The development of this protocol was supported by grants from the European Research Council (Advanced Grant TOPAS to M.J.L.) and the Deutsche Forschungsgemeinschaft (Grants CA 1014/1-1 to D.C. and SFB487 to M.J.L.). T.S. was supported by the Alexander von Humboldt Foundation.

1，样品制备<ol><li>清洗盖玻片 注意:在通风橱中操作。 <ol><li>用干净的镊子把盖玻片（直径为24毫米）插入分隔各个盖玻片盖玻片持有人。 </li><li>把夹持器与盖玻片到烧杯中，并加入氯仿，直到盖玻片覆盖。用铝箔覆盖烧杯以减少在超声波仪浴1小时，在室温下蒸发，并超声处理。就拿盖玻片架从烧杯，让盖玻片干。 </li><li>重复步骤1.1.2用5M NaOH溶液代替氯仿。 </li><li>取盖玻片夹持器进入一个新的烧杯中，并洗涤3次，用蒸馏水。把清洁的盖玻片的玻璃细胞培养板中填充有100％的乙醇。 </li></ol></li><li>校准样品的制备<ol><li>在适当的溶剂中溶解的荧光染料。 </li><li>制备荧光染料测距1:10系列稀释下午1时至1纳米的过滤除菌（0.22微米）的水。 </li><li>取存储在100％乙醇清洗盖玻片和洗涤用过滤灭菌的水。现货20微升在一个单独的清洗盖玻片每个荧光染料稀释。让盖玻片下无菌罩干燥。保护盖玻片从光线和灰尘，直到使用。使用这些样本来估计单分子荧光的强度（参见步骤3）。 </li></ol></li><li>转染<ol><li>培养CHO细胞以1:1的Dulbecco改良的Eagle培养基/营养混合物F-12（DMEM/F12）补充有10％胎牛血清（FBS），100 U / ml青霉素和100微克/毫升链霉素，在37℃，在5％的CO 2 注:使用无酚红的媒体在整个实验中，以尽量减少自体荧光。 </li><li>取清洗盖玻片从100％乙醇溶液中，用无菌磷酸盐缓冲盐水（PBS）冲洗，然后放置1盖玻片到6孔细胞培养PL的各孔中吃了。 </li><li>胰蛋白酶消化，计数和种子的CHO细胞以3×10 5个细胞/孔在含有盖玻片的6孔细胞培养板的密度。让我们在孵化器，以达到大约细胞生长（37℃，5％CO 2）为24小时。 80％汇合时，这是最佳的细胞密度转染。 </li><li>对于每孔，稀2微克所需的质粒DNA（ 例如 。，SNAP-标记的β2 -肾上腺素受体 ​​）和6μL脂质体2000中含有500微升OPTIMEM媒体两个独立的管道。在室温下孵育5分钟。 </li><li>从步骤1.3.4结合的解决方案为一体管和组合，以获得转染混合物。在室温下孵育转染混合物20分钟。 </li><li>在潜伏期（1.3.5），取CHO细胞和预热（37℃）的PBS洗两次。取代的PBS用补充有10％FBS的无酚红的DMEM/F12培养基中以1毫升/孔，但没有抗生素。 </li><li>添加整个转染离子混合物（1ml）中从步骤1.3.5滴加到每个孔中，并轻轻摇动平板来回，以确保完全混合。 </li><li>孵育2〜4小时，在37℃，5％CO2，之后立即进行下一个步骤。 注意:这些转染条件进行了优化，以实现受体密度&lt;0.45粒/ 2微米，这是适用于单分子成像。调整可能使用不同的细胞，构造或试剂时是必需的。 </li></ol></li><li>蛋白标记<ol><li>稀释1微升的荧光团-BG原液在补充有10％FBS的取得1μM的终浓度1毫升DMEM/F12培养基。取转染细胞从孵化器和预热（37℃），PBS洗涤两次。更换PBS以1毫升1μM的荧光团-BG溶液并孵育20分钟，在37℃，5％CO 2的培养箱中。 </li><li>保温后，洗涤细胞3用补充有10％FBS的DMEM/F12培养基次，接着用5分钟温育，每次在37℃下取盖玻片（有标记的细胞），用钳子将其放入成像室。 </li><li>用300μl成像缓冲液洗两次。加入300微升新鲜成像缓冲区并立即着手成像（第2部分）。 </li></ol></li></ol> 2，图像采集 注:使用全内反射荧光（TIRF）显微镜，配有一个油浸高数值孔径的物镜（ 例如 ，100X magnification/1.46数值孔径），合适的激光器（例如，405纳米，488纳米，561纳米和645纳米二极管激光器），电子倍增电荷耦合器件（EMCCD）相机，孵化器和一个温度控制形象化单个荧光分子。 <ol><li>设置所需的显微镜参数， 即得 。，激光线，TIRF角度（此参数控制着穿透渐逝场的化深度），曝光时间，帧速率和每片10影像数。保持器/孵化器和温度控制总是避免温度漂移和结露。 </li><li>把一滴浸油对显微镜的100X目标。放置在成像腔室与标记的细胞到显微镜的试样保持器，并采用明场照明带来的细胞焦点。 </li><li>切换到全内反射荧光照明。保持激光功率尽可能地低，以允许搜索的期望的细胞，但在同一时间最小化的光漂白。 </li><li>选择所需的细胞和精细调整焦点。设置激光功率的电平，使单个荧光团的可视化。获取图像序列和原始图像序列文件保存为。TIFF。 </li></ol> 3，校准（在玻璃和单体/二聚体受体控制单个荧光基团） <ol><li>组装每个校准SAMPLe作为在成像室1.2中所述的方法制备。将每个样品在显微镜上，并选择含有分离良好的受限衍射斑点，漂白在单一步骤中注意样品:这些点代表单个分子的荧光染料。 </li><li>如步骤2中所述收购全内反射荧光图像序列重要提示 :相同的成像参数必须被用于所有的实验，包括那些进行校准。 </li><li>进行检测，并在4.1详细的跟踪分析 - 4.2。提取作为在4.2.6中描述的每个粒子的强度。从这些数据，计算平均值（μ）和单荧光团的强度的标准偏差（σ）。 </li><li> 可选:执行对转染的细胞与单体的细胞表面受体（ 如 CD86）的同样的分析，N末端 ​​具有标记的SNAP 10的任一个或两个拷贝和标记有荧光团-BG衍生物。福卢瓦特上述用于在玻璃上的单荧光团的步骤。如Calebiro，D. 等人描述的估算标记效率。10 </li></ol> 4，图像分析<ol><li>图像序列的准备<ol><li>使用图像处理软件（ 如。，ImageJ的 ），裁剪图像。 </li><li>除个别帧作为单独的。tiff图像在一个新的文件夹，显示每个图像帧号上。 </li><li>通过沿细胞轮廓绘制感兴趣区（ROI）的区域，并使用测量工具在ImageJ的或其他软件类似的工具测量单元面积。使用此值除以颗粒的总数在电影由单元表面面积的开头来计算粒子的密度。 </li></ol></li><li>粒子探测和跟踪 注:使用非商业软件，如U-13的轨道 ，在Matlab工作环境，自动检测并跟踪单个受体颗粒。 注:U 型轨道算法是基于多假设跟踪方法。这种方法通过构建成本矩阵，其中，在一帧给定的粒子对应于一个给定的粒子，在下一帧时，会出现，消失或合并/拆分与/从其他颗粒中的个体的概率被分配链路帧之间的颗粒。 ，全球最小的成本解决方案， 即得 。，一个具有最高概率，最终选定。这也使得跟踪暂时消失的粒子，引起荧光闪烁的典型现象。 U型轨道 （2.1.0）的最新版本具有促进这些分析的执行图形用户界面。 <ol><li>从MATLAB命令提示符下，键入"movieSelectorGUI"打开影片选择界面。按照说明创建一个新的电影数据库从以前保存的单独的图像开始（见4.1.2）。 </li><li>提供在像素大小为nm，以秒为单位的时间间隔，数值孔径，相机的位深度和荧光团的发射波长，所需颗粒探测和跟踪。保存电影资料库。 </li><li>从电影选择界面，运行分析，选择" 单粒子 "作为对象的类型。将出现一个新的窗口，可以定义用于粒子探测和跟踪的参数。开始使用默认的参数。后来，调整这些参数，如果检测和/或跟踪的质量不理想（ 如有些颗粒不检测或轨道是支离破碎） 可选:根据跟踪设置，勾选" 导出跟踪结果矩阵格式 "存储在一个单一的矩阵（字段称为"trackedFeaturedInfo的坐标和各颗粒的振幅"）。对于这些参数的详细说明，请参阅U型轨道文档。 </li><li>运行检测算法。该算法自动确定的位置和强度上的每个衍射限制斑（ 即 ，单一的受体/受体复合物）通过拟合二维高斯函数的标准偏差等于显微镜的周围局部强度最大值的点扩散函数的背景。然后，运行跟踪算法。存储分析的结果在一个。mat文件。 </li><li>使用"movieViewer"套路，包含在U型轨道包装，或类似的自定义的可视化的轨道，并检查检测和质量跟踪。 </li><li>打开。mat文件以查看所跟踪的粒子在每一帧的位置和幅度（ 即 。，强度）。在步骤4.2.4生成的数据被包含在tracksFinal AR的tracksCoordAmpCG字段射线和/或在trackedFeaturedInfo。从粒子的总数计算检测到的粒子密度通过在4.1.3测定细胞表面的面积除以该值。 </li><li> 可选:使用粒子坐标随着时间的推移（见4.2.6）来分析受体粒子的运动。计算用Matlab或类似软件的均方位移（MSD）和扩散系数（D）。每个粒子，每一个时间间隔（Δt）的考虑，使用下面的公式计算出的均方位移（MSD）: <img align="middle" alt="式（1）" fo:content-width="4in" src="/files/ftp_upload/51784/51784eq1.jpg" /> 其中Δt是在帧中的时间间隔，N是所分析的步骤数，x和y是粒子的x和y在由索引指示的帧坐标。使用MSD随时间曲线来评估一个给定的粒子的运动的类型:线性关系说明自由扩散（ 即 。，布朗运动），一个正曲率（ 即 。，曲线看起来像一个抛物线）提出定向运动，负曲率指示局限于运动14。的情况下自由地漫射颗粒，计算出每个粒子由以下方程拟合得到的MSD数据的扩散系数（D）: <img align="middle" alt="公式2" src="/files/ftp_upload/51784/51784eq2.jpg" /></li></ol></li><li>颗粒大小与高斯拟合方法计算 注意:一旦校准样品（单荧光团上的玻璃和/或荧光标记的单体受体）的强度分布是已知的，粒子强度的分布进行混合高斯拟合在图像序列的开始，以确定受体复合物的大小（ 即 ，，受体每个颗粒的数量）10。利用Matlab或s进行这些分析imilar软件。 <ol><li>通过平均粒子从第一帧中的强度到框架前的强度的第一个发生变化（在大多数情况下，由于光漂白的减少）计算每个粒子的强度。 </li><li>根据下面的公式进行混合高斯拟合: <img align="middle" alt="式（3）" fo:content-width="3in" src="/files/ftp_upload/51784/51784eq3.jpg" /> 其中，φ（i）为具有强度粒子I的频率，n是该组件数，αn是有助于向分量n的高度的参数，μ和σ的平均值和参考单个荧光强度的标准偏差（计算方法为在步骤3中所述） 注意 :确定吨他部件（正最大值 ）对每个图像顺序逐步增加N 最大 ，直到另外的组件的最大数量不不再产生在统计上更好的贴合，作为判断F检验。 </li><li> 可选:（ 例如 ，一个400帧序列的最后60帧）上获得对电影的最后一个画面的亮度分布进行混合高斯拟合。更换μ和σ与重复步骤4.3.2本拟合后得到的值，提供精致估计这些参数，和。 </li><li>计算混合高斯拟合的各组分的曲线（AUC）下的面积。计算不同的大小（即，单体，二聚体，三聚体等），通过由整个分布的AUC除以各成分的AUC值的受体粒子的相对丰度。 </li><li>可选的:从不同的小区和相应的颗粒密度（如在4.2.6中描述计算），以生成重复的地方具有不同大小颗粒的分布是相关与粒子密度10使用的数据。 </li></ol></li><li>计算粒度的步骤拟合方法 注:使用步骤拟合分析作为一种替代方法，以确定受体复合物10的大小。依据这种分析是单一荧光团的结果在其瞬间消失的光诱导损伤（光漂白） -包含n个荧光团从而颗粒预期逐步漂白剂产生的强度分布与n个步骤。 <ol><li>从U型轨道或类似检测/跟踪软件（参见4.2.6）生成的。mat文件提取每个粒子的强度分布。 </li><li>使用步骤拟合算法，如1提出文献。 10，计数的漂白步骤，为每个粒子的数目。 </li><li> 可选 :使用结果生成的分布呈现出不同大小的受体粒子的相对丰度和以前的混合高斯拟合的结果描述粒子密度关联起来（见4.3）。 </li></ol></li></ol>

<ol>
	<li>Pierce, K. L., Premont, R. T., Lefkowitz, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seven-transmembrane+receptors.">Seven-transmembrane receptors.</a> Nat Rev Mol Cell Biol. 3, 639-650 (2002).</li><li>Angers, S., Salahpour, A., Bouvier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerization:+an+emerging+concept+for+G+protein-coupled+receptor+ontogeny+and+function.">Dimerization: an emerging concept for G protein-coupled receptor ontogeny and function.</a> Annu Rev Pharmacol Toxicol. 42, 409-435 (2002).</li><li>Ferr&#233;, S., 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=Building+a+new+conceptual+framework+for+receptor+heteromers.">Building a new conceptual framework for receptor heteromers.</a> Nat Chem Biol. 5, 131-134 (2009).</li><li>Milligan, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=protein-coupled+receptor+hetero-dimerization:+contribution+to+pharmacology+and+function.">protein-coupled receptor hetero-dimerization: contribution to pharmacology and function.</a> Br J Pharmacol. 158, 5-14 (2009).</li><li>Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerization+in+GPCR+mobility+and+signaling.">Dimerization in GPCR mobility and signaling.</a> Curr Opin Pharmacol. 10, 53-58 (2010).</li><li>Ulbrich, M. H., Isacoff, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+counting+in+membrane-bound+proteins.">Subunit counting in membrane-bound proteins.</a> Nat Methods. 4, 319-321 (2007).</li><li>Triller, A., Choquet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+concepts+in+synaptic+biology+derived+from+single-molecule+imaging.">New concepts in synaptic biology derived from single-molecule imaging.</a> Neuron. 59, 359-374 (2008).</li><li>Hern, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+dissociation+of+M1+muscarinic+receptor+dimers+seen+by+total+internal+reflection+fluorescence+imaging+of+single+molecules.">Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules.</a> Proc Natl Acad Sci U S A. 107, 2693-2698 (2010).</li><li>Kasai, R. S., 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=Full+characterization+of+GPCR+monomer-dimer+dynamic+equilibrium+by+single+molecule+imaging.">Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging.</a> J Cell Biol. 192, 463-480 (2011).</li><li>Calebiro, D., 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=Single-molecule+analysis+of+fluorescently+labeled+G-protein-coupled+receptors+reveals+complexes+with+distinct+dynamics+and+organization.">Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization.</a> Proc Natl Acad Sci U S A. 110, 743-748 (2013).</li><li>Keppler, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+method+for+the+covalent+labeling+of+fusion+proteins+with+small+molecules+in+vivo.">A general method for the covalent labeling of fusion proteins with small molecules in vivo.</a> Nat Biotechnol. 21, 86-89 (2003).</li><li>Gautier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+engineered+protein+tag+for+multiprotein+labeling+in+living+cells.">An engineered protein tag for multiprotein labeling in living cells.</a> Chem Biol. 15, 128-136 (2008).</li><li>Jaqaman, K., 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=Robust+single-particle+tracking+in+live-cell+time-lapse+sequences.">Robust single-particle tracking in live-cell time-lapse sequences.</a> Nat Methods. 5, 695-702 (2008).</li><li>Saxton, M. J., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-particle+tracking:+applications+to+membrane+dynamics.">Single-particle tracking: applications to membrane dynamics.</a> Annu Rev Biophys Biomol Struct. 26, 373-399 (1997).</li></ol>

The authors declare that they have no competing financial interests.

所描述的协议允许的空间布置，流动性和细胞表面受体复合物的大小在单分子水平的分析。相比，使用荧光蛋白，具有小的有机荧光团，它们是明亮，更耐光的标签，具有允许单个受体粒子的扩展可视化的优点。由于非常低的表达水平得以实现（&lt;0.45受体粒子个/μm2），受体和其他膜蛋白的性质可以在密度不超过生理相关的分析。此外，受体刺激激动剂10或其它操作的影响，例如，旨在再现一种病理状况，可以进行分析。此外，由于标签的策略，SNAP / CLIP标签11,12的灵活性，不同的荧光团可根据自己的具体需求使用-值得注意的是，SNAP的结合和CLIP的标签可以被用来进行两色实验， 例如 ，来观察两个相互作用的蛋白之间的共定位。最后，这个协议可以在几个点通过使用不同的细胞，转染方法和标记策略修改，例如。 关键的步骤包括的背景和自发荧光的最小化（通过使用大量清洗盖玻片，无酚红的媒体，并过滤溶液）的转染条件下（转染后，例如 ，质粒DNA的量和时间等）的优化，以实现极低的表达水平，高效的标签和一个明亮，足够耐光荧光基团的选择。关于荧光团的选择，红/远红光的人通常会给出更好的结果，因为细胞的自发荧光是较高的，在可见光谱和以上550nm的通常几乎可以忽略不计的蓝色/绿色部分。特别要注意，以避免光漂白寻找一个合适的小区和重点调整过程中荧光团。样品点样在玻璃盖玻片上的荧光团，以及单体和二聚体受体的控制（ 例如 ，，CD86与一个或两个SNAP标签）10，应考虑到校准分析和检查标记效率。 这种方法的限制是很大程度上依赖于当前能够达到的时空分辨率。这主要是由从一个荧光团，以及由所使用的照相机的感光度和采集速度收集光子的数目所决定的。为一个单一的，检测到的粒子的定位精度的典型值是20 - 30纳米（用于比较的常规的荧光显微镜的空间分辨率为约200 - 300纳米）。这些值仍然比典型的膜蛋白质的真实尺寸较大（约2 - 8纳米）。因为受体的下降沿在低于微的衍射极限的距离范围（约200 - 300纳米）被检测为单个粒子，适当的控制和统计分析应使用从真实受体-受体相互作用8-10的数中减去随机colocalizations（误报）的最大采集速率实现的。当前EMCCD相机可以超过1千赫，至少在作物的模式（ 即 ，时，传感器中的一部分使用）。然而，采集速度还受到由该到达相机的单个荧光团发射的光子的数目。在实践中，至少有10的曝光时间 - 一般需要20毫秒。出于这个原因，典型的采集速度为10至50赫兹， 即发生变化。中，一帧，每20至100毫秒。在荧光设计，光学元件和检测技术未来的发展可能会允许以进一步提高单分子方法的时空分辨率。

位于细胞表面上的受体感知细胞外环境和各种刺激，如气味分子，离子，小神经递质和大蛋白激素的反应。细胞膜的流体性质允许受体和其他膜蛋白的移动。这是为蛋白质复合物和瞬时蛋白质 - 蛋白质相互作用，如那些用于通过受体组装成的功能单元和信号转导到细胞内发生的形成是必不可少的。例如，G-蛋白偶联受体（GPCR），它构成细胞表面受体1的最大家族，已经提出了以形成di-/oligomers，这似乎是参与信号转导的微调，调节和可能有重要的生理和药理后果2-5。 单分子显微镜具有高spatiotemp直接可视化的巨大潜力口服分辨率位于活细胞，包括其相关联的表面上的单个受体的动态行为，以形成二聚体和更高阶的分子复合物6-10。这比标准的生化和显微镜方法，通常报告的分子数千或数百万的平均行为提供了几个优点。 具有足够明亮，耐光荧光蛋白标记是必不可少的单分子显微镜。该协议需要在近期推出SNAP标签11的优势，以共价连接的小而明亮的有机荧光细胞表面受体。 SNAP是从人类的DNA修复酶的O 6 -烷基鸟嘌呤-DNA烷基转移酶，它可以不可逆地标记有荧光团标记的苄基鸟嘌呤（荧光团- BG）衍生物衍生的20 kD蛋白标记。 CLIP，从SNAP派生的进一步工程化标签，可以代替标记荧光基团-Conjugated苄基胞嘧啶衍生物12。 在报道这个手稿的协议说明如何转染和标签SNAP-11标记的受体与小的有机荧光，并使用全内反射荧光（TIRF）显微镜可视化单一受体或受体复合物的活细胞10的表面上。该报告协议结果在细胞外SNAP-标记的细胞表面蛋白10&gt; 90％标记效率。关于如何使用单分子数据来分析受体复合物的大小和流动性，以及捕捉瞬时受体 - 受体相互作用的进一步信息，提供。整个协议的示意流是在图1，作为一个例子，中国仓鼠卵巢（CHO）细胞，SNAP-标签的G-蛋白偶联受体（GPCRs）的转染，然后用荧光团-BG衍生物作为标记以及其应用的量化y和显示器受体di-/oligomerization描述。这个协议可以扩展到其它细胞表面蛋白和荧光标记（ 例如 ，，CLIP），以及其他的转染和标记方法。

<table><tbody><tr><td>Chloroform</td><td>AppliChem GmbH</td><td>A1585</td><td>CAUTION: toxic and irritating substance as well as a possible carcinogen</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S8045</td><td>CAUTION: strong base and highly corrosive reagent</td></tr><tr><td>Absolute ethanol</td><td>Sigma-Aldrich</td><td>32205</td><td></td></tr><tr><td>Glass coverslip&amp;nbsp;</td><td>Marienfeld-Superior</td><td>111640</td><td>24 mm diameter, 0.13-0.16 mm thickness</td></tr><tr><td>0.2 mm sterile filter</td><td>Sarstedt</td><td>83.1826.001</td><td></td></tr><tr><td>CHO cells</td><td>ATCC, USA</td><td>ATCC CCL-61</td><td>Chinese hamster ovary cell line</td></tr><tr><td>6-well cell culture plate</td><td>Nunc</td><td>140675</td><td></td></tr><tr><td>DMEM/F-12 medium</td><td>GIBCO, Life Technologies</td><td>11039-021</td><td>Phenol-red free medium</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Biochrom</td><td>S 0115</td><td></td></tr><tr><td>Penicillin - streptomycin&amp;nbsp;</td><td>Pan Biotech GmbH</td><td>P06-07 100</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Pan Biotech GmbH</td><td>P10-23100</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen, Life Technologies</td><td>11668-019</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Invitrogen, Life Technologies</td><td>31985-047</td><td></td></tr><tr><td>Fluorophore-conjugated benzylguanine&amp;nbsp;</td><td>New England BioLabs</td><td>S9136S</td><td>SNAP-Surface Alexa Fluor 647. Make a 1 mM stock solution in DMSO. Store at -20&amp;deg;C.</td></tr><tr><td>DMSO</td><td>AppliChem GmbH</td><td>A1584</td><td></td></tr><tr><td>Imaging buffer:</td><td></td><td></td><td>137 mM NaCl, 5.4 mM KCl, 2 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 10 mM HEPES, pH 7.3, sterile-filtered</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; NaCl</td><td>AppliChem GmbH</td><td>A1371</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; KCl</td><td>AppliChem GmbH</td><td>A3582</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>AppliChem GmbH</td><td>A2303</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>AppliChem GmbH</td><td>A3618</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; HEPES</td><td>AppliChem GmbH</td><td>A3724</td><td></td></tr><tr><td>Imaging chamber</td><td>Molecular Probes, Life Technologies</td><td>A-7816</td><td>Attofluor Cell Chamber, for microscopy</td></tr><tr><td>TIRF-M</td><td>Leica</td><td></td><td>Model: DMI6000B</td></tr><tr><td>TIRF objective</td><td>Leica</td><td>11 506 249&amp;nbsp;</td><td>HCX PL Apo 100x/1.46 Oil CORR&amp;nbsp;</td></tr><tr><td>EM-CCD camera&amp;nbsp;</td><td>Roper Scientific</td><td></td><td>Photometrics Cascade 512B</td></tr><tr><td>Temperature controller</td><td>Pecon</td><td></td><td>Tempcontrol 37-2 digital</td></tr><tr><td>ImageJ software</td><td>NIH, USA</td><td></td><td>http://rsbweb.nih.gov/ij</td></tr><tr><td>u-track software</td><td>Laboratory for computational cell biology, Dept. of Cell Biology, Harvard Medical School, USA</td><td></td><td>http://lccb.hms.harvard.edu/software.html</td></tr><tr><td>Matlab software</td><td>The MathWorks, USA</td><td></td><td></td></tr></tbody></table>

high-resolution spatiotemporal analysis of receptor dynamics by single-molecule fluorescence microscopy

单分子显微术正在成为一个强大的方法来分析信号分子，特别是关于那些方面（ 如 。，动力学，不同的国家和人群，短暂的相互作用共存），它们通常隐藏在合奏测量，如行为那些与标准生化或显微镜方法获得。因此，动态事件，如受体 - 受体相互作用，可以实时跟踪与高时空分辨率的活细胞。这个协议描述的基础上以小而明亮的有机荧光团和全内反射荧光（TIRF）显微镜直接可视化单一受体的活细胞的表面上的标签的方法。这种方法允许一个精确定位的受体，测定受体复合物的大小，并捕捉动态事件，如瞬时受体 - 受体相互作用。该协议提供了如何PE的详细说明rform单分子实验，包括样品制备，图像采集和图像分析。作为一个例子，这种方法的应用，分析2的G-蛋白偶联受体， 即 ，，β2 -肾上腺素能受体和γ-氨基丁酸B型（GABA B）受体，报道。该协议可以适于其他膜蛋白和不同的细胞模型，转染方法和标记策略。

所描述的协议可以被应用到各种不同的膜蛋白。作为一个例子，与β2 -肾上腺素能受体和GABA受体乙获得代表性结果列10。由于从单个分子的荧光信号弱，尽量减少背景荧光是第一关键步骤，成功的结果。因此，要广泛使用清洁的盖玻片（ 图2A），以及最大限度地减少样品自发荧光（ 例如 ，通过使用无酚红培养基）是很重要的。下一个步骤是确定单个荧光分子的荧光强度。这可以由成像单荧光点在一个干净的盖玻片（ 图2B）来实现。通常情况下，荧光团的系列稀释来选择用于分析的最佳条件， 即 ，，能产生良好的分离，并均匀分布的单荧光团浓度。其他续ROLS可以由成像控制单体蛋白来进行， 例如 ，标有荧光团-BG衍生物NAP-标记的CD86受体。一旦这些初步的控制和校准已经完成，实际的实验就可以开始。 图3A示出的小区的转染SNAP-标签的β2 -肾上腺素能受体和标记有荧光团-BG衍生物典型的全内反射荧光图像序列的第一帧。这些点代表单个受体或受体复合物。这个图像也显示出适当的颗粒密度为自动检测和跟踪-根据我们的经验，密度超过0.45个/μm2的结果较差跟踪质量，应该避免10 图3B示出应用到相同的检测算法的结果图像序列。每一个蓝色圆圈表示一个粒子检测。跟踪算法的结果列于图3C中 ，其中蓝色花键所代表的单个颗粒的运动轨迹。每个粒子的运动轨迹可以被用于计算其扩散系数。这种方法还允许捕获动态事件作为如图3D所示，其中2颗粒表面上发生短暂的相互作用， 图4示出了扩散系数的测定为两种不同的G蛋白偶联受体， 即分布。，β2 -肾上腺素能和GABA B受 。这种类型的分析使我们能够显示，γ-氨基丁酸乙受体的很大一部分是不动的或具有非常低的流动性10。混合高斯拟合，并加强拟合分析提供受体复合物的大小的精确定量活细胞的表面上（ 图5）上。这种分析还可以揭示复杂的分布， 例如 ，单体和二聚物的共存， 如图5A的示例。图5B和5C提供的颗粒漂白中的一个或两个步骤，并分别正确地分配它们作为单体和二聚体的受体，相应的步骤拟合分析的两个例子。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51784/51784fig1highres.jpg" width="500" /> 图1:工作流程一个典型的单分子实验，在这个协议的详细说明。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51784/51784fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51784/51784fig2highres.jpg" width="500" /> 图2。盖玻片清洗和准备校准样品（A）前，后广泛盖玻片清洁背景荧光的比较。盖玻片通过TIRF显微镜成像（B）荧光团-BG衍生物点在清洗盖玻片增加浓度的TIRF图像。比例尺:10微米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51784/51784fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51784/51784fig3highres.jpg" width="500" /> 图3:典型的单分子图像和探测/跟踪算法的结果。 （A）的 CHO细胞转染的SNAP-标签的β2 -肾上腺素能受体和标记有荧光团-BG衍生物是由全内反射荧光显微镜观察。示出的是所获取的图像序列的第一帧。比例尺:5微米（B）0;检测到颗粒上的原始图像的顶部表示由蓝色圆圈（C）从跟踪算法在相同的图像序列中的应用程序产生的轨迹显示在白色背景上。快照对应于帧中没有的情况。 35，绿色段，合并事件。红色部分，分裂事件。（四）从两个受体粒子获得的代表轨迹（蓝色和绿色，分别），经历一个明显的瞬时相互作用（红色）。两个粒子合并，将共同为几帧，并再次分裂。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51784/51784fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51784/51784fig4highres.jpg" width="500" /> 图4。分析受体MOBILI的TY。单个颗粒的运动轨迹来计算它们的扩散系数。在这个例子中，被报告计算两个不同GPCR的扩散系数的分布。 β2 -肾上腺素能受体的特征是在细胞表面上快速横向扩散，而GABA B受具有有限的移动性。从Calebiro，D. 等 10改性<img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51784/51784fig5highres.jpg" width="500" /> 图5。分析受体复合物的大小。 （A）受体复合物的大小可通过用混合高斯模型拟合粒子强度的分布进行精确估计。该报道的例子显示了这个分析，以表达SNAP标记β2-drenergic受体细胞的应用程序。拟合发现两架Components:1对应于单个荧光的强度（在很大程度上重叠到该校准样品以及样品的局部脱色后获得的分布，这里示出）和一个大约两倍的强度。两种组分可以被分别分配为单体和二聚体，以及两个分量下的区域可以被用来估计单体和二聚体受体的相对丰度。单体受体粒子的漂白在一个步骤（B）的实施例（C具有该特性的两步漂白二聚体受体粒子的）实施例。在（B）和（C）的红色线是步骤拟合算法的结果。这个数字是从Calebiro，D. 等修饰。10 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51784/51784fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy at JoVE.com

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

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., ...

high-resolution-spatiotemporal-analysis-receptor-dynamics-single

Nanyang Technological University

Research

JoVE Journal

Bioengineering

11.3K 次观看.  University of Würzburg, Germany. 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. 

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

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

科研

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 ...

生物工程

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

神经科学

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 ...

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

使用单分子FRET可视化膜受体的构象动力学

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

用荧光显微镜分析膜受体扩散和簇簇的图像处理协议

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...

免疫与感染

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

移动式单分子FRET探头的自动化二维时空分析

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

生物学

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. Under homeostatic conditions, microglia possess a highly ramified morphology. This facilitates their monitoring of the surrounding environment through the continuous extending and retracting of their processes. During brain injury and inflammation, however, microglia become activated and undergo dramatic morphological changes, retracting their ramified processes and swelling their cell body. This facilitates activities such as migration and phagocytosis, which microglia undertake to navigate the brain environment to a less pathological state.
This close relationship between microglial morphology and changes in their activity have enabled considerable insights into various microglial functions. However, such morphological and activity changes are themselves phenomena that can result from any number of intracellular signaling pathways. Moreover, the time-lag between stimulus and response, as well as the highly compartmentalized morphology of microglia, make it difficult to isolate the causative mechanisms that underpin function. To solve this problem, we developed a genetically modified mouse line in which a highly sensitive fluorescent Ca2+-indicator protein is specifically expressed in microglia.
After describing methods for in vivo microglial Ca2+ imaging, this paper presents a structured analysis approach that classifies this Ca2+ activity to rationally defined subcellular regions, thus ensuring that the spatial and temporal dimensions of the encoded information are meaningfully extracted. We believe that this approach will provide a detailed understanding of the intracellular signaling rules that govern the diverse array of microglial activities associated with both higher brain functions and pathological conditions.

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

In this paper, we describe a protocol for in vivo imaging of microglial Ca2+ activity and subsequent analysis of its spatiotemporal dynamics. This method enables thorough characterization of how microglia respond to changes in the brain environment, appropriately capturing the fine spatiotemporal scales at which such events occur.

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. ...