MA是由奥地利科学基金会（FWF，J3086-B11）和感谢的薛定谔奖学金马克斯 - 普朗克协会的财政和行政支持的支持。 GS是由维也纳科技基金（WWTF，LS13-030）的支持。 JH是由维也纳科技基金（WWTF，LS14-031）的支持。

1.生成和平面SLBs中的功能化<ol><li>混合脂质的<ol><li>以10％的DGS NTA镍/ 90％POPC脂质组合物到达在250毫升圆底烧瓶中溶解于氯仿45毫克POPC和6.9毫克DGS NTA-镍。保持氯仿低（只是几个毫升）的体积，以在下一步蒸发它。为1％的DGS NTA-镍/ 99％POPC脂质组合物使用49.5毫克POPC和0.69毫克DGS NTA-倪。 </li><li>除去使用旋转蒸发器慢慢氯仿 - 增加高于环境温度是没有必要的。或者，化学罩内吹飞在惰性气体流如氮气或氩气。而这样做不断转动烧瓶以允许在圆底烧瓶的下半部干燥脂质等于沉积。 </li><li>一旦大部分氯仿被蒸发，附着在烧瓶真空O / N定量除去氯仿。 注：这一步很关键，以避免污染蛋白质和细胞毒性氯仿在稍后阶段的，并确保脂质双层的流动性。 </li></ol></li><li>从干燥的脂质小单层囊泡（SUV的）代 注：小单层囊泡（SUV的）是20纳米和100纳米之间的直径，并且可以通过浴超声处理容易地制备。脂挤出，另一种方法，可以产生SUV和，根据施加于挤压过滤器的孔径，也大单层囊泡（的LUV），它是100纳米至1000纳米的尺寸。然而，越野车最适合用于形成SLBs中。 <ol><li>通过浴超声处理的SUV（在视频显示）： <ol><li> Degass PBS。暂停与250 ml的10毫升圆底烧瓶脱气的PBS将干燥的脂质混合物。 </li><li>填充惰性气体的烧瓶如氮气或氩气，用塞子关闭并密封与高压釜胶带烧瓶中。 </li><li>将密封的烧瓶成水浴超声和超声处理的脂质悬浮液一吨120至170瓦，直到它变成清楚。这需要30至60分钟。 </li><li>监测囊泡形成分光光度的进展：随之而来的相比，PBS稀释乳液的吸收234纳米保持不变（作为脂质浓度的近似指标），但应在550纳米显著下降（由于通过降低光散射较大的颗粒）。 </li><li>以沉淀重非单层囊泡，其与邻接的SLB的形成干扰，沉淀囊泡悬浮液通过离心，以150,000×g离心1小时，在25℃，然后进行8小时，在288000×g离心，4℃。 </li><li>通过注射滤器具有0.2μm的孔径过滤上清液。 </li><li>测量的OD在234纳米和550纳米监测囊泡制剂的清晰度和脂质左的量。 </li><li>存储囊泡在4℃在氩气或氮气。或者，使用囊泡停牌双层编制数月。 </li></ol></li><li>通过脂质挤压的SUV： <ol><li>简言之，通过过滤器传递脂质悬浮液几次为0.1微米的孔尺寸。无论制造方法的，离心囊泡悬浮液两次（1H，150000克，25℃;然后8H，4℃，288000克）以沉淀非单层囊泡其中较重。 注意：当在惰性气体储存在4℃下，囊泡可用于双层制备几个月。 </li></ol></li></ol></li><li>一个SLB的形成和蛋白质充电<ol><li>治疗24×50毫米＃1.5载玻片以3：1的混合物浓硫酸和30％的过氧化氢30分钟。 注：由于混合物的侵略性质特别注意，即穿白大褂和护目镜如图所示的视频！ </li><li>下冲洗流DDH 2 O的出喷瓶的载玻片上。将它们放置在聚四氟乙烯的立场，让他们干为10至30分钟。 </li><li>除去的8或16以及腔室的底部载玻片，填充环氧树脂胶的底部，并小心地放置在胶覆盖的腔室底部的干净和干燥的载玻片上。让硬化约10分钟的粘合剂。从底部取出多余的胶水。 注：载玻片和腔室之间的紧密密封，也可很容易地与使用的牙科双组分硅氧烷造型膏，在10至30分钟，通过使之硬化而实现。这种密封是不一样坚定环氧树脂胶，但经常承受身体劳损。实验后，可以很容易地从腔室，它是可重复使用的，然后在以后的实验中除去。 </li><li>吸管120微升（60μl）的10倍的PBS稀释的脂质悬浮液成的8（16）每个孔 - 孔室（如上所述制备），并让该双层形式20分钟。小心用15ml PBS漂洗双层两次。一旦双层已形成，它必须被浸没在缓冲液和永远不会被暴露在空气中。 </LI&gt; <li>填写每口井都用PBS的方式，然后脱下330μl（8孔室）或165μl（16孔室）。有350μl（175μl）留在井内。添加包含在PBS中稀释至每孔（400或200微升最终）His标签蛋白鸡尾酒的50μL（25μL），并在室温下孵育在黑暗中60分钟。表面蛋白密度可以通过在该温育步骤改变蛋白质浓度被调节。 </li><li>冲洗每个孔两次用15毫升PBS中。 注意：通常情况下，SLBs中应在实验中使用不超过6小时后不再其充电与蛋白质。在此期间，光漂白后的荧光团恢复仍然高达95％，并且可以检测出在双层相关蛋白没有损失。蛋白质密度应在实验前确定（见下文）。 </li><li>任选的步骤：为了检测含有SLB的蛋白质的DGS NTA-Ni表面的非特异性结合，用含有300mM咪唑的PBS洗涤一次的SLB。亲蛋白质应该完全关闭。 </li></ol></li></ol> 2.显微镜设置<ol><li>为建设一个能够检测单个荧光染料的荧光显微镜系统是指在介绍中所述的建议。 </li></ol> 3.功率测量注：荧光基团可以很容易饱和，过剩的激发光。这加速漂白没有进一步增益发射，因此应避免。为了优化取样照射的激发功率密度应直接在样品来测量。这种测量还可以作为今后的实验的参考。此外，评估其中光损失在成像系统中时知道的输入和输出的激光功率之间的比是有帮助的。 <ol><li>移动激励光束到直立位置，以便离开目标在90°角。 </li><li>放置在顶O功率计头f显示光束和测量光束（= P）的功率。文档中的结果。 </li><li>使用潜望镜，把光束进入TIRF地位和形象完整均匀的荧光双层。为了避免漂白和CCD信号饱和处的中性密度（ND）滤波器中的2至3的光密度（OD）为激发光的路径。 <ol><li>测量整个照明场的积分计数（= I总）。而且，测量的照射区域（= 共 ）的大小。测量集成计数（=我中心）将最大的照射中心光点以及光点的大小（= A 中心 ）。 </li></ol></li><li>测量每个像素的背景信号（= B）的任一的照明面积外或没有样品照明。 </li><li>减去从3测得的结果）的背景信号。通过与背景乘以质询（整个照亮区域或中央点）的区域的像素的数量做到这一点每个像素数。 （= I总-共.B和我中心-一个中心 .B）。 </li><li>除以综合和背景被照明的整个领域的集成和背景校正信号校正的中央点的信号。 注：结果表明位于中心点（=（我中心 -一个中心.B）/（I总-共.B））内的总功率的分数。乘这个结果与2）导致的光照亮中心点功率测量的功率P。 </li><li>确定地点在平方微米一个中心的规模 。对于这种划分为微米相机像素尺寸由物镜的倍率，取该结果作为每像素平方微米的平方。 <ol><li>备选地测量图像中的像素大小与使用千分尺滑放置在显微镜的，并采取其结果作为每个像素面积的平方。 MULTiply此值由位于中央点内的像素的数量，以在照射点区域到达。 </li></ol></li><li>除以功率P由中心斑点A 中心的规模照明的中心点，平方微米来计算每平方微米的光照强度。给出一个例子在图6中。 </li><li>在非TIRF光照下测得的功率密度值通常比存在的TIRF照明13，这也取决于具体角度的限制，激光的光被全反射更低。为了在功率密度下，全内反射荧光照明目前到货，图像校准直径为0.1μm无论是在非TIRF与TIRF的荧光珠。的珠的荧光强度中的TIRF和非TIRF模式中测得的比率达到转换因子 C，这使得转换测量的功率密度值，将那些在TIRF照明（方程1）有效。 功率密度 </eM&gt; TIRF = C•功率密度非TIRF（公式1） 与C =珠强度牛逼IRF /磁珠强度非TIRF </li></ol> 4.密度测量<ol><li>确定位于双层个别荧光团的平均信号。装有荧光肽双层连MHC分子的理想选择，因为蛋白质这一目的：标签化学计量是只有一个。如果需要的话漂白上的视场中的双层和所有标签让荧光恢复可视化单一荧光团。 <ol><li>快速连续地记录图像（如应用流采集协议），并在几个帧监控单个分子的流动性。欲了解更多信息，请参见图7。 </li></ol></li><l我&gt;标记双层投资回报率中的中央照明点。测量的ROI，在照明功率密度的方差（其正比于荧光强度）应理想地大于15％以下。 <ol><li>只有在这个投资回报率确定单分子和批量荧光。整合一个投资回报率，其足够大，以捕获单个发射器的点扩散函数的99％或更高的信号。当使用具有16-20微米的像素尺寸的照相机（在制造商的摄像头规格说明），用100倍的倍率及数值孔径等于或大于1.45更大宗旨，以7:3的区域由7像素与强度峰值位于广场中心。 </li><li>确定背景信号，整合相邻7由7像素的正方形，它不含有荧光信号的计数。减去从单分子的信号的背景，以确定单分子氟的修正值escence。只选信号，这仍然是在下一帧可见。 </li></ol></li><li>重复步骤4.2 50到几百倍。平均背景校正的信号。如果需要的话，画出单分子强度值作为直方图。可选：利用开源软件包适当的插件（如ImageJ的）或使用商业软件（如的Metamorph，Molecular Devices）中处理数据。有经验的用户可以写在Matlab或类似软件包自己的分析程序。 </li><li>放置2或更高的中性密度过滤器进入激发路径和采取包含标以相同的荧光团的蛋白质的荧光双层的图像。记录用于单分子强度测量相同ROI中的平均像素强度。记录散装荧光测量的曝光时间。测量无光照背景扣除的相同点。 </li><li>以确定该双内的蛋白质浓度层，由每平方微米像素（例如41.5），100（如果ND​​滤光器为2的OD受雇于）或1000的数目（如果乘在步骤4中确定的背景校正的散装荧光平均像素强度的ND过滤器为3的OD被使用）和用在步骤2中的曝光时间和通过在步骤3中确定的平均单分子信号除以它，使用的曝光时间，在步骤2和每蛋白的荧光团的数量。 </li></ol> 5.测试双层的完整性<ol><li>制备荧光双层并且如上所述将其放置在显微镜工作台上。 </li><li>调整焦距，并采取在TIRF模式双层的图像。申请一个短暂而强烈的漂白脉冲非TIRF模式消融在小圆盘状的投资回报率的荧光。 </li><li>漂白后立即在低强度的TIRF模式双层的图像，然后每五到十秒监测荧光恢复的速度。 </li><li>莫已经对双层的不同区域，并按照步骤2和3以另一图像中的低强度的TIRF模式漂白脉冲之后5分钟。确定强度I 张贴漂白在漂白区，并将其与现有的强度漂白余0处（无漂白应当发生）的试验开始计算动部分（IF）如下： 动的分数中频（％）=（I 0 -余后漂白剂）/（Ⅰ0 -背景）×100， 背景=没有激发光的ROI内测量强度应用 中频应小于10％（最好小于5％）。 </li></ol>

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作者宣称，他们有没有竞争经济利益。

单分子显微镜，为研究它们的天然细胞环境中的蛋白质的活动的独特机会。分子影像变得越来越多，因此向广大科学界有吸引力，但许多生命科学家仍然回避的技术和专业知识的初始投资。从组装自己的成像系统产生的主要好处是，它可以容易地调整到任何人的特定需要。 如本文中所提出的非TIRF激发束可以除了现有的TIRF光路径容易地实现，若荧光团和配体的快速和限定光致漂白（或-activation）是期望的，例如，在执行时FRAP或配体解笼锁实验14 。发射光束分离器的引入允许同时记录的至少两个且在一些情况下，多达四个荧光通道。扩展的列表是在本质上仅由自己的想象。 例如，实现在发射路径的机动发射滤光轮允许多达10种不同的荧光通道之间快速切换。当组合两个机动发射滤光片轮（各装有10过滤槽）串联，多达18个荧光通道能够被读出。全内反射荧光为基础的成像可以补充钙动员测量和一体化氙气或汞激发灯路径后，干扰反射显微镜（IRM）。 IRM是选择的方法来可视化，以使细胞附着在玻璃表面或一个SLB和解释的TIRF基于成像数据时可以容易地提供重要的信息的程度。建立的加宽激发光束与使用简单的分色镜和405的附加的激光波长可以进行光活化定位显微镜（PALM）或随机光学重构显微术（STORM），即2 superresolut离子的方法用下面的可见光15,16衍射极限的位置精度。 然而，实验的成功不仅是适当的硬件的问题。为了充分利用的噪声减少的TIRF基于成像，细胞应该接触到表面在不强加限制在细胞表面上或与它们的质膜的生理学干扰一种时尚。的SLBs官能适当的蛋白质为细胞粘附都超过适合于这个目的，因为所有的SLB包埋配位体是横向移动，并调整它们在双层内横向位置响应于受体结合和偏析动力学。 要在再现的方式制造SLBs中，最好是一开始就使用高纯度的越野车。如本文所述，它是由此临界去除多层囊泡，这些超声处理过程中也产生，两个超速离心步骤，因为这些我nterfere与邻接的SLBs具有高流动性的形成。只有在清洁玻璃表面的高质量形式的SLBs。一旦清洗玻片应立即使用或储存于真空。重要的是，必须的SLBs从未暴露于空气，因为这将导致其破坏。 SLB洗涤涉及，如所示，缓冲漂洗与使用血清吸管的，因为这不仅是一个节省，而且节省时间的过程。 在收获SLB驻留蛋白的纯化和应避免使用洗涤剂。这是因为，洗涤剂会显著减少即使当痕量存在的蛋白质的移动性。规避需要洗涤剂一起，分泌多组氨酸标签配备配体的可溶性表达，建议在哺乳动物或昆虫细胞。如果从大肠杆菌包涵体复性是必需的，必须谨慎施加有效地从包涵体之前，他们的非折叠和除去洗涤剂。 用从他们的模块化和reconstitutive性质，它允许专供解剖细胞活化和细胞粘附的给定受体 - 配体相互作用的作用的SLBs结果的一个主要优点。在这方面，要提到，DGS NTA镍应该存在于SLB在10％以内，以防止蛋白质竞争NTA-NI的结合位点是很重要的。当使用的SLBs窝藏仅1％或2％的DGS NTA镍，在一个给定的多组氨酸标记的蛋白质种类的关联的降低可以注意到，尤其是当共孵育增加的第二多组氨酸标记的蛋白质种类的量（未公开观察）。与SLBs的具有10％DGS NTA镍工作时，这种现象没有得到遵守。 虽然我们已经从未观察到的非特异性进行测试，以含DGS NTA-Ni表面的SLBs任何聚组氨酸标签的蛋白的结合，这种可能性应当首次引入蛋白质时进行测试，为此，我们建议使用SLBs的缺乏DGS NTA镍（蛋白质不应该结合）的。其次，使用含DGS NTA镍的SLB时，蛋白质应彻底清洗SLB含300毫米咪唑的PBS后脱落。 用人的SLBs的另一个显著优点是短暂的相互作用和信号转导事件可以具有增强的时空分辨率17-19进行监测。这至少部分是因为三维装订处理基本上减少到两个成像尺寸，在噪声衰减的TIRF模式尤其录制时。使用的SLBs的是用单分子检测信号，为光激活定位显微镜（PALM）或随机光学重构显微术（STORM）的先决条件，即超分辨率显微镜用低于衍射极限15,16的分辨率兼容。这些特殊成像方式使单分子福斯特共振能量茶nsfer实验设计成可视化在突触环境20个别蛋白质-蛋白质相互作用。这种方法相当详细说明在一个朱庇特的出版物被称为“测量TCR转的pMHC使用基于FRET的显微镜测定法的结合”21。 当解释SLB-基于实验的每个人都应该记住，活细胞的质膜不是所有的属性由SLBs中的特点，而一些缺少的素质可能影响被调查的生理。毕竟，SLB-嵌入式蛋白自由扩散，而不是组织膜微，因为大多数的细胞同行的是5,6,22。从贴壁细胞，其保存活细胞在一定程度的质膜架构来自固定质膜片，已经被成功地用于研究从膜包埋抗原由B淋巴细胞23的摄取。然而，即使这样的膜不与高度动态细胞骨架相互作用，设有没有灵活性，因为它们是由硬质玻璃表面支撑。鉴于这些差异的存在显然需要工程SLBs中，通过可调节的灵活性表面或通过改变它们的刚性应对局部应用光脉冲面都支持它提供手段来划分的蛋白质以定义的方式，哪些。

理解通过该膜蛋白履行其细胞功能的机制常常可以是一个具有挑战性的任务，因为它们的作用方式通常是由低亲合力的相互作用，这是难以检测和评估通过常规的生化方法学确定。膜蛋白可进一步高度富集特定膜结构域5,6或形成动态更高级结构，这原则上可以改变其生物活性的7。 因此，非侵入性的激光辅助单分子荧光显微镜已成为首选来研究复杂的细胞环境中的蛋白质行为的方法。这特别适用于发生在质膜的生化过程，因为这些原则上可以在噪声减少的全内反射（TIRF）模式进行监测。这里样品照射被限制在一个高达200纳米的薄切片在靠近一个玻璃苏rface，这导致在蜂窝背景显著损失，并且由于瞬逝激发光的特性，同样在荧光信号强度8,9-大幅增加。目标是在单分子检测的高位置和时间分辨率时，这两个方面都是重要的。 平面的SLBs不仅与TIRF显微镜兼容。当与适当的配体功能化，他们还提供了直接访问，因为它们发生在免疫细胞和其他细胞研究细胞 - 细胞识别的分子动力学。它们也可以简单地利用到位置足够接近载玻片能动否则非粘附细胞，使这些细胞可以在TIRF照明，这是很理想被成像时，涉及单分子跟踪或单分子基超分辨率传导实验。为此蛋白必须被装载到高移动SLB。所用李的先天优势<html> PID是有蛋白质没有非特异性结合的。此外，SLBs中可视为二维液体的愈合本身固有的能力;在玻璃支撑件内的凹凸被补偿到一定的程度。一个一步一步的协议是为了产生被控蛋白质的利益高度流动双层。平面的SLBs的形成进行说明，其中包含1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC）作为主要成分（90-99％）和合成的和官能化的脂质1,2- dioleoyl- SN -glycero -3 - {〔N-（5-氨基-1-羧基戊基）亚氨基二乙酸]琥珀酰基}镍盐（DGS NTA-Ni）的低丰度（1-10％）。 POPC携带一个饱和脂肪酸和一种单不饱和脂肪酸，并形成高流动性的脂质双层，即使在室温。 DGS NTA镍结合而其头部基团对聚组氨酸尾巴并且用作锚所选择的多组氨酸标签的蛋白（ 图1）。 “&gt;重要的是，显微硬件必须包括一些下面所述外围设备。所提供的信息，并在光学和激光物理的一些基本知识，任何生物学家应该能够建立一个单分子成像设置，样品激发应该理想地在全内反射（TIRF）模式的倒置显微镜进行作为该方案减少了杂散光并过度背景所得从蜂窝自发荧光9除外。对于这一点，一个特殊的TIRF能力的目标（见下文），并且需要激光照射。某些实验中，需要照明倍毫秒范围内，并且在快速连续操作。为了节省照明时间或避免因重复照明常常是有帮助的发射的光分成两个或更多个光谱通道不必要的漂白。这允许同时读出的两个或更多个发射分布，这可以成为当conduc一个显著因子婷实验涉及荧光共振能量转移（FRET）。在这里，从单一的激发光脉冲所产生的排放量既可以从FRET供体进行记录和FRET受体（如致敏发射）。该相机应该获得足够的光子具有足够低的背景在光照时间，以可视化的单荧光团。计算机软件将必须最多同步激发关停和图像采集的任务。全面概述了下方， 如图 2所示： TIRF能力的目标：对于目标为基础的TIRF照明物镜的数值孔径（NA）必须等于或使用标准载玻片大于1.4（N = 1.515）9。放大倍率范围60X之间可以以150倍。目标应该是色校正成像不同的荧光团时，允许共焦。 激光：可用化酶数- [R选项已经显著增加，在过去几年。现代电子调制的连续波二极管激光器是具有成本效益的，并且可以与前所未有频率和速度进行操作。更昂贵的气体离子激光器擅长在激光光束质量，但需要外部关停（见下文），并经常广泛（水）冷却。 激励百叶窗：声光调制器（AOM）允许快速关停快速连续10。用于紧密关闭与机械快门的AOM的组合建议。 AOM的因连续曝光这种方式广泛加热避免光从在关闭位置AOM的泄漏被抵消。 透镜被用于扩大激光束，并且将它们聚焦在物镜的后焦平面。这种方式，激发光离开目标作为一个平行光束（图3），这是需要的TIRF照明样品。移动后焦平面内的焦点从中心到物镜的周边将改变光束离开目标处的角度，但不是在检体中的激光点的位置（图3），这是一个功能整体光束形状。 TIRF照明接着发生在一个关键的角，其可以使用一组反射镜用作潜望镜翻译物镜的焦平面内的激光的聚焦点的调整。透镜应当色校正，并且可以在一组两个或三个透镜中使用。的三透镜系统包括两个透镜充当望远镜扩大激光束（并因此在试样照射点）和第三透镜到加宽光束聚焦到物镜（图4）的后焦面。这两种功能（望远镜和聚焦）也可以通过仅两个透镜的组合来实现（ 见图 4）。 “jove_content”&gt;镜子至少应为95％的反射，以避免激光光的损失。对于每个波束调整一组两个反射镜，通常为这样的布置施加足以精确地调节激光束的任何角度和位置。 分色叠加反射和透射光不同的特定波长的和被用于重叠或分开的两个激光器的光束。 Polychroic过滤器比二向色滤光片更加复杂，并且需要反映传入激发光传输离开发射光从标本。它们被置入物镜和显微镜镜筒透镜之间的滤波器立方体。 清理过滤器 ：根据激光雇工三维激光的类型清理过滤器具有窄传输带宽应放置在激光束离开激光之后。 陷波滤波器旨在有效地吸收激光具有窄带宽和传输所有其它光。它们被放置在排放路径中，以过滤掉任何杂散激光激发光。然而，陷波滤波器在0°入射角只工作。如果带宽的陷波滤波器的非常尖锐，在不同的角度进入的可能不会反映任何更多。阻挡的准直的激光的激光光没有受到影响，但背散射光可能不被有效地从到达照相机阻碍。 配备有适当的过滤器轮机动滤光轮可以容易地放置在激发和发射通路，并允许不同的光强度（当装有ND滤镜）或荧光通道（间简单的切换时，放置在一个氙或汞弧灯的比率的前钙成像或在摄像机前选择用于发射的荧光团）。 50:50立方体分束器Ç一个用于激光束分裂成两个单独的激发束的路径，并也将它们组合在潜望镜的前面。 光束分离器（发射路径）：对于无需任何物理过滤的变化快速图像获取，发射光束被分成一个蓝移和红移通道。原则上，分束器可以通过采用分色楔形或一组反射镜和二向色镜来分离发射光束中的波长依赖性方式来建立。需要两个发射滤光片清理发射频道。 空间滤波器：激发光束的空间滤波，有时需要除去从低质量的激光束发射的非平行光线。空间滤波器由双透镜望远镜的小针孔两个镜头的焦点准确地放置。从激光束的非平行部分所得这样的杂散光变得有效阻止。苏建立TIRF为基础的显微镜时，通道必须满足的条件。空间滤波随较小的针孔，这更难以定位到焦点，并与第一透镜的较小的焦距。以减少所造成的透镜像差的影响是有利的使用，而不是简单的镜片高质量和无限远校正显微镜物镜与低倍率（10倍或20倍）。 潜望镜：甲潜望镜的设置是必要的，以平移的目标，对于TIRF成像的先决条件的后焦平面内的聚焦激光束。它可以由两个两英寸镜，用于调整所述第一反射镜和后用于定位第二反射镜，以反映加宽和聚焦激发束到显微镜（图5）的平移阶段容易地建造。 摄像头：背照式电子倍增电荷耦合器件（EMCCD）常规用于的单分子信号的记录。这是因为它们的高量子效率（高达95％），高采集速度（高达30MHz）和相对低的噪声。在冷却至-80℃降低热噪声，并支撑由若干现有EMCCD相机。所述EMCCD技术的一个限制是，相机噪声增大线性地在相机增益和信号被捕获。这是不符合科学的CMOS（SCMOS）相机，它们是显著更便宜，操作因SCMOS芯片的特殊结构比EMCCD相机也快得多的情况。然而，随着CMOS技术相关联的问题是，图像采集仍然缺少一定程度上的单分子水平的定量读数的，因为每个象素设有不同检测灵敏度。在主要这可以通过像素正常化来补偿，但该过程是决不琐碎11。这就是为什么我们还是义无反顾地重新赞扬SCMOS相机为单分子显微镜，但鉴于这项技术的飞速发展，SCMOS相机可能很快成为首选的摄像头。慢扫描CCD相机摄像机避​​免放大相关的噪音和像素方差在一起，支持他们是否可以在一个所谓的“运动”模式下运行最快的采集速率。在这种模式下，除了感兴趣区域（ROI）的区域中的整个摄像机芯片被屏蔽，这使得它能够在芯片本身采用作为存储设备。后的ROI被暴露首次，所得电荷移入芯片像素线通过像素线，其中该图像被从进一步曝光保护的掩蔽区域。一旦变速ROI的所有行的进掩模区完成时（在亚毫秒范围内），将ROI本身是准备进行下一次的曝光。该循环被重复，直到CCD芯片的所有像素线被充电。该芯片然后读出缓慢显着热度CED读出噪声。例如1000×1300像素的芯片上，20 ROI的50×50像素可以被记录在快速连续。因为图像残留的芯片为一相当长的时间上之前被读出的，重要的是，以确保高质量的掩蔽和冷却相机（例如用液氮），为减少过多的热噪声的装置。一些EMCCD相机还支持动态模式。 软件：激光器，百叶窗，声光调制器和相机曝光时间以及合适的图像存储是不可或缺的任何成功的成像实验。在主营许多定义的操作可以与可用的软件包，来与摄像机进行编程。商业软件包支持的硬件外围设备，它可以与小技术知识来实现​​大量。 脉冲发生器，数据采集（DAQ）板（带模拟和数字输出通道）和示波器：脉冲发生器是很好的选择，以触发脉冲转换成规定的时间和电压脉冲。这样的激光器可以被精确地控制输出功率，并定时在毫秒到亚毫秒范围。数据采集​​板带模拟输出达到相同的，而且很容易通过PCI插槽，集成到计算机主板。脉冲持续时间，幅度和频率用示波器验证。 整个激发光束路径的外壳：为了避免由于空气对流整个激发光束路径应该从它的实验室环境括波动激发轮廓。导电TIRF显微镜当这一措施是特别重要的。光学组件也防止灰尘和激光照射人眼。外壳可以从黑卡板，它可以在艺术用品商店购买方便地构建。 要确定SLB荧光恢复Photobleac后的流动性兴（FRAP）测量12被执行。为FRAP两个激发光束路径的存在是可取的（ 见图 3）。第一光束路径被设计成图像的双层的荧光。这可以在TIRF配置和低的光强度来实现。第二束路径应允许一个短但强烈漂白脉冲，并应在非TIRF模式来配置，使得它离开物镜沿着光轴。圆形孔可以放入激发光束路径（ 见图 8）为将具有限定边缘的完美的圆形漂白轮廓。为了这个图象孔径到物体面，其最佳位置将是在透镜3的焦平面（ 见图 3）。然而，由于该透镜的长焦距，也可以产生足够的质量孔径图像，当光圈置于略微偏移的位置。 其执行T中之后他成像实验的原始数据必须进行适当的分析。几个步骤一步协议提供，覆盖的主要设置（例如照明的功率和时间，TIRF角）的调整，获取和分析数据。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>#1.5 glass slides</td>
			<td>VWR</td>
			<td>631-0853</td>
			<td></td>
		</tr>
		<tr>
			<td>250ml round bottom flask</td>
			<td>VWR</td>
			<td>201-1357</td>
			<td></td>
		</tr>
		<tr>
			<td>50:50 beam splitter cubes</td>
			<td>Thorlabs</td>
			<td>BS013</td>
			<td></td>
		</tr>
		<tr>
			<td>Acousto-opitcal modulator C1205-2</td>
			<td>Isomet</td>
			<td>-</td>
			<td>only necessary if CW lasers, which cannot be modulated, are used</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-NHS</td>
			<td>Life technologies</td>
			<td>A-20000</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555-NHS</td>
			<td>Life technologies</td>
			<td>A-20009</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-NHS</td>
			<td>Life technologies</td>
			<td>A-20006</td>
			<td></td>
		</tr>
		<tr>
			<td>autoclave tape</td>
			<td>VWR</td>
			<td>489-1312</td>
			<td>or any other heat-stable sticky tabe</td>
		</tr>
		<tr>
			<td>Avanti Mini-Extruder</td>
			<td>Avanti Lipids</td>
			<td>610000</td>
			<td>alternative to the bath sonicator</td>
		</tr>
		<tr>
			<td>bath sonicator QSONICA Q700</td>
			<td>QSONICA</td>
			<td>Q700</td>
			<td></td>
		</tr>
		<tr>
			<td>beam splitter</td>
			<td>Cairn</td>
			<td>P280/210/MLS</td>
			<td></td>
		</tr>
		<tr>
			<td>B&#252;chi rotary evaporator</td>
			<td>VWR</td>
			<td>531-0837</td>
			<td></td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Andor</td>
			<td>iXon Ultra 897</td>
			<td>see manuscript text for alternatives</td>
		</tr>
		<tr>
			<td>concentarted hydrogenperoxide</td>
			<td>Roth</td>
			<td>9683.1</td>
			<td></td>
		</tr>
		<tr>
			<td>concentrated sulfuric acid</td>
			<td>Roth</td>
			<td>X944.2</td>
			<td></td>
		</tr>
		<tr>
			<td>epoxy glue</td>
			<td>Uhu</td>
			<td>45705</td>
			<td>Uhu plus sofortfest 2min</td>
		</tr>
		<tr>
			<td>filter wheel</td>
			<td>Sutter instruments</td>
			<td>Lambda 10-2</td>
			<td></td>
		</tr>
		<tr>
			<td>LabTek chambers</td>
			<td>VWR</td>
			<td>734-2062</td>
			<td></td>
		</tr>
		<tr>
			<td>laser&#160;</td>
			<td>Toptica</td>
			<td>-</td>
			<td>different variants (wavelengths 375 - 785 nm) are available</td>
		</tr>
		<tr>
			<td>lens</td>
			<td>Thorlabs, Newport</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>DGS NTA-Ni (lipid)</td>
			<td>Avanti Lipids</td>
			<td>790404C</td>
			<td>already in Choroform-solved delivered version of the nicekl salt variant is recommended</td>
		</tr>
		<tr>
			<td>POPC (lipid)</td>
			<td>Avanti Lipids</td>
			<td>850457C</td>
			<td>already in Choroform-solved delivered version is recommended</td>
		</tr>
		<tr>
			<td>microscope Zeiss Axiovert 200</td>
			<td>Zeiss</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>mirrors</td>
			<td>Thorlabs</td>
			<td>BB1-E02</td>
			<td></td>
		</tr>
		<tr>
			<td>optical filters</td>
			<td>AHF, Chroma, Semrock</td>
			<td>-</td>
			<td>filter sets are available for many different combinations of dyes</td>
		</tr>
		<tr>
			<td>oscilloscope</td>
			<td>BK Precision&#160;</td>
			<td>2120C</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline (PBS)</td>
			<td>Life technologies</td>
			<td>14190-136</td>
			<td>degas before usage</td>
		</tr>
		<tr>
			<td>periscope</td>
			<td>Thorlabs</td>
			<td>RS99/M</td>
			<td></td>
		</tr>
		<tr>
			<td>picodent twinsil 22</td>
			<td>Picodent</td>
			<td>13001000</td>
			<td>alternative to the epoxy glue</td>
		</tr>
		<tr>
			<td>power meter Lasermate-Q</td>
			<td>Coherent</td>
			<td>-</td>
			<td>any powermeter sensitive in the used spectral range will do</td>
		</tr>
		<tr>
			<td>pulse generator</td>
			<td>Stanford Research Systems</td>
			<td>SRS DG535&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>spatial filter</td>
			<td>Thorlabs</td>
			<td>KT310/M</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe filter 0.2&#181;m</td>
			<td>Millipore</td>
			<td>GVWP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>TIRF-capable apochromat objective 100x, NA 1.46</td>
			<td>Zeiss</td>
			<td>440782-9800-000</td>
			<td></td>
		</tr>
		<tr>
			<td>trichloromethane/chloroform</td>
			<td>Roth</td>
			<td>3313.1</td>
			<td></td>
		</tr>
		<tr>
			<td>tubes forultracentrifugation polycarbonate 1.00 mm, 11 mm x 32 mm&#160;</td>
			<td>Thermo Fisher</td>
			<td>45237</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall ultracentrifuge&#160;</td>
			<td>Thermo Fisher</td>
			<td>RC M150GX</td>
			<td></td>
		</tr>
		<tr>
			<td>ultracentrifuge rotor</td>
			<td>Thermo Fisher</td>
			<td>S120-AT2</td>
			<td></td>
		</tr>
		<tr>
			<td>UV spectrophotometer Nanodrop 2000c</td>
			<td>Thermo Fisher</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>vacuum pump</td>
			<td>VWR</td>
			<td>181-0248</td>
			<td></td>
		</tr>
	</tbody>
</table>

single molecule fluorescence microscopy on planar supported bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

单分子荧光显微镜系统的结构相当详细在图2中所概述。个别零件，如光学元件和其他硬件组件在引言中解释。光激发光束路径，这会引起以限定的方式来TIRF和非TIRF照明，示出并在图3至5进行说明。请注意，在第一潜望镜镜定位在前方的50:50分束器的位置千分尺移动工作台（图5）。这个光束分离器叠加用于成像的TIRF光束（以绿色表示）和非TIRF束（以红色表示）用于漂白或本地孔径定义的光介导的样品活化（如图3所述）。将合并的光路（图5，橙色表示）经由第二潜望镜镜反射成倒MICR的背面端口oscope需要。 如在图4中解释和在图2中所概述的，两个或三个凸透镜的实施扩大了的激光束轮廓和聚焦激光束到物镜的后焦平面带来两个准直光束照射在TIRF样品和，分别在非TIRF。 所需的计算上的检体的功率密度测量强度值的例子示于图6中。从被照明和中央区域（用于成像）测得的强度中减去平均背景信号，在一个区域内的被确定场来看，这是不被激光束（ 此处7089计数）照亮。被照明（1684计数）的背景校正的平均强度和中央区域（4830计数），然后由相应的区域大小相乘，以产生积分强度（1.471×10 8个计数用于照明面积和3.424×10 7的计数为中央区 ）。中央和照明区域内的积分强度的比例产生的总功率照射该中心区域（0.23或23％）的级分。如图中电影，整体功​​率，必须在与使用中的非TIRF照明模式功率计的目标来确定。在我们的例子中它被调整到5毫瓦，从而引起的5毫瓦x 0.23 = 1.15毫瓦的功率的中心区域内。自41.5像素量在我们的例子中，以1微米2，中央区域具有7089个像素/41.5像素×微米2 = 170.8μm2以下的尺寸。由该面积（170.8微米2）除以照射中心区域（1.15毫瓦 ）的光的功率产生一个平均功率的0.7千瓦/厘米2的中心区域内的密度。 图7提供的图像和快速连续获得单荧光团的相应的强度的结果（每秒100帧，即带有10毫秒的时间帧）。为强度定量7的矩形区域的由7（= 49）个像素，其覆盖荧光信号的平均信号，被确定。此外，背景的平均信号的七个矩形区域内通过在单分子信号的接近七个像素来确定。背景校正的平均信号乘以像素的区域（= 49）内的编号，以产生单分子信号（标记为粗体）。 代表性的FRAP实验旨在评估SLB-相关的荧光标记的蛋白的迁移率示于图8。注意在图8A中的 repeti略去使用加宽低强度成像光束和单次使用第二个更狭窄的孔径定义的高强度的光束快速荧光消融在零秒时间点。扣除背景的黄色圆圈，已归事先于漂白脉冲的初始平均强度值内平均强度， 绘制在图8B对记录的速度和程度的荧光已经恢复的时间。如图所示，初始双层强度的90％以上（由绿线表示）已在75秒以下的荧光消融回收。其结果是嵌在所示的SLB蛋白质的小于10％是不可移动。 <img alt="图1" src="/files/ftp_upload/53158/53158fig1.jpg" /> 图一SLB系统1.纲要。 （A）的 SLBs中制成POPC（90-99％）和合成脂质DGS NTA-N中的I（1-10％）。它们自发形成时干净的玻璃表面被充电以越野车，包括相应的脂类。（B）中一旦形成，这些的SLBs可以容易地装饰有扩展要么与一个C-或含12个组氨酸（12H N-末端标记，为可溶性蛋白例如，共刺激分子B7-1和粘附分子ICAM-1）或蛋白二聚体扩展包含两个标签6个组氨酸每个（2x6H，例如αβMHCII类二聚体）。 <a href="https://www.jove.com/files/ftp_upload/53158/53158fig1large.jpg" target="_blank">请点击此处查看本一个更大的版本数字。</a> <img alt="图2" src="/files/ftp_upload/53158/53158fig2.jpg" /> 图2的激发和发射光束路径概述。气体离子激光器（如氩离子或氪+）可以被用于快速关停与使用声光莫操作<html> dulators。激光二极管在许多时下的波长可与代表一个很好的选择，因为他们可以通过电子调节，在微秒的愤怒。注意，激光束能够容易地调整两个（分色）镜和分割，结合使用简单束分离器来产生两个或更多的光束路径。在这个例子中的TIRF成像光束（监视事件）和漂白束（要么漂白荧光团或光激活中的孔定义的方式笼分子）被使用。既激发路径可以彼此独立地进行操作，通过使用附加的百叶窗。潜望镜允许用于平移物镜的后焦平面内的聚焦激光束，以产生样品的TIRF照明。荧光图像可以频谱之前采集分裂与使用的发射分束器与使用超灵敏相机（例如EM-CCD或科学CMOS摄像头）的。.COM /文件/ ftp_upload / 53158 / 53158fig2large.jpg“目标=”_空白“&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53158/53158fig3.jpg" /> 图3.通过物镜的后焦平面内平移所述聚焦束调节的TIRF照明。作为所示的激光束的焦点是通过光束的易位偏移从中心到周边的物镜的后焦平面内（使用潜望镜镜布置）。其结果是一个平行光束离开目标在倾斜照明直到一个临界角处达到其全内反射发生的情况。渐逝场是在哪里发生全反射玻璃，媒体接口生成的。 <a href="https://www.jove.com/files/ftp_upload/53158/53158fig3large.jpg" target="_blank">请点击此处查看该网络的放大版本</a><html>古尔。 <img alt="图4" src="/files/ftp_upload/53158/53158fig4.jpg" /> 图4.简单的光学系统，以将激光束聚焦到物镜的后焦面。需要三个或两个透镜以加宽的激光束，并将其集中在TIRF物镜的后焦平面。两个透镜系统包含除三透镜系统更少的透镜相关的错误，并可能更适合用于产生TIRF成像光束。目标是定义梁加宽和照射点边缘锋利，当三个镜头可能是可取的，因为，将需要研究采用光激活或-bleaching。 <a href="https://www.jove.com/files/ftp_upload/53158/53158fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> /53158fig5.jpg“/&gt; 图5.注释在荧光显微镜的背面的光束路径的照片。正如已在图2中的一个手杖概述容易实现两个激发束，一个在TIRF（以绿色表示），另一个在非TIRF模式（在所示红色）。这些成为覆盖有使用50:50分束器（BS）的。凸透镜（L1和L2）被定位到相应的光束将它们聚焦在物镜的后焦平面（未示出）。甲潜望镜由两个反射镜（M1和M2）通过进入显微镜的端口引导两个光束。所述第一反射镜（M1），可以通过使用一个平移段（TS）的移动（通过转动千分尺螺钉所示）移动至束分成TIRF位置中的一个。一旦TIRF已经建立，所述第二光束（用于光漂白或-activati​​on，这里显示为红色）可以独立于TIRF光束调节至离开目标非TIRF模式。 <a href="https://www.jove.com/files/ftp_upload/53158/53158fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53158/53158fig6.jpg" /> 图6.测量照明斑的中心区域内的功率。随着被指示选择感兴趣适当的区域，这反映背景，整个照明区域（IA）和中央区域（CA），其中事件将稍后被记录。减去那些记录，对IA和CA背景值，并通过与各区域（=积分强度）内本像素的数目乘以校正平均强度整合的强度。划分CA的积分强度由该IA的对在光束功率照射该CA的比例到达（在这个例子中：23％）。通过乘以总束功率（这里确定照射在CA的光的功率：5米W）的比例照亮的CA（此处：0.23）。为了确定中心区域的大小相乘的区域（这里：7089像素）与像素与面积的尺寸转换系数（此处为：1 / 41.5微米2的像素-1）。要到达激发光照射CA的平均功率密度，划分功率（这里：1.15毫瓦）由CA的大小。（此处：170.8微米2） <a href="https://www.jove.com/files/ftp_upload/53158/53158fig6large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53158/53158fig7.jpg" /> 图7.定量的单分子信号。（A）单个荧光团上SLB的时间过程。感兴趣区域（ROI，黄色）A 7×7象素尺寸的区域被放置在信号周围定量。背景从相邻7×7像素确定投资回报率（绿色）。（二）量化的平均像素强度列。为了确定单个分子的信号，背景值（绿色ROI）中减去从信号值（黄色ROI），并乘以49（C）一种荧光单分子的强度是对时间作图。请注意，省略了90毫秒的时间点作为荧光团在漂白过程中。 <a href="https://www.jove.com/files/ftp_upload/53158/53158fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/53158/53158fig8.jpg" /> 图8.荧光漂白恢复（FRAP）分析，以确定SLB的完整性后。 （A）FRAP是在一个双层承载IEK / MCC-的Alexa Fluor 647实验的每10名图像显示进行。（B）FRAP所示的实验定量（A）。值表示漂白前平均强度（I）中（A）所示的黄色ROI中由初始强度（I O）划分。红色的数据点显示在（A），绿线代表0.9恢复原来的强度。 <a href="https://www.jove.com/files/ftp_upload/53158/53158fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Single Molecule Fluorescence Microscopy on Planar Supported Bilayers at JoVE.com

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

single-molecule-fluorescence-microscopy-on-planar-supported-bilayers

Research

JoVE Journal

Bioengineering

13.8K 次观看.  Vienna University of Technology. Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution. 

视频: Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

警队复用单分子蛋白水解测量磁性镊子

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

科研

生物工程

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用：细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

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 ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

生物膜荧光探针力：受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

无标记单分子检测使用微型环光学谐振器

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

定量荧光显微镜为基础的单脂体测定，用于检测单个脂质体之间的成分不均匀性

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...