当的曝光时间之间的40 - 80毫秒。在采集的图像1 - 2 Hz的采样频率。囊泡动力学可能是在更高的频率（10赫兹）更好地理解采样。观察常规时间一般为2分钟的。 </li><li>加入500μlKRH解决方案和记录细胞的TIRFM模式。这是静止状态。节省时间序列图像。 </li><li>着眼于静息的相同的条件（激光功率，时间曝光，帧号）根据相同的小区和记录。经过五年的框架，加入500μl的氯化钾，KRH的解决方案，并保持在氯化钾室。这是受激状态;节省时间序列图像。 </li></ol></li></ol> 3.图像分析和数据处理注意：要分析的图像，宏已经开发了在实验室中，基于该图像分析软件的现有功能;类似宏可在网上（见表材料和设备提供URL）。 <ol><li> 荧光强度量化 <ol><li>使用“序列的荧光强度”宏观为荧光强度定量中的图像的感兴趣区域（ROI）的区域中，在电影的过程。 </li><li>打开时间序列图像。转到宏观菜单，然后选择“序列荧光强度”。在“分析”窗口出现“选择ROI”。 </li><li>选择的选择的工具之一，在菜单中创建的投资回报率。放置3感兴趣区在细胞膜的区域无斑点（背景ROI）。聘请THIs“背景的ROI”来评估漂白和设定的阈值的融合事件的分析（ 图3A）。 </li></ol></li><li> 漂白校正和阈值判定（图3B） <ol><li>评估漂白，打开荧光强度行“背景的投资回报”，（ 图3BA）。正常化在每个帧的初始强度值（F 0）（F / F 0）（ 图3BB）的荧光强度值。平均化的值。 </li><li>突出的平均数据，并使用图表菜单选项的线图。 </li><li>从数据分析菜单中选择“趋势线”，打开图分析对话框。选择回归的类型。设置“指数”的回归。然后选择“在图表上显示方程”。在图形窗口中，指数方程出现的参数值将被自动分配，（ 图3BC </strong&gt;的）。 </li><li>指数修正适用于每个帧如下的强度值： FN（修正）= FN / EXP（N *一） FN =在帧n实验测量荧光强度; N =帧数;一个=漂白因子（恒定表达强度的损失是由于光漂白速率; &#160; 图3BD）。 </li><li>以设定阈值，打开一个规格化和校正“背景的ROI”，计算平均荧光信号，其标准偏差（SD）。平均值加上3 SD代表的阈值（ 图3BE）。使用此阈值进行数据分析。 </li></ol></li><li> 使用半自动程序的融合活动选择 <ol><li>打开用图像分析软件的时间序列图像。应用高斯滤波器活动图像序列。 </li><li>使用“计算对象”工具或宏允许选择分析图像的一个对象，其像素具有一个限定的范围内的平均荧光强度。设置强度范围手动，使用阈值函数（去酒吧的菜单，设置测量→门槛，突出感兴趣的区域）。适当的阈值是在本地的荧光背景信号的30％。 </li><li>应用宏“过滤器对象”选择符合以下条件的唯一对象： <ol><li>应用范围选项（最小和最大含）方面。一方面报告长轴和等同的对象的椭圆的短轴之间的比率。看点永远是≥1。适当的值是分= 1，最大值= 3。 </li><li>适用范围的直径。 直径报道在2度间隔连接两个轮廓点并穿过对象的质心测量的直径的平均长度。设置在像素的范围内（或者在微米，如果使用校准系统）。 </li><li>定义prelimin最佳范围元实验：手动选择感兴趣的点，然后使用积曲线函数测量它们的直径。 </li></ol></li><li>选择“显示对象”：选择的对象会出现叠加到TIRFM图像（ 图4B）。 </li><li>包括在分析中只有那些表现出短（一到三个帧）点的瞬态荧光强度增加，紧接着的信号（瞬时点）的显着损失。采用圆形选择大约一个光斑直径径向创建一个ROI围绕选定的囊泡/点（实验的ROI）。手动执行此步骤。 </li><li>与感兴趣区选择，计算在电影的过程中的每个ROI的平均荧光强度。 </li></ol></li><li> 数据分析（图3C-D）的 <ol><li>导出的时间过程中的每一个“实验的ROI”到电子表格测得的荧光变化; （ 图3DA）。正常化在每一帧的初始荧光强度（F / F 0），（ 图3分贝 ）的强度值。 </li><li>所报告的步骤3.2.4（ 图3Dc的 ）中的每个帧的指数应用校正强度值。 </li><li>计算的融合事件的总数（峰数），每个聚变发生的时间（峰宽）和荧光峰（峰高和AUC）的振幅申请使用的电子表格或数学软件包逻辑功能。使用逻辑公式聚变事件分析的一个例子报道于图的3DD和3DE。 </li><li>假设荧光强度超过阈值（平均背景荧光强度±3SD），为小泡的融合到质膜的增加，将所得的峰为融合事件。 </li><li>计算峰宽作为最后的和每个峰的第一x值之间的差异。这个值乘以1 /（采样频率）。考虑这个值š囊泡融合和粘附在质膜囊泡重新酸化和回收（ 图的3DD）之前的时间。 </li><li>计算的全细胞的AUC作为值超过阈值的总和。考虑这个值作为在记录时间由于自发（休息）净荧光改变或诱发（刺激）突触活动。 </li><li>计算峰高为每个峰和阈的最大y值之间的差。考虑这个值作为指示融合型（单与同时/顺序融合或瞬态主场迎战全融合）的。 </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balice-Gordon+R.J.+Heterogeneity+in+Synaptic+Vesicle+Release+at+Neuromuscular+Synapses+of+Mice+Expressing+SynaptopHluorin.">Balice-Gordon R.J. Heterogeneity in Synaptic Vesicle Release at Neuromuscular Synapses of Mice Expressing SynaptopHluorin.</a> J. Neurosci. 28 (1), 325-335 (2008).</li><li>Tsuboi, T., Rutter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+forms+of+&#34;kiss-and-run&#34;+exocytosis+revealed+by+evanescent+wave+microscopy.">Multiple forms of &#34;kiss-and-run&#34; exocytosis revealed by evanescent wave microscopy.</a> Curr Biol. 13, 563-567 (2003).</li><li>Miloso, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+Acid-Induced+Neuritogenesis+of+Human+Neuroblastoma+SH-SY5Y+Cells+Is+ERK+Independent+and+PKC+Dependent.">Retinoic Acid-Induced Neuritogenesis of Human Neuroblastoma SH-SY5Y Cells Is ERK Independent and PKC Dependent.</a> J. Neurosci. Res. 75, 241-252 (2004).</li><li>Jaskolski, F., Mayo-Martin, B., Jane, D., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin-dependent+Membrane+Drift+Recruits+AMPA+Receptors+to+Dendritic+Spines.">Dynamin-dependent Membrane Drift Recruits AMPA Receptors to Dendritic Spines.</a> J Biol Chem. 284 (18), 12491-12503 (2009).</li><li>Treccani, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+and+corticosterone+rapidly+increase+the+readily+releasable+pool+of+glutamate+vesicles+in+synaptic+terminals+of+prefrontal+and+frontal+cortex.">Stress and corticosterone rapidly increase the readily releasable pool of glutamate vesicles in synaptic terminals of prefrontal and frontal cortex.</a> Mol Psychiatry. 19 (4), 433-443 (1038).</li><li>D'Amico, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surface+density+of+the+glutamate+transporter+EAAC1+is+controlled+by+interactions+with+PDZK1+and+AP2+adaptor+complexes.">The surface density of the glutamate transporter EAAC1 is controlled by interactions with PDZK1 and AP2 adaptor complexes.</a> Traffic. 11 (11), 1455-1470 (2010).</li><li>Perego, C., Di Cairano, E. 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Neurochem. 75 (3), 991-1003 (2000).</li><li>Kume, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dibutyryl+cyclic+AMP+induces+differentiation+of+human+neuroblastoma+SH-SY5Y+cells+into+a+noradrenergic+phenotype.">Dibutyryl cyclic AMP induces differentiation of human neuroblastoma SH-SY5Y cells into a noradrenergic phenotype.</a> Neurosci Lett. 443 (3), 199-203 (2008).</li><li>Diaspro, A., Chirico, G., Usai, C., Ramoino, P., Dobrucki, J., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching.">Photobleaching.</a> Handbook of Biological Confocal Microscopy. , 690-699 (2006).</li><li>Rossano, A. J., Chouhan, A. K., Macleod, G. 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The authors declare that they have no competing financial interests.

本文提出了一种协议，以图像和分析囊泡动力学分泌细胞，采用荧光基因编码向量和TIRFM。成功的成像通过TIRFM关键要素是细胞模型和细胞转染与囊泡释放和回收的基因编码的光学指标的选择。 TIRFM非常适合于细胞生长附着于玻璃盖和足够平坦以使膜融合事件的稳定可视化。囊泡最好应分散在细胞使它们的贩卖，融合和胞吞作用可以被成像和量化在单囊泡水平。不幸的是，神?...

神经元之间的化学突触传递是通信中的神经系统的主要机制。它依赖于神经递质通过囊泡融合和检索的动态循环的释放在突触前部位。许多参与囊泡动力学的蛋白质已被鉴定;然而，它们对这一现象的具体贡献仍有待阐明1。 我们的理解是由以下事实最广泛使用的测定法外/胞吞作用并不总是最合适的部分的限制。相关囊泡融合一些研究和动态依靠电生理技术。这种技术提供了一个最佳的时间分辨率和优良调查囊泡至质膜的初始熔化，但无法检测到许多支持突触功能的基本分子事件。电子显微镜，在另一侧，提供了最好的morphologica每个奇异步骤，但是事件的动态方面的升说明不能被捕获，因为样品必须固定以便进行分析。 的新的光记录技术2,3，结合在荧光分子探针发展4-6进步的出现，使得在活细胞中胞吐过程的可视化，从而提供约突触的结构和功能的信息的新的水平。 最初的研究开发活动相关的苯乙烯染料（FM1 - 43和相关有机染料）7,8。国家的最先进的成像技术采用了绿色荧光蛋白（GFP）（pHluorin）拴管腔小泡蛋白9的pH敏感的变体。这些探针通常被关闭，因为低pH值管腔的囊泡当存在时。融合与质膜后，囊泡内部暴露于中性细胞外空间中，pH<html>突然增加，缓解pHluorin的质子依赖淬火和荧光信号迅速出现。如pHluorin的变化的速度比的融合事件，通过监测荧光的增加，囊泡融合与膜可以测量和分析。因为表面pHluorin标签的分子被内吞，所述荧光信号随后返回到基础水平，因此也可以使用相同的构建体并监控囊泡再循环9。 而囊泡标记的pH传感器可以确保只有那些囊泡真正融合与质膜的可视化，成像在高空间和时间分辨率是必需的，以详细描述了所涉及的外/内吞过程的步骤。的光学技术，它提供了必要的时空分辨率为全内反射荧光显微镜（TIRFM），荧光显微镜10的应用程序。 “&gt;全内反射发生在玻璃盖玻片和样品。当光路到达玻璃盖玻片用的入射角比临界角大的界面，激发光不被传递到样品，但完全反射回去。在这些条件下，一个瞬逝光波的形式在界面处，并传播与较少的光密度（样品）的平台。由于渐逝场的强度呈指数衰减的距离从接口（具有穿透深度约100nm）只在最接近于盖滑移的荧光团可被激发而更远离边界都没有。在转染的细胞用GFP-构建体，该深度对应于表示在质膜上的蛋白质或在囊泡结构接近它。如在细胞内部的荧光团可以不被激励时，所述背景荧光最小化，并具有非常高的信号/背景的大鼠的图像IO形成11。 有几个特点使选择的TIRFM技术监测囊泡动态。完美的对比度和高的信号 - 噪声比允许非常低的信号从单个囊泡导出的检测。在各帧的基于芯片的图像采集提供必要检测高动态过程的时间分辨率。最后，细胞的最小曝光的样品中任何其他飞机大大地减少光毒性，并能持久延时录制12。 数据分析仍然这种技术的最具挑战性的和关键的方面。监视囊泡融合的最简单的方法是测量报告荧光蛋白在细胞表面的积累，随着时间的推移13。作为融合的增加，净荧光信号也增加。然而，这种方法可能会低估的过程中，特别是在大的细胞和在静止的条件下，因为内吞作用和漂白过程偏移由于囊泡的胞吐作用的增加的荧光强度。另一种方法是按照每个单个融合事件14。这后一种方法是很敏感的，并且可以揭示关于融合的机制的重要细节。然而，它要求手动选择单一活动，因为完全自动化程序遵循囊泡和登记他们的荧光信号的波动并不总是可用。囊泡动力学的观察要求采样单元在高频率。这会产生大量的数据，可以几乎不进行手动分析。 本文的建议是优化TIRFM成像技术用于监测基底和刺激的神经递质释放，在SH-5YSY神经母细胞瘤细胞系，并描述，一步一步，在实验室中开发的程序来分析数据，既在全细胞和单囊泡水平。

<table border="0" cellpadding="0" cellspacing="0" width="1414">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Equipment</td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td style="width:371px;"></td>
			<td style="width:468px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Axio Observer Z1</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:119px;">491912-9850-000</td>
			<td>inverted microscope 
			<a href="http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-observer-for-biology.html#introduction">http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-observer-for-biology.html#introduction</a></td>
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			<td height="21" style="height:21px;width:259px;">Multiline Argon Laser Lasos 77</td>
			<td style="width:199px;">Lasos</td>
			<td style="width:119px;">00000-1312-752</td>
			<td>multi-line (458/488/514 nm), 100mW argon-ion laser 
			<a href="http://www.lasos.com/products/argon-ion-laser">http://www.lasos.com/products/argon-ion-laser</a></td>
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		<tr height="23">
			<td height="23" style="height:23px;width:259px;">Laser TIRF slider</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:119px;">423681-9901-000</td>
			<td><a href="http://www.zeiss.com/microscopy/en_de/products/imaging-systems/single-molecular-imaging-laser-tirf-3.html">http://www.zeiss.com/microscopy/en_de/products/imaging-systems/single-molecular-imaging-laser-tirf-3.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">100x Objective</td>
			<td style="width:199px;">Zeiss</td>
			<td>421190-9900-000</td>
			<td>Oil, NA 1.45 Alpha-Plan 
			<a href="https://www.micro-shop.zeiss.com/?l=en&amp;p=us&amp;f=o&amp;a=v&amp;m=a&amp;id=421190-9900-000&amp;ss=1">https://www.micro-shop.zeiss.com/?l=en&#38;p=us&#38;f=o&#38;a=v&#38;m=a&#38;id=421 
			190-9900-000&#38;ss=1</a></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">CCD Camera RetigaSRV Fast 1394&#160;</td>
			<td style="width:199px;">QImaging</td>
			<td style="width:119px;"></td>
			<td><a href="http://www.qimaging.com/products/datasheets/Retiga-SRV.pdf">http://www.qimaging.com/products/datasheets/Retiga-SRV.pdf</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">LAMBDA 10-3 optical filter changer with SmartShutter</td>
			<td style="width:199px;">Sutter Instrument Company</td>
			<td style="width:119px;"></td>
			<td><a href="http://www.sutter.com/IMAGING/lambda103.html">http://www.sutter.com/IMAGING/lambda103.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;"></td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Software</td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image ProPlus 6.3 Software</td>
			<td style="width:199px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>spot selection, ROI selection, fluorescence intensity determination 
			<a href="http://www.mediacy.com/index.aspx?page=IPP">http://www.mediacy.com/index.aspx?page=IPP</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Excel</td>
			<td style="width:199px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td>photobleaching correction, whole-cell and single-vesicle analyses 
			<a href="http://office.microsoft.com/it-it/excel/">http://office.microsoft.com/it-it/excel/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism 4.00&#160;</td>
			<td>GraphPad Software, Inc.</td>
			<td style="width:119px;"></td>
			<td>statistical analysis 
			<a href="http://www.graphpad.com/scientific-software/prism/">http://www.graphpad.com/scientific-software/prism/</a></td>
		</tr>
	</tbody>
</table>

tirfm and ph-sensitive gfp-probes to evaluate neurotransmitter vesicle dynamics in sh-sy5y neuroblastoma cells: cell imaging and data analysis

突触囊泡融合，通过和检索的动态循环释放神经递质的化学突触。 实时监测突触活动和解剖的不同步骤外-内吞作用的单囊泡水平对于理解在健康和疾病突触功能至关重要。 遗传编码的PH敏感的探针直接靶向突触小泡和全内反射荧光显微镜（TIRFM）提供必要遵循囊泡动力学的时空分辨率。通过全内反射所产生的渐逝场只能激发置于一薄层（&lt;150纳米），其上的细胞粘附在玻璃盖之上的荧光团，正是外 - 内吞作用的过程发生。所得高对比度的图像非常适合囊泡跟踪和融合事件的定量分析。 在这个协议中，SH-SY5Y人N-euroblastoma细胞被提出作为一种有价值的模型为研究，因为他们的平坦面神经递质释放在通过TIRFM单囊泡水平，并分散囊泡的存在。被提供的方法用于生长的SH-SY5Y以贴壁细胞和与突触-pHluorin染它们，以及执行TIRFM和成像技术。最后，战略目标选择，计数和分析，在全细胞和单囊泡融合水平的事件呈现。 为了验证成像过程和数据分析方法，pHluorin标记的囊泡的动力学下休息了分析和刺激（去极化钾浓度）的条件。膜去极化增加的融合事件的频率，并导致记录在全细胞的净荧光信号的并行加注。单囊泡分析揭示聚变事件行为（上升峰的高度和宽度）的修饰。这些数据表明，第在钾去极化不仅诱导大量神经递质释放，而且修改囊泡融合和再循环的机制。 用适当的荧光探针，该技术可以在不同的蜂窝系统可以采用解剖构和刺激分泌的机制。

描述的全内反射荧光成像和数据分析程序的目的是研究在蜂窝系统囊泡动态。这种技术可以被用于确定信号分子和药物对聚变事件和神经递质的囊泡动力学17的影响。使用GFP标记质膜蛋白，所述TIRFM分析已被用来表征在胶质GFP标记谷氨酸转运和上皮细胞18,19的构贩卖。 为了验证报告的成像过程和数据分析策略，融合事件被记录在基础和刺激条件（钾引起的去极?...

Watch this Scientific Journal Video about TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis at JoVE.com

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤：细胞成像和数据分析

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

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10.7K 次观看.  Università degli Studi di Milano. This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

视频: TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

科研

神经科学

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

嗅鞘细胞移植周围神经损伤后，以评估功能恢复

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

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 ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

背根神经节分离与原代培养对神经递质释放的研究

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

iPSC-巨噬细胞死亡神经母细胞的体外定量成像检测

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized cells use this process to execute vital body functions such as insulin secretion from &#946; cells and neurotransmitter release from chemical synapses. Owing to its physiological significance, exo-/endocytosis has been one of the most studied topics in cell biology. Many tools have been developed to study this process at the gene and protein level, because of which much is known about the protein machinery participating in this process. However, very few methods have been developed to measure membrane lipid turnover, which is the physical basis of exo-/endocytosis.
This paper introduces a class of new fluorescent lipid analogs exhibiting pH-dependent fluorescence and demonstrates their use to trace lipid recycling between the plasma membrane and the secretory vesicles. Aided by simple pH manipulations, those analogs also allow the quantification of lipid distribution across the surface and the intracellular membrane compartments, as well as the measurement of lipid turnover rate during exo-/endocytosis. These novel lipid reporters will be of great interest to various biological research fields such as cell biology and neuroscience.

This protocol presents a fluorescence imaging method that uses a class of pH-sensitive lipid fluorophores to monitor lipid membrane trafficking during cell exocytosis and the endocytosis cycle.

使用 pH 敏感荧光脂质类似物 ND6 测量膜脂质周转率

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

肌动蛋白动力学、离合器耦合和牵引力分析，促进生长锥体前进

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...