Name: studying the stoichiometry of epidermal growth factor receptor in intact cells using correlative microscopy
Uploaded: 2015-09-11T15:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy at JoVE.com

We thank E. Arzt for his support through INM, M. Koch for help with the ESEM, and DENS Solutions for providing the microchips. Research in part supported by the Leibniz Competition 2013.

1.准备标签和固定试剂<ol><li>准备使用高压灭菌的磷酸盐缓冲盐水标记缓冲液（BSA-GEL-PBS），pH值7.4（PBS）中。补充用1％BSA（牛血清白蛋白组分V，无生物素）和0.2％明胶（GEL，从冷水性鱼类的皮肤）和涡/摇匀。 注:可存储为〜5日于4℃。 </li><li>从高压灭菌磷酸盐缓冲盐水，pH 7.4中制备洗涤缓冲液（BSA-PBS）中。补充用1％BSA和涡/摇匀。注:可存储为〜5日于4℃。 </li><li>制备的50mM硼酸盐缓冲液（BB）从200mM的硼酸原液，将pH调节至8.3用200mM四硼酸钠。注:可在4℃下存放〜12个月。 </li><li>从0.2M的二甲胂酸钠储备溶液制备的0.1M二甲胂酸盐缓冲液（CB），将pH调节至7.4，添加了0.1M的蔗糖。 注意:打开原液可保存〜5日于4℃。 </li><li>制备0.1M甘氨酸在PBS（GLY-PBS）中。 注:可存储为〜2个月的，在4℃。 </li><li>从16％的PFA新鲜的或打开药瓶，原液，EM级准备在CB 3％多聚甲醛（3％PFA）。 注意:打开原液可保存〜5日于4℃。 </li><li>准备2％戊二醛的CB（2％GA）从新鲜的（或打开小瓶）25％GA原液，EM级。 注意:打开原液可保存〜5日于4℃。 </li><li>准备300nM的表皮生长因子生物素标记的解决方案<ol><li>稀6微米的EGF生物素原液中的BSA-GEL-PBS中，使用每个微芯片100-200微升的体积。使用在几个小时。 </li></ol></li><li>准备10纳米STR-QD标签解决方案<ol><li>离心链霉亲和QD原液4分钟在8000 XG，取上清液，在BB稀释1:10。不同类型的量子点可以被使用，例如，QD655，或QD800。稀释在BSA-GEL-PBS终浓度，使用每个微芯片75-100微升。使用内几个小时。 </li></ol></li></ol> 2.准备氮化硅薄膜微芯片<ol><li>清洁的显微镜载玻片，作为用于光学显微镜，用干净的房间组织和高效液相色谱（HPLC）级乙醇。 </li><li>在层流工作台具有低尘水平，打开一个直径100毫米的培养皿中，并奠定了清洗显微镜玻璃放入盘中;离开盘开。 </li><li>用清洁的镊子，优选涂覆有碳纤维或其它涂层，以4-10微芯片与薄（为50nm）的氮化硅膜，一前一后，他们的收纳箱和到载玻片上。抓住芯片轻轻地在身体两侧，避免接触脆弱的上侧，由薄氮化硅膜。请特别注意始终保持膜的一面朝上，不要去碰它。 注:该芯片应该是矩形，并具有平坦的边缘垂直于它们的氮化硅覆盖面，使他们能够容易地抓握。 </li><li>关闭培养皿的盖子，并把它带到通风橱。得到一个新的洁净室的组织和它存放在培养皿旁边。通过填充一个用50-70毫升丙酮，和其他与乙醇类似体积，既HPLC级的准备两个干净的200毫升玻璃烧杯中。传送从载玻片上制成的微芯片的第一到洁净室的组织。 </li><li>从组织，通过一个用于去除保护抗蚀剂层转移的微芯片1进入第一烧杯用丙酮。等待2分钟，同时轻轻移动烧杯几次，以确保微芯片以及漂洗。 </li><li>转移微芯片直接与乙醇的烧杯中，没有让他们干出来的。等待2分钟。然后将烧杯转移到层流工作台中。得到一个新的洁净室组织。 </li><li>转移到芯片的组织，让他们干几分钟。传送微芯片在玻璃上滑动，在培养皿，关闭盘，并把它的等离子清洗机。 </li><li>存放于微芯片到等离子体清洁玻片。运行5分钟的清洗程序来呈现氮化硅膜的亲水性的表面上。 注:施加于我们的等离子体清洁器的合适的设置是:70毫乇，11.5 SCCM的O 2和35.0 SCCM Ar原子，50瓦向前射频（RF）指标，5瓦的RF范围和最大气流。反射的RF。 </li><li>把玻璃载片与微芯片放回陪替氏培养皿，并关闭它的盖子。请注意，以保持无菌微芯片从现在开始。 </li><li>检查光学显微镜为潜在的膜破裂，或剩余的灰尘颗粒在微芯片的窗口区域。 </li><li>把培养皿于层流工作台中。 </li><li>放置以下项中的层流工作台中:含有灭菌试管/瓶0.01％聚-L-赖氨酸溶液（锁相环，分子量为150,000-300,000，无菌过滤，和细胞培养测试），高效液相色谱法级水，Dulbecco氏改良的Eagle培养基（DMEM），补充有10％胎牛血清，DMEM无血清，一个新的洁净室的组织，无菌24孔平板（12孔板也可使用），无菌96孔板，无菌枪头，移液管，而扁平，圆形末端，镊子。 </li><li>取24孔板，并填写一个孔与PLL和两口井用HPLC级水，使用约。体积每孔1ml。取96孔板，并填写每一个微芯片两口井与血清补充DMEM，一个很好用无血清培养基，使用约。体积每孔加入200μl。 </li><li>从现在开始，使用镊子，用无菌技巧来处理芯片。实现这一目标，例如，通过简单地燃烧，随后冷静的镊子的技巧。 </li><li>首先从载玻片微芯片转移到洁净室的组织。 从组织，转移多达六个微芯片到PLL填充井，孵育微芯片5分钟。 不E:对于一个实验，用更多的微芯片，补口井的24孔板。 </li><li>接着冲洗微芯片放置它们，一个接一个，进入第一充水井，并从那里进入第二水填充井。不要将微芯片在水中时间超过一分钟左右，以避免拆除PLL的。 </li><li>最后单独地将它们放在96孔板的DMEM培养基填充孔中。存储所述96孔板在CO 2培养箱中直至细胞悬浮液制备。 </li></ol> 3.编写微芯片上的细胞<ol><li>执行COS7细胞的传代培养，以制备细胞悬浮液以约的浓度。 5×10 5细胞/ ml。确保细胞是单分散。 </li><li>取96孔平板与微芯片和添加细胞悬浮液的一个液滴到每个微芯片。等待5分钟。 </li><li>开始研究微芯片的窗口区域倒置显微镜卓OSE正确的时刻用于其转移至一个新的井用无血清DMEM。 注:细胞的一个很好的密度是每2500 平方微米面积约一个单元实现的，对应于150×400微米大小的窗口24 COS7细胞。更高的细胞密度有阻碍细胞的最大平坦化的风险。请注意，经过小区已安顿下来到SIN膜，它需要3-5分钟，以产生足够的附着力抵抗转移到新井过程中的浮动关闭。 </li><li>微芯片传送到下一个井当足够的细胞已粘附到该窗口。保持微芯片总是与细胞朝上，避免本方的任何动人。 </li><li>转让完成后，再次检查每个微芯片的窗口。如果有太多的细胞被洗掉和剩余的细胞的数量不足时，再磨碎的细胞悬浮液（分手新形成的细胞块），并重复步骤3.1-3.5。 </li><li>当所有的传输芯片有足够的在他们的窗口区域粘附的细胞，转移的芯片到最后一个孔用无血清的DMEM。将 96孔板放回的 CO 2培养箱中让细胞恢复和变平为几个小时，最好O / N。 </li></ol> 4. EGFR标签，固定和荧光显微镜<ol><li>用于以下步骤中，使用96孔和24孔板中。预填孔与各自的解决方案，并使用一个行或列每微芯片。在24孔板中的通风橱中的固定步骤，CB，PFA和GA下使用。始终传输芯片与细胞朝上，避免本方的任何动人。 </li><li>通过将微芯片与新鲜的BSA-GEL-PBS中的井冲洗微芯片，然后将96孔板在培养箱中在37℃下5分钟。 </li><li>孵育3分钟，在37℃为300nM的EGF生物素溶液。因为细胞就会开始EGF标记EGFRs的内吞，请尽快到STEp 4.4。 </li><li>冲洗3次，用PBS，和1时间带的CB（现为96孔板再次是在RT）。 </li><li>孵育在3％PFA中10分钟。 </li><li>冲洗芯片1时间CB和3次用PBS。 </li><li>孵育在GLY-PBS中进行2分钟，漂洗2次，1次PBS洗涤。 </li><li>孵育在10nM的STR-QD 10分钟。 </li><li>冲洗4次用BSA-PBS中。 </li><li>淹没在玻璃底35mm培养皿预先填充BSA-PBS在RT的微芯片。 </li><li>获得QD标记的细胞的微分干涉对比（DIC）图像和荧光图像。 <ol><li>使用40X空中目标的细胞标记QD655的概览图片。使用油浸物镜（例如，63X），以允许所发射的荧光的光的最有效的收集;这被成像标记的细胞QD800时尤其需要。 </li><li>使用暴露该量子点，以340和380纳米之间的荧光激发光，并且允许发射的光的集合以上420nm处专门的滤波器立方体表最量子点（在这种情况下，过滤器的立方体A）。 </li><li>最大限度地减少光的强度，以避免过于强烈的光线可能会损坏样品。曝光时间设置为至少300毫秒，使所有量子点的信号被捕获，并指出，量子点的荧光有闪烁现象。 </li><li>调整光线强度，放大提供明亮的图像而看不到图像的噪声，同时最大限度地降低光线强度。 注意:可替换地，如果图像系列是从相同区域采取较短曝光时间都可以使用。这些系列可以加工以产生最大的投影图像，从而导致平均，从而降低噪音的产生的图像的。 </li></ol></li><li>将成像的微芯片背面（细胞朝上）到96孔板填充有BSA-PBS的孔中。 </li><li>微芯片冲洗1次CB。 </li><li>孵育在2％GA 10分钟。 </li><li>冲洗1次在CB和3次用BSA-PBS中。 </li><li>存储芯片，直到扫描电镜我maging，在BSA-PBS预填充新的96孔板的孔中。对于每一个微芯片填补了排四井用HPLC级水。 注:样品可以保持在4℃下进行数周，但是，最好的结果是当在两天内进行的完整的成像序列来实现的。量子点就会慢慢开始从样品中分离，故无建议等待10天以上的扫描电镜成像之前完成。 注意:荧光图像被存储以供将来分析。将细胞中的图像的位置可以很容易地用电子显微镜图像通过定位在SiN窗口的角并测量相对于该角部的位置进行相关。 注:这是最好打印出高质量的荧光图像和记录的扫描电镜图像时使用它们用于定向的目的。 </li></ol> 5.湿法ESEM-STEM全细胞的<ol><li>准备扫描电镜湿干<ol><li>挂载气体二次电子ðetector（GSED）和珀耳帖阶段，将含有干检测器（位于样品下）。 </li><li>开始通过级冷却水的流动，​​并且将温度设置为3℃。 </li><li>设置了阶段的xy坐标到零，并调整工作距离到约6毫米。 </li></ol></li><li>在ESEM成像，保持96孔板与微芯片冷却，例如在冷冻的冷却元件，在一个聚苯乙烯泡沫塑料箱。 </li><li>用于装载样品放入预冷ESEM-STEM阶段，取96孔板从冷却容器，并将其放置靠近打开的ESEM阶段。将一个新的洁净室组织在它旁边。 </li><li>通过浸四次，每次约1秒钟，成井充满了HPLC级水冲洗快速的微芯片（面朝细胞）。 </li><li>印迹在洁净室组织的微芯片简要的背面，并将微芯片放入预冷的阶段。确保该细胞保持湿，这可以是由一个反射面承认（在一个沉闷的表面干燥结果）。 </li><li>湿润样品表面3微升冷却HPLC级水（一定不要用枪头触摸表面），并固定在舞台的样本。 </li><li>放置三个附加3微升水滴在舞台上靠近样品。 </li><li>关闭样品室</li><li>通过循环的压力在800至1500帕五次吹扫样品室。结束与800帕的循环。 </li><li>切换的电子束（30千伏）上，并检查下的最低倍率与两个检测器， 即 GSED和STEM检测器的样品要搜索的微芯片的窗口的位置。 注意:如果水层太厚，窗口可能不可见。在这种情况下，开始降低压力至760 Pa和等待几分钟。窗口第一变得与GSED可见。 </li><li>通过移动SPECIM定位在图像的中心在SiN膜窗口烯阶段和降低压力至750-720帕。 注意:当水层足够薄时，窗口也变得与干检测器可见。 </li><li>从现在开始，使用STEM探测器的暗场段来记录影像。首先，收购一个或两个图像显示在整个窗口区域中的所有单元格。 </li><li>与光显微镜图像比较这些ESEM图像，定位在SiN窗口的角。 </li><li>关联通过比较倍率，旋转，和可能的镜像的坐标都显微镜模式的帧。在找到两个图像相同的细胞。 </li><li>选择非常小的灰尘颗粒或细胞区域具有小的特征来调整成像设置，如eucentric高度，精聚焦和透镜散光的校正。在至少50.000X的倍率工作，使用电子探针电流约为0.6 nA的，30微秒的像素停留时间，和1024×884像素的图像尺寸。 注:其他设置也可能bË使用，只要避免增加了电子剂量。 注意:工作非常干净即使当，芯片表面将在实践中总是包含一些污物颗粒，因为该协议中没有清洁的室内环境中执行。 </li><li>从QD标记的细胞记录的STEM图像，指的是荧光图像，并选择感兴趣的细胞。 注:这是最好的开始与细胞表现出强烈的QD信号。 </li><li>记录该单元的STEM概述图像。 </li><li>去到外围区域的单元格边框可见并放大以达到50.000X放大。 </li><li>显示单个量子点记录的图像，使用20〜30微秒像素停留时间。为了避免仅使用高倍率一次辐射损伤，图像中的每个区域。 注意:通常，在此阶段两个，焦点和散光的矫正，需要一些微调。 </li><li>的高倍放大图像，缩小和AC足够数量的记录之后叠纸的另一STEM概述形象，描绘了刚刚录制的高倍率为暗矩形区域。 注意:这些概览图像以后将用于关联ESEM和荧光图像和到蜂窝范围内确定的高倍率的图像的精确位置。 </li><li>进行到下一个感兴趣的细胞，入手的概览图像，记录一系列的高倍率的图像，并用的概述图像结束。 </li><li>在一个环境扫描电镜成像会议结束后，开始发泄室之前增加压力，以850-900帕。 注意:此增加了压力提高回收带有静止润湿表面的微芯片的机会;然而，这并不总能实现。从未被干燥了的样品可以恢复在BSA-PBS和，如果需要的话，在稍后的时间点重新调查在ESEM。 </li></ol>

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The authors have nothing to disclose.

学习的空间分布和化学计量的膜蛋白，如表皮生长因子受体，在全细胞提供了有关其功能和它们的11月14日的可能变化重要的上下文信息。它是具有挑战性的研究单体，二聚体，和簇的分布直接在亚细胞水平使用常用生化方法，其信息丢失有关蛋白在细胞或细胞15之间差异的定位。此处所描述的标记和显微镜方法允许蛋白复合物上的几纳米的长度尺度的可视化，使得它的化学计量可以单独，完整细胞4-7的范围内进行研究。这是不可能的（超分辨率）光镜23，因为其分辨率不足以区分从事蛋白质复合物的分子。荧光共振能量转移（FRET）是一种合奏平均技术&lt;SUP&gt; 24，并 ​​且不一定检测到二聚体和更高阶簇作为短程能量转移25的结果。接近结扎测定26实际上并不测量距离，因此不是能够与高蛋白质浓度的蜂窝区之间进行区分，不可避免地导致一些检测到的接近性通过随机机会的，从一个蜂窝区具有类似蛋白密度，但含有蛋白质的物表现出标签之间明显的距离。所需的高的分辨率来可视化蛋白质复合物的成分是由透射电子显微镜（TEM）来实现的。然而，全细胞的常规TEM是由薄样品/分段切片通过质膜27，或质膜撕开或折断28,29产生随机形成小片膜的要求的限制。质膜因此不成像为一个整体，导致缺乏的可能关键的细胞环境的信息。 出现其它实验挑战蛋白质化学计量在细胞中的研究从所施加的蛋白标记。常用的免疫标记承担该受体和标签之间的一对一的化学计量只能用单价探针来实现的难度;不是这种情况下，当第一和第二抗体是必需的。以获得有关蛋白质化学计量的信息，则要求一个探针结合唯一地一个表位上的受体，并具有一个结合位点的荧光探针或在另一侧的高原子序数纳米颗粒。其次，精度达到的抗体-标记被限制在帐户其大尺寸30至30纳米，从而使相邻单体蛋白质，同源二聚体，或更大的集群之间的辨别是不可行的，至少对于EGFR家族的受体。更小的特定标签是已知的文学和CA折射率n即使是细胞内的标签31应用，但这些并不常用。整个标签（EGF-生物素 - 链霉亲和QD）的长度是一个相似的几何尺寸为表皮生长因子受体，并足够小，以便能够检测出二聚体。此外，所描述的两个步骤的标记过程避免了标记诱导的受体群集。 我们的方法是不是很困难，没有太多超过标记蛋白的荧光显微镜。然而，一个实验需要进行非常小心，因为工序数相加并在以下步骤之一的错误可能会导致在实验中彻底失败。处理芯片并不比TEM网格，但一些训练空芯片的处理比较繁琐的建议。环境扫描电子显微镜，STEM全水合细胞可能是最困难的方面，并且至少需要熟练的操作和实践好几天才能根据需要可视化的量子点，以达到约3纳米的分辨率。辐射坝所研究的样品的年龄存在风险。的压力和温度应仔细观看，以保持水层。此外，水层的厚度可小区之间变化。最好是监视使用检体上方的气态电子探测器的水层的存在，如别处6所述。蜂窝区域应该只被成像一次或两次，以避免损坏。 该方法的一个关键限制是，大约超微结构高分辨率的信息不存在。其他技术，例如，低温透射电镜，需要研究蛋白质结构和细胞超微结构15。其次，研究活细胞蛋白的时间推移显微镜的倍数的动态相互作用不在考虑辐射损伤是可行的，并要求光镜23国家的最先进的。目前，我们的方法不能够确定自升二聚物的绝对水平的abeling效率是未知的，但我们希望在以后添加这些数据。但它仍然是可能的判断二聚体存在与否。从文献中已知它，即使是在15％左右的标记效率是足以检测二聚体与足够的统计显着性32。 它容易可以经由他们的配位体来研究其他膜结合的受体，通过特异性抗体的生物素化的Fab片段，或通过使用所描述的两步标记协议等小型的接头7。既然我们已经显示了两种不同的量子点的颜色（大小），可以使用和金纳米颗粒标记是在以前的工作6中使用 ，它似乎是可行的，以在相同的实验中标注的多个蛋白质种类。多种蛋白质种类的化学计量的研究的主要挑战是确保了类似的标记效率为两个种类或至少所得到的相对化学计量的对尊重归一化香港专业教育学院贴标效率。然而，在该研究中使用的两个不同的量子点似乎没有多大区别标记效率（参见图2和4）。涉及两步标记与量子点，和相关的荧光显微镜和ESEM-STEM所描述的方法提出了以研究膜蛋白的复杂的相互作用在整个细胞的完整质膜一个可行的方法。

一种新的方法是利用干1-3个 ，或相关的荧光显微镜和STEM 4-7近期推出，图像全细胞标记的蛋白质在液体状态。这种方法能够研究膜蛋白和蛋白复合物，包括在整个细胞的完整质膜单分子水平的化学当量的空间分布的。这里，我们描述涉及受体蛋白质的荧光量子点（QDs）的两步特异性标记的协议8，和相关的光学显微镜和ESEM-STEM。作为例子，我们研究了EGFR，属于该蛋白激酶超家族的膜蛋白。当配体结合，受体激活，然后形成一个与另一个激活的EGFR二聚体;随后的信号级联驱动细胞增殖9。突变导致异常EGFR表达或活性，在许多类型的癌症10发挥核心作用。学习在克在全细胞这个膜受体的空间布置提供了有关其功能和它们的11月14日的可能变化重要的上下文信息。但它仍然具有挑战性探测EGFR单体，二聚体，和簇的分布上直接与biochemical-或传统的显微镜方法15一个（子）细胞水平。我们的方法能够可视EGFRs具有几纳米，使得蛋白质在一个复杂的位置可以被确定的空间分辨率的。最重要的是，所得到的大约化学计量分发信息涉及的蛋白在完整细胞的天然状况。 为了实现在标签和受体之间的短距离，EGFR直接经其配体的EGF标记，用生物素 - 链霉键合的QD。这种标签可以与荧光 - 和电子显微镜来检测两者。我们已经用这种标签COS7细胞的相关成像液封闭在微流体室中先前1,16,17。然而，对帐户链霉每QD分子的倍数，该标签可能诱发受体成簇，导致低的标记效率或相当昂贵的实验，这是不可能完成关于该蛋白质的所研究的化学计量。可避免标签诱导的受体聚类完全通过采用这里给出的两步标记过程，产生一个探测器，包括生物素化EGF与其中只有一个链霉抗量子点（STR-QD）耦合。将细胞温育第一与生物素化EGF和再固定。固定步骤决定了蛋白质的过程被停止（根据固定的速度）的时间点。 STR-QD此后施加作为标记协议的第二个步骤。群集不发生的，因为蛋白质的动态膜运动是受阻一旦蛋白被固定。此外，STR-QD溶液，这是实验的最昂贵的组件，可以在一个优化的低浓度被应用，和标签的该第二步骤不是时间关键，即QD可以耦合在几分钟。 研究的EGFR的化学计量为分布在整个细胞中，细胞样品的制备在硅微芯片18。施加两步QD标记和荧光显微镜图像的记录之后，将细胞用纯水漂洗，并用ESEM-STEM 19成像在水合状态。所得相关图像提供有关蜂窝EGFR分布，以及在水合细胞的天然环境的化学计量信息。类似的方法被用来研究在乳腺癌细胞中HER2蛋白在最近的研究7。

<table><tbody><tr><td>Inverted fluorescence microscope</td><td>Leica</td><td>AF6000 + DMI6000B</td><td></td></tr><tr><td>ESEM</td><td>FEI Company</td><td>Quanta 400 FEG</td><td></td></tr><tr><td>DMEM (1x) + GlutaMax + glucose + pyruvate</td><td>Gibco</td><td>31966-021</td><td></td></tr><tr><td>DPBS (1x) - Calcium chloride - Magnesium chlorid</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Fetal bovine serum, Performance Plus (FBS)</td><td>Gibco</td><td>16000-036</td><td></td></tr><tr><td>Cellstripper</td><td>Mediatech, Inc.</td><td>25-056 Cl</td><td></td></tr><tr><td>Water, chromasolv Plus for HPLC</td><td>Sigma-Aldrich</td><td>34877-2.5L</td><td></td></tr><tr><td>PBS Roti-Stock 10x PBS</td><td>Carl Roth</td><td>1058.1</td><td></td></tr><tr><td>Ethanol, Rotisolv HPLC grade</td><td>Carl Roth</td><td>P076.2</td><td></td></tr><tr><td>Albumin Fraction V, biotin-free (BSA)</td><td>Carl Roth</td><td>O163.2</td><td></td></tr><tr><td>Gelatin, from cold water fish skin</td><td>Sigma-Aldrich</td><td>G7041-500G</td><td></td></tr><tr><td>Glycine</td><td>Carl Roth</td><td>T873.1</td><td></td></tr><tr><td>Epidermal Growth Factor</td><td>Molecular Probes</td><td>E3477</td><td>Biotin-XX Conjugate (biotin EGF)</td></tr><tr><td>Sodium teraborate decahydrate</td><td>Sigma-Aldrich</td><td>S9640-500G</td><td></td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B6768-500G</td><td></td></tr><tr><td>Qdot 655 streptavidin conjugate</td><td>Molecular Probes</td><td>Q10121MP</td><td></td></tr><tr><td>Qdot 800 streptavidin conjugate</td><td>Molecular Probes</td><td>Q10173MP</td><td></td></tr><tr><td>Silicon microchips with silicon nitride membranes of 50 nm thickness and dimensions of 50 &amp;micro;m x 0.40 mm</td><td>DENS Solutions</td><td>Custom made</td><td></td></tr></tbody></table>

studying the stoichiometry of epidermal growth factor receptor in intact cells using correlative microscopy

这个协议描述的表皮生长因子受体（EGFR）在COS7成纤维细胞，和随后的相关荧光显微镜和完整细胞中水合状态的环境扫描电子显微镜（ESEM）的标签。荧光量子点（QDs）偶联对EGFR经由两步标记方案，提供了一种有效的和具体的蛋白标记，同时避免了受体的标签诱导聚类。荧光显微镜提供的表皮生长因子受体的细胞位置概览图像。扫描透射电子显微镜（STEM）的检测器，以检测与纳米级分辨率的QD标签。所得相关图像提供蜂窝EGFR分布，并且在水合完整细胞的自然背景下单分子水平的化学计量的数据。环境扫描电镜-STEM图像揭示了受体到场为单体，二聚体为，并在小簇。有两种不同的量子点标记，例如，一发射655纳米和800表明类似特征的结果。

图1和 2示出的QD655标记，膜结合EGFR的可视化在完整的，完全水合的COS-7细胞的代表性图像。 图 1中的DIC图像给出了细胞的膜形貌的印象，而在图1b的相应的荧光图像示出的表皮生长因子生物素温育。 图1A 3分钟后的EGFR的分布和 B分别从两个缝合在一起记录一个40X空中目标图像。表皮生长因子激活的EGFR被分布在整个蜂窝表面。在大多数细胞中有轻微的荧光（ 图1B）的增强可以看出，在小区边缘，表明EGFR 20的局部增加发生。使用类似的协议进行的对照实验证实特异性EGFR标记（数据未显示）。这些控件包括:1）没有CE的先前孵化控制标签LLS用EGF生物素，也就是说，只用STR-量子点孵育，和2）的温育与非生物素化EGF和STR-量子点。 带有矩形标记三种细胞进一步研究了环境扫描电子显微镜，STEM。在图1C中描绘的扫描电镜-STEM图像- E显示了这些细胞的低倍概述。这些透射图像反映全细胞在水合状态用一层薄薄的水驻留过放置在硅微芯片用氮化硅（SiN）观察窗的单元。在真空室中所包含的显微镜水蒸气，和样品的温度和压力之间的平衡，调节到保持薄层的液体在细胞上。各个单元可以容易地识别是考虑其在SiN膜窗口的形状和位置的。低倍率ESEM-STEM图像揭示了精细结构，诸如丝状伪足，从小区边缘向相邻小区中延伸，部分结构里面的薄小区区域。厚中央蜂窝区域包括核出现白色因为通过样品传输是不可能的用于所使用的能量（以30keV）的电子。 高分辨率ESEM-STEM图像记录在薄，周边区域。在一个薄的液体层的空间分辨率为ESEM-STEM纳米颗粒在细胞的薄区域是在先前的研究6确定为等于3纳米。 图 2A中所示四个高分辨率的图像- D组记录在图1C的小矩形的位置- E.所用的放大倍率，足以分辨单个量子点，表现为明亮，子弹形棒，分别绑定到一个单独的表皮生长因子受体。该QD655具有6×14 2 nm的典型尺寸。 图2A（对应于矩形区域中指示Figu细胞上重新1C）显示了在膜的QD标签折叠对角线交叉的图像。这种膜结构具有比周围区域的膜更高的EGFR密度。在不同的位置，两个标签均在附近。两个例子为20和24纳米的距离都标有箭头。这些对标签被解释为属于EGFR二聚体。 图2B给出了从一个小区（ 图1D，左矩形）的边缘的一例，EGFR单体和二聚体可被视为良好。 图2C（图1C，右矩形）被记录的区域与低的荧光信号， 即，降低了 EGFR的密度。然而，表皮生长因子受体在这里还发现了二聚体为好。此外，两个簇10或11 EGFRs存在（见椭圆）。 图2D示出了膜折叠（图1E）与更小的簇包含5-6 EGFRs的另一个例子，除了几个单体和二聚体。 作为能够从该数据中，一对相关功能 21克（x）的获得是为在图2中所有的标签位置来确定的信息种类的例子。注意，此分析不是该协议的一部分，并且该过程被描述在别处6,7。克（x）是一个计量颗粒的机会被从一个参考颗粒一定的径向距离x之内找到。克（X）= 1表示的随机分布，和一个较大的值是证据聚类。在图2A-D的四个图像自动检测共210标签的位置，和g（x）的使用5nm的块大小和平滑滤波器，10纳米的带宽计算的。了g（x）的曲线（图3）示出了25nm的优选的中心到中心的量子点的距离。 EGFRs因此不随机取向但其中一个显著分数，位于此优选距离。从approximatË分子模型6（图3插图）我们估计，量子点和EGF的结合口袋之间的中心到中心的距离等于〜附着于EGFR二聚体的两个量子点之间14纳米，和中心到中心的距离为〜27纳米（该值可能是由几纳米，以改变由于连接体的柔软性）。优选的标签距离从而与预期的标签距离为该方法的精确度之内的EGFR二聚体一致。了g（x）的曲线是比单位为距离可达300纳米时，指示的簇的存在，与数据一致。这一分析表明，表皮生长因子受体的化学计量，可以研究与我们的方法。 图4示出了类似的结果，如图2，不同的是用EGF-QD800进行标记，和一个63X油浸物镜被用于荧光图像。这个数据证实，这些典型的结果还发现，当表皮生长因子受体标记与量子点，在不久的红色光谱，发射完成的。 图4A是一个代表性的细胞的荧光图像， 图4B示出在ESEM-STEM概览模式相同小区和图4C是一个150000倍放大图像，记录在电池的上膜边界（参见矩形在图4B）。类似于图2A和 D，图像捕获QD标记的表皮生长因子受体上的膜折叠并显示单体，二聚体，和群集受体。需要注意的是电子致密QD核出现稍小和圆比量子点的核发射在655nm处，用〜5纳米的更小的磁芯尺寸 22是一致的。 <img alt="图1" src="/files/ftp_upload/53186/53186fig1.jpg" /> 图1相关的DIC，荧光EGF-QD655和ESEM-STEM上完全水合COS-7细胞标记的表皮生长因子受体 </st荣&gt;生长在SiN膜窗口区域中的细胞，使质膜的地形印象的（A）中的DIC图像。 （二）荧光图像显示单元位于中央的SiN膜窗口（由虚线矩形标出）。 （C - E）从标有A和B的矩形单元三ESEM-STEM低倍放大的图像。这些细胞概览图像用来确定随后记录高分辨率的图像的精确位置。四高分辨率图像（ 图2A-D所示）的位置都标有白色矩形。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53186/53186fig1large.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/53186/53186fig2.jpg" /> <stro纳克&gt;图2.高分辨率ESEM-STEM图像描绘膜结合EGFRs。（A）的显微照片，在标注于图1B中的区域的75,000X放大倍率。许多单体是可见的。一些二聚体标以箭头。 （B）和（C）的左侧的50,000X放大倍率获取的图像，分别向右，膜的部位标注在图1C。 EGFRs的集群进行了概述。记录50,000X放大倍率标注在图1D位置（D）的形象。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53186/53186fig2large.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/53186/53186fig3.jpg" /> 图3.对相关函数g（x）作为径向距离的函数x确定间杂色<html>在图2A-D检测到的所有210标签CLE距离。在25峰毫微米表示一个中心到中心的量子点距离具有发生比随机，由此AG（X）= 1表示的随机机会的可能性要高得多。虚线是将眼表示g的随机分布的（x）的一个指南。插图是EGFR二聚体结合表皮生长因子，并通过生物素连接链霉亲涂量子点的近似分子模型。链霉亲和的模型，表皮生长因子，表皮生长因子受体从1stp（链霉），1EGF（EGF）的CPK模型得到，1NQL，2JWA，1M17，1IVO和2GS6（EGFR）的结构，在RCSB蛋白质蛋白质数据库，通过Jmol的版本创建12.2.15。生物素模型所画RCSB配体Explorer版本1.0。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53186/53186fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> /53186/53186fig4.jpg"/&gt; 图4.典型的COS-7细胞标记EGF-QD800和成像与相关荧光和环境扫描电镜-STEM（一 ）荧光图像，（B）低倍概述ESEM-STEM图像。 （C）的记录，在b中的矩形的位置在150,000X放大率高分辨率图像。单体，二聚体，和聚集EGFRs检测类似与EGF-QD655标记。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53186/53186fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy at JoVE.com

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

studying-stoichiometry-epidermal-growth-factor-receptor-intact-cells

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.3K 次观看.  INM-Leibniz Institute for New Materials. This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level. 

视频: Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

在氧化应激期间细胞粘附和纤维蛋白扩散动力学

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

科研

生物化学

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

用于蛋白质磷酸化定量的优化单分子下拉测定

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

生物学

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Proliferation is a critical part of cellular function, and a common readout used to assess potential toxicity of new drugs. Measuring proliferation is, therefore, a frequently used assay in cell biology. Here we present a simple, versatile method of measuring proliferation that can be used in adherent and non-adherent cells.

使用点击化学测量血管平滑肌细胞增殖

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

通过总内部反射荧光显微镜与数量和亮度分析相结合，活细胞中细胞表面受体的显微化动力学

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Presented here is a protocol for labeling membrane proteins in mammalian cells and coating the sample with graphene for liquid-phase scanning transmission electron microscopy. The stability of the samples against the damage caused by radiation can also studied with this protocol.

用于液相电子显微镜的化学固定哺乳动物细胞的石墨烯外壳

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using ...

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate multiple functions, including adhesion, proliferation, migration, and differentiation. Mechanical forces can be transmitted from the cell via the adhesion receptor integrin to ligands in the ECM. The amount and spatial organization of these cell-generated forces can be modulated by growth factor receptors, including epidermal growth factor receptor (EGFR). The tools currently available to quantify crosstalk-mediated changes in cell mechanics and relate them to&#160;focal adhesions, cellular morphology, and signaling are limited. DNA-based molecular force sensors known as tension gauge tethers (TGTs) have been employed to quantify these changes. TGT probes are unique in their ability to both modulate the underlying force threshold and report piconewton scale receptor forces across the entire adherent cell surface at diffraction-limited spatial resolution. The TGT probes used here rely on the irreversible dissociation of a DNA duplex by receptor-ligand forces that generate a fluorescent signal. This allows quantification of the cumulative integrin tension (force history) of the cell. This article describes a protocol employing TGTs to study the impact of EGFR on integrin mechanics and adhesion formation. The assembly of&#160;the TGT mechanical sensing platform is systematically detailed and the procedure to image forces, focal adhesions, and cell spreading is outlined. Overall, the ability to modulate the underlying force threshold of the probe, the adhesion ligand, and the type and concentration of growth factor employed for stimulation make this a robust platform for studying the interplay of diverse membrane receptors in regulating integrin-mediated forces.

TGT surface is an innovative platform to study growth factor-integrin crosstalk. The flexible probe design, specificity of the adhesion ligand, and precise modulation of stimulation conditions allow robust quantitative assessments of EGFR-integrin interplay. The results highlight EGFR as a 'mechano-organizer' tuning integrin mechanics, influencing focal adhesion assembly and cell spreading.

用于量化生长因子介导的整合素力学和附着力的张力计系绳探头

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate ...