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 border="0" cellpadding="0" cellspacing="0">
	<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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></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>shoul</td>
			<td>Molecular Probes</td>
			<td>E3477</td>
			<td></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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon microchips with silicon nitride membranes</td>
			<td>DENS Solutions</td>
			<td>Custom made</td>
			<td></td>
		</tr>
		<tr>
			<td>of 50 nm thickness and dimensions of 50 &#181;m x 0.40 mm</td>
			<td></td>
			<td></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/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/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/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/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

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

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

不对称受体模式研究细胞滚动轨迹

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

科研

生物工程

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

轴突伸展生长：神经生长Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

创伤显微注射和分析<em&gt;在体内</em&gt;表皮的伤口本地化响应记者在<em&gt;果蝇</em&gt;胚胎。

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

静电生长因子释放到微纤维支架

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...