这项研究是由美国心脏协会和启动资金由美国韦恩州立大学AA支持的国家科学家开发格兰特（13SDG14420049）。我们非常感激医生罗纳德W.霍尔茨和玛丽·比特纳博士提供有关牛肾上腺准备和丹尼尔·阿克塞尔罗德博士与pTIRFM设置和对稿件的意见非常有用的援助建议。SYT-1 pHluorin是与使用下吕Miesenbock博士（牛津大学）的许可。 GCAMP5G通过Addgene获得的。我们感谢詹姆斯·泰勒，Tejeshwar饶，雷切尔L.艾克曼，以及Praneeth Katrapti与优化各个步骤的描述的程序的帮助。

1。 PTIRF系统设置该pTIRFM技术是建立在一个倒置显微镜平台上。激光束通过一个侧照射口和非偏振型侧朝向过滤器立方体定向，然后聚焦到60X 1.49 NA TIRF物镜的后焦平面中。两个附加镜头（1.6X和2X）在显微镜和EMCCD相机之间的发射路径给约80纳米的最终像素大小。激光束被用软件控制的电流计镜进行外延和全内反射（TIR）的照明之间快速切换引导。下面描述的协议假定光学器件的TIRF已经定位在空气中的表为荧光团的激发和成像的最佳位置。它描述了如何偏振光学加入到现有的TIRF显微镜设置以使细胞膜变形成像。我们实验室的光学设置了一个原理图生成两个TIR和polarizat离子型TIR示于图1A中 。该协议假定使用488-nm激光的荧光囊泡蛋白的激发和561 nm激光对碳菁染料的成像，做到了。 <ol><li>电源上的所有组件显微镜，激光和计算机。 </li><li>引导光束从488nm的激光的1.49 NA透镜如图1A的后焦面（BFP）。如果激光束被聚焦在BFP，就会出现准直的和出现在天花板上的目标的正上方的小定义良好的光斑。 </li><li>调整X检流计镜（位于相当于样品平面反射镜），使激光束移动的离轴和出现从在逐渐更陡的角度的目标的客观标准。 </li><li>接下来，验证TIR的实现。添加10微升荧光微球，以1毫升体积的PSS在玻璃底培养皿中。荧光只从底部微ÕF中的盘子，最接近TIR接口，应该被检测到。检测漂浮微球表示入射光和客观法线之间的角度是不够的。 下面的部分描述的建立所必需的基于偏振的成像光学元件。 </li><li>使用对准488nm的光束作为引导，调节原料561 nm的激光束的位置，使得它沿一个相同的光路行进。在561 nm激光将用于碳菁染料的成像，做到了。 </li><li>插入一个发散透镜和反射镜的561 nm激光的下游。使用反射镜，调整光束，使其coaligned与488nm的光束的光斑被聚焦在天花板上时，检流计反射镜是在"0"位置。 </li><li>在光束路径紧接激光孔的下游，中央四分之一波片（QW）。使用一个QW，要么是无色或调谐至激发波长。一个量子阱将圆极化任何线性偏振的光，进入以45度角与光轴。一些光学元件，例如偏振立方体，具有朝一个偏振轴的衰减的偏差。量子阱可以被旋转，以补偿这种偏差。这种调整将导致椭圆偏振的光束。甲QW是必要的，因为激光束本身是高度线性极化（垂直方向），因为它出现从孔径。 </li><li>放置一个偏振立方体（PC1）的椭圆偏振束的下游。偏振立方体反映了电场的垂直（y）分量，并传递水平（x）的组成部分。偏振立方体被放置在一个小的平移阶段，以促进偏振束路径随后对准。 </li><li>使用第二个偏振立方体（PC2）和反射镜（ 图1A），以重新组合的光束。 </li><li>放置一个快门的第一偏振立方体（PC1）和垂直之间的合作mponent镜。将水平分量镜和第二偏振立方体（PC2）之间的第二活门。这些百叶窗是由成像软件使得用户能够光束偏振之间迅速地选择控制。 </li><li>用一个偏振滤光器，以验证该电场取向在每个光束路径。阿偏光过滤器将只允许在与过滤器的轴线传输。 </li><li>确认激光束的垂直和水平分量被彼此和488nm的光束对准。做出必要的调整。三束电子束都应该集中在BFP和出现准直从物镜到天花板上的同一点。这点可以被标记由X，方便日后对齐。 </li><li>将浸油的客观下降。放置装有荧光微球的培养皿（参见上面的步骤4）上的目标。 </li><li>通过移动在X电反射镜进入TIR。在公路运输，垂直部件指光束的t为S-偏振和梁的水平分量是现在的p-偏振分量。通过旋转QW板调整光束之间的相对强度。查看每个极化渐逝场与罗丹明样品，其被预测为随机定向。旋转QW板相匹配的平均像素强度。如果需要的话，在一个偏振光束路径添加中性密度滤光器减弱其强度。 </li></ol> 2。嗜铬细胞分离下面提供了一个程序，从小腿肾上腺隔离健康嗜铬细胞。它是由灯芯，信 ​​通， 等 。19和奥康纳改编自先前公布的协议，Mahata 等 20分离后，将细胞使用的是电穿孔电穿孔系统与感兴趣的质粒（次）。这个系统的结果在细胞中表达所需的荧光蛋白的20-30％。通常情况下，细胞70％存活的选roporation过程。 PSS和媒体组件的详细信息列于表2，图3和4中提供。 <ol><li>细胞制备的前两天，把35毫米光学质量玻璃底（0.17毫米厚），菜用1毫升其次1.25 ml牛胶原蛋白聚-D-赖氨酸（0.1毫克/毫升）。离开菜风干在组织培养罩。 </li><li>从屠宰场获得牛肾上腺。保持腺体上冰，而他们运回实验室。牛肾上腺可能被包裹在一层厚厚的脂肪。用手术剪去除多余的脂肪和暴露肾上腺静脉开口。灌注与介系词-PSS静脉反复，直到腺体清除血液。 </li><li>接着，慢慢地吸液管加入2ml TH-PSS溶液注入静脉开口，直到腺体肿胀。孵育充气腺体，在37°C下15分钟。重复对TH-PSS治疗，并再次孵化。 </li><li>通过围绕与S腺体的边缘外肾上腺皮质切cissors和剥离腺体分开，露出髓质。用手术刀轻轻刮去，并从皮层组织分离的髓质。 </li><li>直言不讳髓质有一对手术刀5-10分钟的。 </li><li>最后消化步骤是必要的区隔个别嗜铬细胞。倒入肉末细胞悬液成旋转瓶。部分地填充该烧瓶用的TL-PSS（15毫升）以2:1的比例TH-PSS（30毫升）中，并温育30分钟，在37℃以约100转，而旋转。 </li><li>通过一个400微米的筛网过滤从未消化的组织分离出单个的嗜铬细胞。收集滤液。离心200×g离心沉淀细胞，重悬在冰冷的PSS。 </li><li>通过250μm的筛网，过滤沉淀的细胞，并最终在电穿孔缓冲液重悬（参见步骤2.9）。 </li><li>使用血细胞计数器计数细胞，并用于转染的准备。 </li><li>根据转染细胞的电穿孔系统制造商的建议。精确的条件，电必须根据经验来确定。注意:这提供了该制备牛嗜铬细胞的转染效率和细胞存活率之间的最佳平衡的设定为1100伏，40毫秒，1个脉冲。我们预计，随着这些条件下，细胞的20-30％，转染。约30％的细胞不电穿孔过程中存活下来。 </li><li>板细胞轻轻聚-D-赖氨酸和胶原蛋白处理的培养皿1毫升温热电穿孔媒体。 </li><li>将菜在37℃培养箱（5％CO2）。加入1 ml 2倍的抗生素媒体至6小时后，每道菜。第二天的媒体更改为正常的媒体。 </li><li>嗜铬细胞电穿孔后2-5天，通常成像。 </li></ol> 3。 pTIRF图像采集<ol><li>开机成像系统，并开始采集软件。 </li><li>验证激光对齐。检查逝使用微场分布。 </li><li>准备好全局和局部灌注系统。清洁液水库过滤的去离子H 2 O和填充基础和刺激的PSS解决方案。 </li><li>前成像嗜铬细胞，验证p偏振和s-偏振激发照亮视场的同一区域，且光照强度都大致相当。要做到这一点，填补用2毫升的PSS含罗丹明为10毫米的终浓度玻璃底菜。依次激发罗丹明样品与p-偏振和s-偏振的光并捕获图像。在实践中，从做了P和S的排放量进行归一化的P和S罗丹明排放的平均值。看到的图像分析与讨论部分作进一步的细节。 </li><li>现在进行染色嗜铬细胞一样。从含铬细胞培养皿冲洗培养基和替换用2 ml基础-PSS的。加入10μl10 mM的DID（以乙醇稀释）直接将含有细胞的培养皿中。轻轻摇动培养皿2-10秒，取出DID + PSS解决方案。 </li><li>与基底的PSS洗碟3至4倍，以除去任何残留的那样。现在这些细胞被染色，并准备使用。 </li><li>加一滴浸油的目标。将载有关于目标的最根本染色嗜铬细胞培养皿。 </li><li>无论是通过目标或者相机上观看的菜，发现它是转染了目的蛋白质并用做了一个细胞。良好的染色的细胞表现出一名P发射的生动突出了小区的边缘;在S发射应该在整个细胞体积大致均匀的（ 即附着在玻璃和成像中的TIRF的小区的区域）。 </li><li>位置的局部灌注针头，使其大致为100μm离细胞。 </li><li>专注于细胞膜和启动自动对焦硬件立即细胞刺激/图像采集之前。&lt;/ LI&gt; <li>开始图像采集。灌注细胞，持续10秒与基底溶液中，然后进行60秒，去极化56毫米氯化钾溶液。获取的图像，同时561 nm的p-偏振分量之间迅速地关闭，561 nm处S-偏振（监测P和变化做了S排出量）和488 nm激发（图像转染的囊泡探头）。在实验过程中获得的图像的实例示于图2和图3。 </li></ol> 4。图像分析计算图像处理可以与ImageJ的执行，但在一个灵活的编程语言如IDL或Matlab利用脚本可以通过自动化的任务大大增加分析的吞吐量。两种类型的图像，必须计算从原始图像排放得到的拓扑信息-在P / S和P +2 S P / S在本地膜变形报告而P +2σ总和在总膜染料在某一区域报告该字段的。 <ol><li>所有图像应扣除背景。选择一个离细胞的投资回报率，衡量投资回报率的平均值像素强度，再减去该值从图像堆栈中的所有像素。 </li><li>计算P / S，由随后的"S"的发射帧（像素到像素）除以每个"P"的发射帧，P / S与p-和s-偏振分量激励的相对强度，在偏差变化光学系统，以及干涉条纹。为了减少这些影响，归一化从DID排放得到与含有10mM若丹明18的溶液中得到的比值的P / S的比率。 </li><li>个人DCV值的胞吐作用是显而易见的，从在荧光小泡蛋白，如突触-1 pHluorin（ 图2B）的强度的突然变化。通过在240纳米半径的ROI集中于在P / S的局部增加，平均的象素确定的变化的P / S和P +2σ比在胞吐作用的部位。当融合发生在没有P / S的明显增加，在ROI被集中在定影DCV的区域。 </li><li>计算机模拟可以进行解读的P / S和P +2σ膜强度变化的一个扩张融合孔的简单几何形状的术语。对于模拟的详细信息，请参阅Anantharam 等 18 </li></ol>

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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=Primary+culture+of+bovine+chromaffin+cells.">Primary culture of bovine chromaffin cells.</a> Nat. Protoc. 2, 1248-1253 (2007).</li><li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+signal-to-noise+Ca2++composed+of+a+single+green+fluorescent+protein.">A high signal-to-noise Ca2+ composed of a single green fluorescent protein.</a> Nat. Biotechnol. 19, 137-141 (2001).</li><li>Akerboom, J., 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=Optimization+of+a+GCaMP+calcium+indicator+for+neural+activity+imaging.">Optimization of a GCaMP calcium indicator for neural activity imaging.</a> Neurosci. 32, 13819-13840 (2012).</li></ol>

作者什么都没有透露。

基于偏振的TIRFM检测膜拓扑快速，微观的变化。实施该技术是比较简单的，特别是如果全内反射荧光光学系统已经到位。所有需要的是一个四分之一波片，两个偏振立方体，和百叶窗分离P-偏振分量和s-偏振的照明路径。这些组件的桌子上的精确位置通常是由可用空间决定。 为了获得膜的拓扑信息，所使用的荧光探针应相互插入一个固定的或已知的方位。的技术中，如本文中所描述，假设使用羰花青染料，但其它染料，如FM1-43 14，也可以使用。膜染色过程本身很简单。培养的细胞只需要很短的曝光（小于10秒），以没得到膜的旺盛标签少量DII /的。加入过量的染料导致光散射AGGREG阿泰上降低影像质量的玻璃。过温育细胞与染料导致染料内在这对图像质量和可能的细胞存活力的有害影响。如果一个细胞染色好，就会有明显的区别在P和S发射图像。 如图2A所示 ，在P发射将生动地突出显示该膜的那是盖玻片倾斜（ 即小区覆盖区的边缘），而S的发光强度将大致均匀的细胞足迹区域。如果P和S之间的明显区别是看不出来的，那么它有可能是细胞粘附较差的底物或细胞膜是不健康的，并且该单元不用于实验。 我们以前的研究说明pTIRFM利用DII作为荧光膜探针。这里，我们利用这样做是因为它适合于采用GFP / pHluorin作为其它荧光团的双色成像实验。我们激发做无线TH一个561纳米的激光，而不是640 nm激光（这是更接近其激发峰），因为荧光漂白剂更迅速地在640nm。尽管在561 nm的激光是在没公布的激发光谱，荧光被有效地发射的尾巴。在561 nm激光的另一个优点是，它激发两个DII，也使我们能够使用相同的偏振设置两个探针。请注意，在膜中的荧光基团的取向会影响膜的拓扑结构的解释。例如，如果荧光团进行了垂直于膜面取向的，其过渡偶极子（而不是平行或几乎平行的，因为是与DII或者没有的情况下），那么非平面的膜的区域，则可更容易由S突出，而不是在P排放。 我们还描述的技术用于分离牛肾上腺嗜铬细胞和电穿孔。牛嗜铬细胞是优异的Secretory模型。它具有易于在文化，响应各种分泌剂的维护，并具有可随时可视化与传统光学显微镜大型分泌囊泡的优势。表达荧光蛋白，将细胞铺在胶原处理过的培养皿之前，电穿孔悬浮液中。在我们的经验，表达效率要高得多与电穿孔比用任一的Ca 2 +的磷酸盐或脂质体转染试剂。电穿孔的缺点是，它需要更多的细胞（如多不进程存活）和昂贵的商业试剂。对于原因尚不清楚对我们来说，不是每一个膜结合一样。另外，在烹调的细胞中，只有一小部分被转染。发现转染与染色以及用做细胞可以是费时这就是为什么优化染色和电是非常值得的努力情况。 总之，这是一个rticle描述pTIRFM是如何实现的监测融合在嗜铬细胞致密核心囊泡的过程中发生的快速和局部的膜的变形。虽然只有一个应用进行了讨论，该技术是非常适合的，涉及改变膜的形状，包括细胞内吞作用，细胞分裂和细胞运动等生物过程的研究。

胞吐作用的正确执行，需要在双层膜的形状极端的变化，必须精确地策划。在融合前，质膜的颗粒下局部弯曲时，部分地减少对双层膜的相互作用的能量势垒。后来，随着膜融合，产生高曲率的区域，必须稳定。最后，熔融膜必须弯曲了他们的初始形态，扩大融合孔，使内容发布1。因为单靠膜是不太可能发生如此戏剧性的和协调的变化，蛋白质是必要的调解活动。但他们如何行动在融合过程来影响改变膜拓扑保​​持非常开放的问题。 优良的体外模型存在可视化膜曲率。利用负染EM，例如，一直重要的整形目前分子模型的两个主要贩卖P上的操作roteins -突触结合蛋白和动力素（综述参见查普曼2和亨肖3）。胞吐的大多数实时检测不直接检测曲率。相反，曲率是从实验上腔货物放行4-8或改变膜面积9-11动力学的报告推断。 PTIRFM通过桥接实现直接，实时改变膜微观形貌测量在体内和体外研究之间的差距。 PTIRFM，在非成像模式，开创了由阿克塞尔罗德和他的同事测量NPD-PE的内部模型膜12结合的方向。该技术随后应用于可视化标记DII和FM1-43 13-18活细胞的动态变化。在pTIRFM，2渐逝场的偏振被用于顺序地激发一个膜包埋探针:p偏振（在入射面）和s偏振（垂直于入射平面）。在成像之前，探针 - 在这种情况下，没有 - 简要地加入到细胞外介质，并允许在被研究的细胞的细胞膜，以相互插入。在区域，其中所述膜是不平行的玻璃罩（如在膜的皱褶或凹陷）时，DID也将是不平行的。因此，这些区域将是可激发的p-偏振的光束。在p-偏振的光束会少有效地激发做膜的地区大多是平行于玻璃罩的。像素到像素的图像比例（P / S）汇报从根本将因此特别强调膜变形的区域连续p和s极化激发射。从理论上讲，在P / S比率的图像是敏感的，即使小的玻璃罩质膜偏差，用计算机模拟18所预测的变化幅度的角度。P / S的图像也独立于盖玻片表面的荧光团距离的和当地的荧光浓度。本地的荧光团浓度，而不是通过图像上的像素到像素的总和为P发射的和2倍的S排出量（P 2 S）的报告提供。在P 2 s测量是缩进的精确几何形状敏感，具有一定的，很少，或没有变化成为可能。这可以通过计算机模拟证明，一个融合孔从早期（ 即用细颈）到更高的状态16,18的模式转变。在P通过一个狭窄的颈部连接于质膜的稠囊泡2 S（和更确实接近的界面）被预测为更大，例如，比稠囊泡具有宽得多的孔隙（其中的确是渐逝场的最暗部分内）。 在这篇文章中，我们将讨论pTIRFM是如何实现的，并用来研究膜形状时融合孔的扩张所发生的迅速，局部变化。虽然只有一个应用程序是明确的二scussed，该方法推广到其他各种细胞生物学过程的直接或间接涉及膜重塑。

<table border="0" cellpadding="0" cellspacing="0" width="817">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:277px;">iXon3 EMMCD Camera</td>
			<td style="width:179px;">Andor</td>
			<td style="width:147px;">897</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IX81 Inverted Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Side-mounted Filtercube Assembly</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">43 Series Ar-Ion Laser</td>
			<td>CVI Milles Griot Laser Optics</td>
			<td>543-AP-A01</td>
			<td>Tunable to 488nm</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sapphire 561 LP Diode Laser</td>
			<td>Coherent</td>
			<td></td>
			<td>561nm</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scanning Galvo Mirror System</td>
			<td>Thorlabs</td>
			<td>GVS102</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">VC3 Channel Focal Perfusion System</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA VC3X4PP</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">QMM Quartz MicroManifold</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA QMM-4</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">10 PSI Pressure Regulator</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA PR10</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Manipulator</td>
			<td>Burleigh</td>
			<td>TS 5000-150</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mounted Achromatic Quarter-Wave Plate</td>
			<td>Thorlabs</td>
			<td>AQWP05M-600</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">420-680nm Polarizing Beamsplitter Cube</td>
			<td>Thorlabs</td>
			<td>PBS201</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Six Station Neutral Density Wheel</td>
			<td>Thorlabs</td>
			<td>FW1AND</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Stepper-motor Driven SmartShutter</td>
			<td>Sutter Instruments</td>
			<td>IQ25-1219</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HQ412lp Dichroic Filter</td>
			<td>Chroma</td>
			<td>NC255583</td>
			<td>Joins 488nm and 561nm beams</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coated Plano-Concave Lens</td>
			<td>Edmund Optics</td>
			<td>PCV 100mm VIS 0</td>
			<td>Diverges 488nm beam</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coated Plano-Concave Lens</td>
			<td>Edmund Optics</td>
			<td>PCV 250mm VIS 0</td>
			<td>Diverges 561nm beam</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coated Plano-Convex Lens</td>
			<td>Edmund Optics</td>
			<td>PCX 125mm VIS 0</td>
			<td>Focuses both beams</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coated Plano-Convex Lens</td>
			<td>Edmund Optics</td>
			<td>PCX 50mm VIS 0</td>
			<td>Cemented to filter cube assembly</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">z488/561rpc Dichroic</td>
			<td>Chroma</td>
			<td>z488/561rpc</td>
			<td>Filtercube dichroic</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">z488/561_TIRF Emission Filter</td>
			<td>Chroma</td>
			<td>z488/561m_TIRF</td>
			<td>Filtercube emission</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">UIS2 60x Objective</td>
			<td>Olympus</td>
			<td>UPLSAPO 60XO</td>
			<td>NA 1.37</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Neon Transfection System</td>
			<td>Invitrogen</td>
			<td>MPK 5000</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MetaMorph Imaging Software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DiI Membrane Dye</td>
			<td>Invitrogen</td>
			<td>V-22885</td>
			<td>For Alignment Purposes</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TH Liberase</td>
			<td>Roche</td>
			<td>5401135001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TL Liberse</td>
			<td>Roche</td>
			<td>5401020001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DNAse I Type IV from bovine</td>
			<td>Sigma</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hemocytometer</td>
			<td>Fisher</td>
			<td>0267110</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DiD Membrane Dye</td>
			<td>Invitrogen</td>
			<td>D-7757</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rhodamine</td>
			<td>Invitrogen</td>
			<td>R634</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">.22 &#956;m Membrane Syringe Filter Unit</td>
			<td>Millipore</td>
			<td>SLGS033SS</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fluoresbrite Polychromatic Red Microspheres</td>
			<td>Polysciences</td>
			<td>19507</td>
			<td>0.5 microns</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Immersion Oil</td>
			<td>Sigma</td>
			<td>56822</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LabVIEW</td>
			<td>National Instruments</td>
			<td></td>
			<td>Controlls galvo mirrors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Spinner Flask</td>
			<td>Bellco</td>
			<td>1965-00250</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging plasma membrane deformations with ptirfm

为了获得新的见解胞吐作用的动态，我们组的重点是必须的囊泡与质膜融合过程中精确调节的变化，脂质双分子层的形状。这些快速和局部的变化是由脂类和控制膜曲率特殊蛋白质之间的动态相互作用实现的。如果没有这样的互动，不仅有囊泡融合带来灾难性的后果，但许多涉及膜的形状控制的其他细胞功能。近年来，一些具有膜成形性的蛋白质的身份已被确定。还有什么缺少的是何时，何地，以及如何作为融合和内容发布进步的路线图。 我们的分子事件，使膜重塑的理解历来由缺乏的是膜曲率敏感或具有时间分辨率为TRAC分析方法的限制K表的快速变化。 PTIRFM满足这两个条件。我们讨论pTIRFM是如何实现的可视化和解释快速，亚微米变化，嗜铬细胞膜的过程中致密核心囊泡（DCV）融合的方向。我们使用的嗜铬细胞是从牛肾上腺隔离。将膜染色的亲脂性羰花青染料，1,1&#39; - 双十八烷基-3,3,3&#39;，3&#39;-tetramethylindodicarbocyanine，4 - 氯苯或没有。没在膜平面内插层具有"固定"的取向，因此是敏感的渐逝场的极化。在做染色细胞膜兴奋依次用561 nm激光（p-偏振分量，S-POL）的正交极化。 488 nm激光是用于可视化囊泡成分和时间融合的时刻。胞吐作用是通过局部灌注细胞去极化的KCl溶液触发。分析是使用自定义编写的软件，了解如何做离线执行排放强度的变化与融合孔的扩张。

传统和偏光TIRFM成像技术在同样的空气表的贯彻落实。该光学元件的构造是类似的，与激发光被极化的主要差异（ 图1A）。偏振光优先激发荧光基团与偏振方向的吸收偶极子。因此，对于pTIRFM是在监测膜拓扑结构改变有效，所使用的探针，必须在膜中相互插入一个固定的方向。荧光羰花青染料（DII，DID）相互插入到脂质双层中的取向的方式与在膜平面内（ 图1B）过渡偶极子。做了标记的膜的P偏振光照明（ 图1C）选择性地激发盖玻片斜荧光团（红在图1B和1D）。 嗜铬细胞旺盛的膜标贴是AB后实现rief孵育那样。 图2A示出了被污染以及细胞膜的一个例子。一个健康的，贴壁细胞会表现出P波和S排放量有明显的差异。在P发射图像显示是对于细胞的其他部分更加美好的单元格边框。在S发射图像显示在整个小区占地面积大致均匀的荧光。计算出的像素到像素P / S和P 2 S映象是膜曲率和染料浓度，分别敏感。所示的嗜铬细胞也已转染了突触-1 pHluorin（SYT-1）来标记分泌囊泡（ 图2B）。 嗜铬细胞被刺激以56毫米的KCl去极化细胞膜和胞吐触发。一些明亮的荧光SYT-1 pHluorin点突然变得明显，因为DCV值的保险丝（ 图2B，右图）。一个白色的方框绘制围绕一个融合事件（ 图2B，右图）。这种融合活动我s在图2C和2D分析。 图2C示出一帧一帧的变化SYT-1-pHluorin，P / S和P 2章图像强度。 SYT-1的荧光强度迅速减弱的蛋白扩散远离融合点（ 图2D）。代表囊泡融合/质膜复杂的缩进减小在相对 ​​较慢的速度（注意图2D图形）。下图（ 图2E）描绘了这些测量的一种解释。 在图2中示出的快速和局部的膜变形是刺激诱 ​​发的Ca 2 +内流的结果。这显示在图3A中 。一个嗜铬细胞转染基因的Ca 2 +指示剂GCaMP5G 21,22和56毫米氯化钾刺激。膜去极化导致GCaMP5G荧光显著增加（ <stro<html>纳克&gt;图3A和3B），标志着增加subplasmalemmal的Ca 2 +水平。该电池的30×20像素的区域被选择和帧接一帧的变化GCaMP5G，P / S和P +2σ像素强度示于图（ 图3B）和曲线图（ 图3C）。的变化的P / S之前的时间"0"表示的帧是显而易见的（ 即胞外分泌前车架）。白色箭头表示该膜的变形（增加P / S）是伴随着P中2 S排出量的减少。胞质GCaMP5G蛋白是由稠合的DCV排除的区域。还请注意，在图3C中 ，左侧面板的突然减少GCaMP5G强度在时间0。长寿命的增加，P / S和减少P中2作者提出一个融合孔即扩张缓慢（ 图3D）。 / files/ftp_upload/51334/51334fig1.jpg"/&gt; 图1。插图为pTIRFM技术。 A）中所示的示意图，结合传统与基于偏振的全内反射荧光成像。四分之一波长（QW）板被放置在一个561纳米蓝宝石激光椭圆偏振激光束的前面。偏振立方体（PC1）用于偏振分离成线性垂直（V）和水平（H）组成。的垂直和水平分量成为P-偏振分量和s-偏振的激发光束，分别在TIR表面。在p-偏振分量和s-偏振分量的路径各自独立地关闭了（S1和S2）。他们重组了镜子和第二偏光立方体（PC2）。他们加入了488纳米光束（也独立关门，S3），通过由镜子和非偏振分光镜（DC）的一个波束控制单元。镜片（L）被用来展开和聚焦光束。合并的488纳米和561纳米的光束被引导到显微镜的通过电流计的​​侧光照端口米的镜子（GM）。它们聚焦到物镜的后焦平面（BFP）。从荧光发射的光子被捕获在一个EMCCD相机连接到PC。b）概标签是由短暂孵育细胞与染料和反复洗涤以除去多余的进行。没在其跃迁偶极矩大致在膜平面上。C）偏振的发生率（p-偏振）的平面内的入射光产生的倏逝场是主要极化垂直于界面，如图膜插层D）与p-偏振光一个DID-标记的膜的照度会选择性地激发荧光团的那些是玻璃罩斜（RED）。如果这是一个s偏振光的倏逝场，这些荧光团是平行于玻璃罩会被激发，而不是（黑）。 <img alt="图2" src="/files/ftp_upload/51334/51334fig2.jpg" /> 图2。 </s<html> TRONG&gt; 监控细胞膜的变形与pTIRFM。 A）随着计算的P / S和P 2 S排出量原图像P和S发射图像显示。比例尺，3.2微米B）表达SYT-1 pHluorin一个嗜铬细胞去极化与氯化钾。一些明亮的荧光斑点（右图）表示个体DCV值的融合。比例尺，3.2微米。C）帧一帧图像的融合SYT-1 pHluorin DCV的。次（上图），以秒为单位。融合DCV前时间0指定的帧。相应的P / S和P 2 S排出量的图像也显示。 ，比例尺为1微米D）图中C。虚线图像是在时间0 -融合架E）的结果在C和D的一种可能的解释如下所示。<html>空白"&gt;请点击这里查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/51334/51334fig3.jpg" /> 图3变形是刺激诱 ​​发的Ca 2 +内流的结果。 A）转染GCaMP5G一个嗜铬细胞去极化与56毫米氯化钾。因此而增加的GCaMP5G荧光意味着增加subplasmalemmal的Ca 2 +水平。 乙 ）以上的图像帧接一帧的图像单元中A.时报30×20像素区域的以秒为单位。白色箭头指示的P / S的增加和P +2度S减小的区域。胞质GCaMP5G蛋白是由定影DCV排除的区域。比例尺，1微米。C）图中B所示的图像D）示出的结果中B和 C的一种可能的解释。3highres.jpg"目标="_blank"&gt;请点击这里查看该图的放大版本。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> iXon3 EMMCD相机</td><td>安道​​尔</td><td> 897 </td><td></td></tr><tr><td> IX81倒置显微镜</td><td>奥林巴斯</td><td></td><td>侧置式Filtercube大会</td></tr><tr><td> 43系列氩离子激光器</td><td> CVI米勒斯Griot公司激光光学</td><td> 543-AP-A01 </td><td>可调谐至488纳米</td></tr><tr><td>蓝宝石561 LP半导体激光</td><td>连贯</td><td></td><td> 561纳米</td></tr><tr><td>扫描振镜反射镜系统</td><td> Thorlabs公司</td><td> GVS102 </td><td></td></tr><tr><td>张3通道焦距灌注系统</td><td> ALA的科学仪器</td><td> ALA VC3X4PP </td><td></td></tr><tr><td> QMM石英MicroManifold </td><td> ALA的科学仪器</td><td> ALA QMM-4 </td> &lt;TD&gt; </td></tr><tr><td> 10 psi压力调节器</td><td> ALA的科学仪器</td><td> ALA PR10 </td><td></td></tr><tr><td>机械手</td><td>伯利</td><td> TS 5000-150 </td><td></td></tr><tr><td>安装消色差四分之一波片</td><td> Thorlabs公司</td><td> AQWP05M-600 </td><td></td></tr><tr><td> 420-680 nm的偏振分光棱镜</td><td> Thorlabs公司</td><td> PBS201 </td><td></td></tr><tr><td>六站中性密度轮</td><td> Thorlabs公司</td><td> FW1AND </td><td></td></tr><tr><td>步进电机驱动SmartShutter </td><td>萨特仪器</td><td> IQ25-1219 </td><td></td></tr><tr><td> HQ412lp二向色滤光片</td><td>浓度</td><td> NC255583 </td><td>加入488 nm和561 nm的光束</td></tr><tr><td>涂平凹透镜</td><td>爱特蒙特光学</td><td> PCV100毫米VIS 0 </td><td>发散488 nm的光束</td></tr> &lt;TR&gt; <td>涂平凹透镜</td><td>爱特蒙特光学</td><td> PCV250毫米VIS 0 </td><td>发散561 nm的光束</td></tr><tr><td>涂平凸透镜</td><td>爱特蒙特光学</td><td> PCX125毫米VIS 0 </td><td>着重两光束</td></tr><tr><td>涂平凸透镜</td><td>爱特蒙特光学</td><td> PCX50毫米VIS 0 </td><td>巩固过滤立方体组装</td></tr><tr><td> z488/561rpc分色</td><td>浓度</td><td> z488/561rpc </td><td> Filtercube分色</td></tr><tr><td> z488/561_TIRF排放过滤器</td><td>浓度</td><td> z488/561m_TIRF </td><td> Filtercube发射</td></tr><tr><td> UIS2 60X目的</td><td>奥林巴斯</td><td> UPLSAPO 60XO </td><td> NA 1.37 </td></tr><tr><td>霓虹灯转染系统</td><td> Invitrogen公司</td><td> MPK 5000 </td><td></td></tr><tr><td>的MetaMorph成像软件</td><td>分子器件</td><td></td><td></TD&gt; </tr><tr><td> DiI荧光染料膜</td><td> Invitrogen公司</td><td> V-22885 </td><td>对于对齐的目的</td></tr><tr><td> TH LIBERASE </td><td>罗氏</td><td> 5401135001 </td><td></td></tr><tr><td> TL Liberse </td><td>罗氏</td><td> 5401020001 </td><td></td></tr><tr><td>从牛的DNase I IV型</td><td>西格玛</td><td> D5025 </td><td></td></tr><tr><td>血球</td><td>费舍尔</td><td> 0267110 </td><td></td></tr><tr><td>做染料膜</td><td> Invitrogen公司</td><td> D-7757 </td><td></td></tr><tr><td>罗丹明</td><td> Invitrogen公司</td><td> R634 </td><td></td></tr><tr><td> 0.22μm的滤膜过滤注射器单位</td><td>密理博</td><td> SLGS033SS </td><td></td></tr><tr><td> Fluoresbrite多色红微球</td><td> Polysciences的</td><td> 19507 </td><td> 0.5微米</td></tr><tr><td>浸油</td&gt; <td>西格玛</td><td> 56822 </td><td></td></tr><tr><td> LabVIEW的</td><td>美国国家仪器公司</td><td></td><td>控制振镜</td></tr><tr><td>旋转烧瓶</td><td> BELLCO </td><td> 1965-00250 </td><td></td></tr></tbody></table> 表1中。 pTIRF显微镜设备。 <table><tbody><tr><td> 内容 </td><td> 准备-PSS </td><td> 基肥-PSS </td><td> STIM-PSS </td></tr><tr><td>氯化钠</td><td> 145毫米</td><td> 145毫米</td><td> 95毫米</td></tr><tr><td>氯化钾</td><td> 5.6毫米</td><td> 5.6毫米</td><td> 56毫米</td></tr><tr><td> 氯化镁 </td><td> - </td><td> 0.5毫米</td><td> 0.5毫米</td></tr><tr><td> 氯化钙 </td><td> - </td><td> 2.2毫米</td><td> 5毫米</td></tr><tr><td> HEPES </td><td> 15毫米</td><td> 15毫米</td><td> 15毫米</td></tr><tr><td> pH值</td><td> 7.4 </td><td> 7.4 </td><td> 7.4 </td></tr><tr><td>葡萄糖</td><td> 2.8毫米</td><td> 5.6毫米</td><td> 5.6毫米</td></tr><tr><td>青霉素 - 链霉素</td><td> 1X </td><td> - </td><td> - </td></tr></tbody></table> 表2。灌注和嗜铬细胞准备的解决方案。 <table><tbody><tr><td> 内容 </td><td> 电穿孔媒体 </td><td> 普通电镀媒体 </td><td> 2X抗生素媒体 </td></tr><tr><td> 1X DMEM/F-12 </td><td> 1毫升/板</td><td>为2ml /板</td><td> 1毫升/板</td></tr><tr><td> FBS </td><td> 10％ </td><td> 10％ </td><td> 10％ </td></tr><tr><td>胞嘧啶阿糖胞苷（CAF） </td><td> - </td><td>从10毫库存1微升/毫升</td><td> - </td></tr><tr><td>青霉素</td><td> - </td><td> 100单位/ ml </td&gt; <td> 200单位/ ml </td></tr><tr><td>链霉素</td><td> - </td><td> 100微克/毫升</td><td> 200微克/毫升</td></tr><tr><td>庆大霉素</td><td> - </td><td> 25微克/毫升</td><td> 50微克/毫升</td></tr></tbody></table> 表3。嗜铬细胞培养基。 <table><tbody><tr><td> 内容 </td><td> TH-PSS </td><td> TL-PSS </td></tr><tr><td> LIBERASE TH </td><td> 2毫升</td><td> - </td></tr><tr><td> LIBERASE TL </td><td> - </td><td> 0.85毫升</td></tr><tr><td>准备-PSS </td><td> 98.0毫升</td><td> 84.0毫升</td></tr><tr><td> DNA酶</td><td> 8.75毫克</td><td> 7.4毫克</td></tr></tbody></table> 表4。 TH-PSS和TL-PSS解决方案。

Watch this Scientific Journal Video about Imaging Plasma Membrane Deformations With pTIRFM at JoVE.com

Imaging Plasma Membrane Deformations With pTIRFM

基于偏振的全内反射荧光显微镜（pTIRFM）实现实时检测细胞膜动力学。本文介绍pTIRFM的膜重塑的过程中调节胞吐作用的研究落实。该技术推广到在细胞生物学中的其他进程直接或间接地涉及改变膜的形状。

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

imaging-plasma-membrane-deformations-with-ptirfm

Research

JoVE Journal

Biology

13.5K 次观看.  Wayne State University. 基于偏振的全内反射荧光显微镜（pTIRFM）实现实时检测细胞膜动力学。本文介绍pTIRFM的膜重塑的过程中调节胞吐作用的研究落实。该技术推广到在细胞生物学中的其他进程直接或间接地涉及改变膜的形状。 

视频: Imaging Plasma Membrane Deformations With pTIRFM

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

科研

生物学

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

测绘在细胞膜分子扩散，多目标跟踪（MTT）

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

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, ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

外周相关蛋白血浆膜分型的测定

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.