Name: fluorescence recovery after merging a droplet to measure the two-dimensional diffusion of a phospholipid monolayer
Uploaded: 2015-10-15T15:00:00.000+00:00
Duration: 7 min 54 s
Description: Watch this Scientific Journal Video about Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer at JoVE.com

这项工作是由韩国科学技术院资助的K-谷RED＆B通过韩国国家研究基金会项目于2014年和基础科学研究计划（NRF- 2012R1A6A3A040395，NRF-2013R1A1A2057708，NRF- 2012R1A1A1011023）的支持。

注意:请参阅材料安全数据表（MSDS）使用丙酮，氯仿之前，这是致癌物质。 1.准备磷脂单层膜在平水 - 气界面<ol><li>磷脂单层的形成<ol><li>的磷脂溶液的制备<ol><li>清洗4毫升，使用丙酮，乙醇和去离子水的至少三次的聚四氟乙烯包覆帽小瓶，并彻底吹氮气到小瓶除掉的水。 </li><li>溶解1毫克磷脂（例如，dioleoylphosphatidylcholine，DOPC），以1毫升氯仿中的小瓶，得到1mg / ml的浓度的影响。在通风橱中的安全执行此过程。使用更低或更高的磷脂溶液浓度如果必要的。 </li><li>添加染料标记的磷脂（例如，若丹明约82mol，若丹明DPPE）小于1摩尔％的磷脂SOLU的化，为了可视化磷脂单层用荧光显微镜。在通风橱中的安全执行此过程。 </li><li>包住小瓶用聚四氟乙烯胶带，以防止溶剂蒸发，并将样品在冰箱中储存于-20℃。 </li></ol></li><li>磷脂沉积到空气 - 水界面<ol><li>清洁培养皿（55毫米直径和12毫米高）用乙醇和去离子水的至少三次。 </li><li>填培养皿，用10毫升去离子水，以形成气 - 水界面。 </li><li>传播用微注射器的磷脂溶液的几微升到一个干净的界面，以实现所需的表面压力，并等待至少30分钟，使溶剂蒸发完全，做实验之前。 注意:朗缪尔槽可以用来代替培养皿如果表面压力的精确控制是必要的。 </li></ol></li></ol></li><li>苏rface压力测量<ol><li>测量磷脂单层用威廉米悬片张力的表面上的压力。等待至少30分钟，以获得滤纸润湿不够的，如果滤纸用作威廉米悬片。详细的协议是在库恩等人可用 。20。 </li><li>调整磷脂精确地控制表面压力的沉积量。加入4微升DOPC溶液（1毫克/毫升）的上30.25厘米2 -area界面如果面压力的约5达因/厘米是必需的。 注意:如果滤纸没有完全润湿，表面压力显着地在实验的最初30分钟，由于滤纸的重量变化而变化。 </li></ol></li><li>最小化该单层的对流流动的<ol><li>使用包含3毫米油藏两个薄频道的锥形装置中，连接到培养皿的一大段，以抑制的对流单层，其可干扰形态学成像和表面压力的测量。 </li><li>确保以匹配锥形装置的内部和外部之间的水位，以确保磷脂到空气 - 水界面磷脂沉积之前自由移动在整个区域。 </li></ol></li></ol> 2.准备磷脂单层膜在液滴的曲面<ol><li>使用微量拉马逐渐变细的玻璃毛细管的过程<ol><li>放置一个玻璃毛细管（外径1mm时，ID0.78毫米，长100mm），在微量拉马的持针器。 </li><li>设计一个程序用于拉动，毛细管具有适当的参数值（热:斜坡，拉:60，威赛:70，延迟:70及压力200）和拉毛细管具有根据制造商的协议的设计的程序。 注意:毛细管结束与微米直径几需要形成一个100微米液滴由申请颖以〜10kPa的压力。如果毛细管的前端太小，高得多的压力，&gt; 600千帕，需取得液滴，而这是难以控制液滴的大小与一个过大的毛细管前端。 </li></ol></li><li>磷脂吸收到液滴表面 注意:为了形成磷脂单层在液滴的弯曲界面，没有染料的磷脂溶液标记磷脂是用来获得的强度相反，向平坦单层，制备由步骤（1）此外，这两个过程2.2.1和2.2 .2允许在这里。如果在液滴表面的表面压力的精确控制是必要的，程序2.2.2强烈推荐。然而，如果不是，过程2.2.1，这是一种比步骤2.2.2更简单的方法，是有用的。 <ol><li>涂覆磷脂过程在前端<ol><li>清洁用丙酮，乙醇和去离子水的至少三次玻片。 </li><li> PL王牌在清洁的载玻片锥形毛细管，和倾斜毛细管以促进接触载玻片与前端。 </li><li>降的磷脂溶液（1mg / ml）的几个液滴的涂敷是用玻璃注射器附着于载玻片的毛细管的顶端，并等待至少30分钟后，完全蒸除溶剂。 注意:强烈建议过程2.2.1.3期间粘附毛细管在载玻片的末端，因为前端的周边太小只有通过毛细管力来保持磷脂在前端的液滴。 </li></ol></li><li>的囊泡溶液的制备<ol><li>清洁的小瓶，并从药瓶中除去水，如在过程1.1.1.1引入。 </li><li>通过施加氮气轻轻擦干一个2毫升体积的磷脂溶液（1毫克/毫升），并变干的小瓶1小时在室温下，以消除任何剩余的溶剂。在通风橱中的安全执行此过程。 </li><li>添加2毫升去离子水到小瓶含干燥的脂类，并孵育在烘箱小瓶在60℃下1小时。 </li><li>摇动小瓶几次，和超声（HF-频率:最多40千赫，功率:370瓦）30分钟，得到囊泡。 </li><li>执行挤压和冻融过程，以获得单分散单层囊泡。用于制备囊泡的详细协议在Mayer等人21进行说明。 注意:液滴界面的表面压力通过形成包含单分散单层囊泡液滴的后调整的等待时间精确地控制。使用悬滴法22，有必要根据时间来测量表面压力的变化，做实验之前。 </li></ol></li></ol></li><li>液滴的形成，它包含一个磷脂单层在弯曲表面<ol><li>在锥形毛细管填充用10微升去离子水从再修改的Ë2.2.1。另外，使用10微升的囊泡解决方案从程序2.2.2。 </li><li>毛细管连接到一个自动微喷射器，以提供压力，以形成液滴。 </li><li>安装，其连接到微注射器显微以精确地控制毛细管的位置的毛细血管。 </li><li>准备一个明场显微镜（显微镜1，物镜:10X NA 0.3）成像毛细管的侧面用CCD相机。显微镜1从而使得能够观察沿z轴微滴的精确位置，并估计液滴的大小。 </li><li>前端移动到一个位置，前端的横向视图由显微镜1采用显微良好可视化，并应用一个可变压力（约100百帕斯卡）与毛细管的前端，直到适当的尺寸（约100液滴的微米直径）形成。 注:建议自动微量进样器和微操作是使用研发，如果很好地控制液滴的液滴尺寸和位置是必要的。另外，也可以使用手动的。 </li></ol></li></ol> 3.成像荧光恢复滴合并后注:本协议的基本原则是相同的了FRAP技术，除了降合并过程。在A.洛佩斯等人 15和D·阿克塞尔罗德等人 16 FRAP提供的详细协议和相关理论。 <ol><li>监测和控制的液滴的位置<ol><li>准备一个倒置显微镜设置（显微镜2，物镜:10X NA 0.3，管镜头:焦距长17厘米），使两者在荧光显微镜与罗丹明DPPEs（激发波长560纳米，583纳米发射）适当的过滤器设置和明亮的视野显微镜。在这里使用的CCD照相机的可视化液滴的顶视图用荧光显微镜米颂歌和明亮的视野显微镜模式。 </li><li>动涂有磷脂到一个平面空气 - 水界面沿z轴的液滴，但不合并液滴的是，使用显微。用显微镜1形象化液滴的侧视图。 </li><li>定位液滴在平坦单层的顶视图的中心，使用显微。使用显微镜2的明视场显微镜模式可视化的平坦单层顶视图。 </li></ol></li><li>合并液滴到扁平空气 - 水界面<ol><li>进一步将液滴朝平口，直到滴合并到接口，使用显微。如果合并过程被成功完成后，具有圆形形状，其是由白色背景包围的暗区，是利用显微镜2的荧光观察模式。 </li><li>记录系列，根据时间的荧光图像的合并液滴之后，使用微指令的荧光模式应付2.使用速度更快的帧速率比这里单层的扩散时间尺度。在DOPC单层，它需要几分钟扩散到200微米暗区完全。 </li></ol></li></ol> 4.确定由图像分析的扩散系数注意:为了从一系列图像确定扩散系数，对图像分析定制程序如下所述构建。这一计划的详细源代码在朱庇特网站上提供。 <ol><li>检测感兴趣的圆形区域的<ol><li>检测感兴趣区域的中心的<ol><li>获得一系列在一个恢复过程，其中包含一个圆形暗区和一个白色背景已记录的荧光图像，并设置系列作为参考图像的第一图像。此处，R D是暗区中的参考图像的半径。 </li><li>回旋参考图像与白圈谁本身强度均匀的整个区域。使用命名为&#39;CONV2&#39;为卷积所示的定制的程序行124下面的源代码嵌入功能中，白色圆的半径比R D所略小。 </li><li>找到的位置，指示在卷积计算的最小值，并将此位置的参考图像中的感兴趣区域的中心。 </li></ol></li><li>确定感兴趣的区域的半径<ol><li>卷积参照图像用白色圆圈包含的中心位置，由一个过程4.1和均匀的强度超过整个区域来确定。此处，R S为白色圆的半径。使用迭代如"关于&#39;或&#39;，而&#39;与圆的方程式，使白色圆圈中所示的定制程序线102至109的源代码。 </li><li>卷积参考图像与另一白圈具有略微较大的半径（5％-10％），R L，比R 5。使用相同的方法与过程4.1.2.1使圆中所示的定制程序线113至120的源代码。 </li><li>获得的4.1.2.1和4.1.2.2两个卷积计算之间的差值。 </li><li>重复从R 5 = R D / 2至R S = 2R D中的上述程序，由与像素电平增加的R 5和R L两者。使包含从程序4.1.2.1全部源代码4.1.2.3重复迭代。 </li><li>求R S的半径，表示在两个卷积计算之间的值的最大差异。使用命名为"最大"找到半径嵌入函数指示在值最大差值，如图定制程序行148的源代码，以149.此R 5因此指示暗区中的参考图像中的半径。 </li></ol></LI&gt; </ol></li><li>分数强度的计算<ol><li>置，它包含一个中心位置和半径，由程序4.1作为感兴趣区域确定的圆周。 </li><li>根据时间计算感兴趣区域的平均强度的一系列荧光图像。卷积每个帧与所述感兴趣区域与由感兴趣区域的面积除以它来计算平均强度。细节是在定制的方案，这是在朱庇特网站上提供的源代码可用。 </li><li>计算分数强度，定义为下面的公式，其中F（t）是平均强度在圈内是感兴趣的区域，根据时间， 的 F i是在圈内的初始强度，和F o为强度一个白色背景。 F（T）=（F（T） - 的 F i）/（FØ - 的 F i）（公式1）。 </li></ol></li><li>装修的Fractional强度FRAP理论<ol><li>适合的小数强度与下面的方程，其中τ是一个特征扩散时间和I 0，I 1被修正的贝塞尔函数，使用拟合程序，并获得τ <img alt="公式（2）" src="/files/ftp_upload/53376/53376eq2.jpg" /> （式2）。 </li><li>获取基于该关系，τ扩散系数= 2/4 D，其中 D为扩散系数，a是暗区的半径。 注意:在装配过程中，也可以沿时间轴移的小数强度分布时恢复过程已经开始，记录的图像之前。 </li></ol></li></ol>

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S., Jacobson, K., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+diffusion+in+phospholipid+multibilayers+measured+by+fluorescence+recovery+after+photobleaching.">Lateral diffusion in phospholipid multibilayers measured by fluorescence recovery after photobleaching.</a> Biochemistry. 16 (17), 3836-3841 (1977).</li><li>Peters, R., Beck, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+diffusion+in+phospholipid+monolayers+measured+by+fluorescence+microphotolysis.">Translational diffusion in phospholipid monolayers measured by fluorescence microphotolysis.</a> Proc. Natl. Acad. Sci. (U. S. A.). 80 (23), 7183-7187 (1983).</li><li>Kuhn, H., Mobius, D., Bucher, H., Crawley, J. N., 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=.">.</a> Physical Methods of Chemistry. 1, Part 3B, 651-653 (1972).</li><li>Mayer, L. D., Hope, M. J., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicles+of+variable+sizes+produced+by+a+rapid+extrusion+procedure.">Vesicles of variable sizes produced by a rapid extrusion procedure.</a> Biochim. Biophys. Acta - Biomembr. 858 (1), 161-168 (1986).</li><li>Rotenberg, Y., Boruvka, L., Neumann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+surface+tension+and+contact+angle+from+the+shapes+of+axisymmetric+fluid+interfaces.">Determination of surface tension and contact angle from the shapes of axisymmetric fluid interfaces.</a> J. Colloid Interface Sci. 93 (5), 169-183 (1983).</li><li>Jeong, D. W., Kim, K., Lee, S., Choi, M. C., Choi, S. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+recovery+after+merging+a+surfactant-covered+droplet:+a+novel+technique+to+measure+the+diffusion+of+phospholipid+monolayers+at+fluid/fluid+interfaces.">Fluorescence recovery after merging a surfactant-covered droplet: a novel technique to measure the diffusion of phospholipid monolayers at fluid/fluid interfaces.</a> Langmuir. 30 (48), 14369-14374 (2014).</li></ol>

The authors declare no competing financial interest.

FRAM共享了很多与FRAP基本原则，尤其是用于测量荧光恢复漂白特定区域之后，但FRAM采用合并液滴到平面界面，以形成漂白区，而不是施加强光的处理。因此，该合并过程是在FRAM最重要的步骤，特别是，在深色染色在合并液滴后的平坦单层的形状决定的扩散率测量的精确度。具体而言，基于当前的方法中，我们只设置一个半径为R的圆作为感兴趣的区域来计算深色染色的平均强度，在过程4中所示的，如果有一个过分偏离圆形形状，这会扰乱的扩散系数的精确测量。因此，有必要获得一个圆形暗部，但是，非圆形状偶尔由于几个原因而形成的。首先，如果有存在于宠物一对流里盘，暗区域的形状变成沿流动方向拉长。正如在步骤1.3中，一个锥形装置有助于抑制对流流动，而这种伸长会被最小化。其次，如果毛细管的前端期间合并过程触摸平界面，暗区域形成有各种形状，因为毛细管端中断合并过程。通过获得的液滴是仅挂在毛细管的最末端，这一中断可以被防止。特别是，疏水化处理的毛细管有助于液滴坐在毛细管的最末端。因此，上面的故障排除方法和修改，使我们能够更精确地测量扩散性能。 因此，这口井troubleshot过程允许FRAM超过FRAP有几个显著优势。首先，FRAM需要简单的设备相比，FRAP。为FRAM，就足够了使用简单的荧光万分之一应付代替昂贵的共焦显微镜的高功率激光器，其波长必须与染料的吸收光谱相匹配。此外，有必要使用附加装置来调整大小到漂白区，在FRAP，而FRAM通过调节液滴的大小，或在液滴表面上的表面压力控制区域的大小容易。第二，有可能在FRAM使用各种种类的染料。 FRAP仅使用染料分子，其漂白工艺比扩散过程快得多。如果在漂白过程中发生的染料的扩散，这是很难准确地估计扩散性。 FRAM的，但是，并不需要一种用于漂白染料和这从而使得能够使用各种类型的荧光成像染料的。此外，FRAP需要额外的高功率激光器和滤波器设定来改变染料种类，而仅需要更换过滤器集在FRAM。最后，中，由于单层在液滴表面和平面接口可以独立形成，从而能够相互扩散的研究或通过合并A型单层到B型单层两个不同的脂质单层之间的混合，而FRAP只测量单层的自扩散。 尽管有这些优点，合并的液滴在一个平面空气 - 水界面的过程可以潜在地限制此技术由于以下几个问题，可能是令人关注的，如两个单层之间的面压力不匹配，合并过程的时间尺度，并增加了平坦单层合并液滴后的表面压力。这些问题，但是，不影响扩散测量显著的原因如下。第一，即使在两个不同的单分子膜的表面压力有很大的不同，一个均衡过程是在恢复过程中非常早的阶段完成。例如，如果吨他表面单层在液滴表面的压力比上述单层在平坦空气 - 水界面的更高，暗区扩大，直到表面压力达到平衡。由于这个过程是在10毫秒完成时，表面压力将它影响扩散过程显著之前进行平衡。其次，如果合并过程的时间尺度是足够快，它并不限制扩散测量的。幸运的是，一个典型的合并过程也在10毫秒完成。最后，即使多个合并液滴到平面接口不增加，因为槽的表面积的表面压力的远远大于液滴的表面面积大。根据槽和液滴，在协议中所述的尺寸，每一分子的面积通过合并的液滴增加小于0.015％。因此，表面压力由于每分子面积的变化的增加小于0.1％，这是negligiblË因为它比在表面压力的典型误差小得多，如通过威廉米悬片张力测量。 总之，我们引入了一个新方法，通过合并液滴单层至平坦界面来测量磷脂单层的横向扩散性。这种技术需要相对简单的设备，并还允许使用各种各样的染料物种。的FRAM因此可能适用于任何表面活性剂，包括磷脂，嵌段共聚物，蛋白质和纳米颗粒甚至在一个流体 - 流体界面扩散测量。此外，我们预计，FRAM提供一个研究相互扩散或两种不同的表面活性剂之间的混合新的途径。

<html>磷脂是细胞膜的主要成分和细胞器的膜，以及这些膜的层的流动性经常通过改变膜蛋白1的活性影响细胞功能- 3。例如，膜&#160;脂质稳态通过调节膜的流动性，这是由膜蛋白检测实现的，并且流动性的异常水平引起严重的疾病，如脂肪肝和胆汁淤积4。此外，磷脂单层在空气 - 肺泡流体界面的流动性在其功能具有重要意义。高流动性的肺表面活性物质的磷脂单层有利于吸入过程中扩散层，而低流动性使呼气5,6期间留在肺泡内的层。它估计磷脂层的流动性，以了解他们的角色是重要的。 _content"&gt;甲直接测量流动性的是粘度，和磷脂层的粘度已通过使用微米大小的胶体7，磁性针8,9，和磁性微按钮6,10,11测量。然而，这些技术被限制为相对刚性的薄膜，并且不能测量低粘性的薄膜。对于这种情况，扩散性将是一个替代量化磷脂层的流动性。几十年来，各种技术来测量磷脂层的扩散性能已经开发，如荧光猝灭法12，脉冲场梯度NMR 13，和荧光相关光谱（FCS）14，其中一个最有代表性的方法是荧光漂白恢复（FRAP）15,16后，由于测量过程和相关的简单性理论，一些研究磷脂层的扩散性能使用的FRAP已经执行<suP&gt; 17 - 19。然而，FRAP通常需要昂贵的共聚焦显微镜的设置用高功率激光。 在这里，我们提出了一个新的技术合并（FRAM）之后测量磷脂单层，称为荧光恢复的横向扩散。 FRAP和FRAM之间的关键区别在于，漂白步骤被替换为滴聚结。合并涉及的非荧光单层到荧光标记的平坦单层液滴留下一个圆形的黑色污渍上一个明亮的平板单层，从而建立与漂白工序相同的初始状态。然后，我们观察荧光恢复在深色染色用时间来衡量的横向扩散。由滴聚结这个新的"漂白"步骤提供显著优势FRAP:FRAM只需要一个荧光显微镜，而不是昂贵的共焦显微镜用高功率激光所必需的FRAP。一世加氮，FRAM具有广泛选择的荧光染料的，因为它不涉及漂白过程。最后，将两个不同的单层，液滴单层和平坦单层，可以独立地准备，从而允许相互扩散被测定，而FRAP仅测量单层的自扩散。 FRAM提供了多种从非常粘稠的材料扩散测量的近无粘性材料。原则上，FRAM可以测量10 6微米2 /秒，这与现有技术的最大扩散，当扩散出现在整个区域100微米100微米的时间期间用于拖放聚结过程（约10毫秒）。此外，慢扩散过程可以容易地测量，除非单层是固体。 FRAM可以只要适当荧光标记的分子可用于任何种类的表面活性材料。

<table><tbody><tr><td>Acetone</td><td>OCI corporation</td><td>Acetone 3.8L Extra Pure</td><td>Purity: &amp;ge; 99.5%, Please consult material safety data sheets (MSDS) before use.</td></tr><tr><td>Ethanol</td><td>OCI corporation</td><td>Ethanol 3.8L Extra Pure</td><td>Purity: &amp;ge; 94%, Please consult material safety data sheets (MSDS) before use.</td></tr><tr><td>Dioleoylphosphatidylcholine, DOPC</td><td>Avanti Polar Lipids</td><td>850375C</td><td>Please consult material safety data sheets (MSDS) before use. Chloroform is carcinogenic.</td></tr><tr><td>Chloroform</td><td>LiChrosolv</td><td>Chloroform ultrapure (A3633)</td><td>Purity: &amp;ge; 99.8%, Please consult material safety data sheets (MSDS) before use. Carcinogenic</td></tr><tr><td>Rhodamine DPPE</td><td>Avanti Polar Lipids</td><td>810158C, 810158P</td><td>Avoid direct light exposure to prevent photobleaching</td></tr><tr><td>Wilhelmy plate tensiometer</td><td>R&amp;amp;K ultrathin organic film technology</td><td>Wilhelmy tensiometer</td><td>http://www.rieglerkirstein.de/index.htm</td></tr><tr><td>Micropipette puller</td><td>Sutter instrument</td><td>P-1000</td><td></td></tr><tr><td>Micro-injector</td><td>Eppendorf</td><td>Femtojet</td><td></td></tr><tr><td>Micromanipulator</td><td>Eppendorf</td><td>Micromanipulator 5171</td><td></td></tr><tr><td>Microscope 1</td><td>Objective lens: Olympus</td><td>Objective lens: UPlanFl 10x</td><td>Objective lens: 10X NA 0.3</td></tr><tr><td>Microscope 2</td><td>Objective lens: Olympus, Tube lens: Thorlabs</td><td>Objective lens: UPlanFl 10x</td><td>Objective lens: 10X NA 0.3, tube lens: focal length 17 cm</td></tr><tr><td>CCD for Microscope 1</td><td>Jai</td><td>CV 950 camera</td><td></td></tr><tr><td>CCD for Microscope 2</td><td>Andor</td><td>iXon3 EMCCD</td><td></td></tr><tr><td>Filter set</td><td>Chroma technology</td><td>Catalog Set 49004: ET545, T565, ET605</td><td>Prepare suitable for dye molecules</td></tr><tr><td>Sonicator</td><td>DAIHAN Scientific</td><td>Wiseclean WUC-D06H</td><td></td></tr></tbody></table>

fluorescence recovery after merging a droplet to measure the two-dimensional diffusion of a phospholipid monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

随时间的恢复过程中合并涂覆有DOPC单层液滴到平整DOPC单层后获得的一系列荧光图像，如在图1中列出的DOPC单层在扁平空气-水界面中掺入低量的此若丹明DPPE，并因此使得能够可视化了明亮的色彩和新添加到平坦界面的暗部背景。还有，观察到在固定的表面压力为23达因/厘米一个恢复过程。根据时间分数强度的变化甲拟合方程1的示于图2。此配合的R 2值为0.999，并且该配合还甚至在较低或较高的表面压力效果很好。从该配合得到的DOPC单层的扩散系数为27.54微米2 /秒，在表面压力为23达因/厘米。 为了进一步验证了FRAM的，溶解氧的扩散系数PC和dipalmitoylphosphatidylcholine（DPPC）单层根据表面压力测量。 如图3，FRAM捕获DPPC单层扩散系数的迅速下降在〜9达因/厘米的表面压力的，其中的LC（液体冷凝） - LE（液体膨胀）相变发生10，和的值扩散系数也同意很好地与先前由Peters等19测量。此外，与表面压力的扩散系数的指数衰减中观察到DOPC单层，这种倾向是由Caruso等人的 12测量的 1几乎相同。 <img alt="图1" src="/files/ftp_upload/53376/53376fig1.jpg" /> 图1（A）的FRAM的示意图（合并后的荧光恢复）技术。作为唇的id单层涂布液滴合并到扁平空气 - 水界面，而不荧光的脂质单层脂质标记被插入平面型脂质单层。 （B）的荧光显微镜图像与时间DOPC单层的恢复过程中，在表面压力为23达因/厘米（比例尺= 100微米）。在DOPC单层的暗区是由在几分钟内扩散过程完全恢复。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53376/53376fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53376/53376fig2.jpg" /> 图2.分数强度与时间。黑色圆圈和灰色虚线表示从图像分析（程序4.1和4.2）和等式1的拟合获得的小数强度随时间的分数强度的值，在程序4.3所示，分别为。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53376/53376fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53376/53376fig3.jpg" /> DOPC（三角形）和DPPC（空方）单层的表面压力的函数图3.扩散系数。与亮灰色颜色，并与暗灰色的线表示先前报道Peters等的扩散系数的值。和Caruso等人，分别。这个数字已经被修改郑某等人。23。从。郑某等人的Langmuir，30（48），14369-14374授权转载。版权所有2014年美国化学学会。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53376/53376fig3large.jpg" target="_blank">请她</a>E要查看该图的放大版本。

Watch this Scientific Journal Video about Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer at JoVE.com

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

合并一个液滴后荧光恢复来衡量磷脂单层的二维扩散

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

fluorescence-recovery-after-merging-droplet-to-measure-two

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.0K 次观看.  KAIST. We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

视频: Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

科研

生物工程

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

生物化学

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

基于荧光的测量磷酸酯/磷酸二醇 4-磷酸盐膜之间的交换

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) of macromolecules, and can be applied to both in vitro and in vivo biological systems. Multi-fluorescence recovery after photobleaching is performed by photobleaching a region of interest within a fluorescent sample using an intense laser flash, then attenuating the beam and monitoring the fluorescence as still-fluorescent molecules from outside the region of interest diffuse in to replace the photobleached molecules. We will begin our demonstration by aligning the laser beam through the Pockels Cell (laser modulator) and along the optical path through the laser scan box and objective lens to the sample. For simplicity, we will use a sample of aqueous fluorescent dye. We will then determine the proper experimental parameters for our sample including, monitor and bleaching powers, bleach duration, bin widths (for photon counting), and fluorescence recovery time. Next, we will describe the procedure for taking recovery curves, a process that can be largely automated via LabVIEW (National Instruments, Austin, TX) for enhanced throughput. Finally, the diffusion coefficient is determined by fitting the recovery data to the appropriate mathematical model using a least-squares fitting algorithm, readily programmable using software such as MATLAB (The Mathworks, Natick, MA).

In this article we will describe the procedure for measuring diffusion coefficients using multi-photon fluorescence recovery after photobleaching. We will begin by aligning the laser along the optical path to the sample and determining the proper experimental parameters, then continue generating and finally fitting fluorescence recovery curves.

通过双光子荧光恢复漂白后测量扩散系数

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) ...

生物学

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

在支撑脂双层配体纳米簇阵列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

聚合物拥挤脂质膜上的单分子扩散和组装

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip:蛋白质 - 磷酸肌醇相互作用的无标记研究

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

由荧光相关光谱测量的一次淋巴细胞的细胞膜分子扩散

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

免疫与感染

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...