我们感谢哈拉尔德·赫斯和Eric Betzig访问的PALM显微镜，证明了原理性实验，理查德·费特共享固定的协议，试剂和鼓励。我们感谢，鲁道夫·斯特凡Eimer迈克尔·戴维森，SEYDOUX杰拉尔丁，Leube，基思Nehrke，基督教Frøkjr - 延森，奥德阿达·恩圭马·马克哈马隆德DNA结构。我们也感谢卡尔·蔡司公司提供蔡司PAL-M，一个β版本的的蔡司Elyra的小一至小PALM显微镜。

1。高压冷冻<ol><li>准备冷冻保护通过添加0.2克BSA入1毫升您的标本的一个合适的培养基中（例如，用于细胞培养的培养基，M9 线虫 ）。在37°C水浴孵育管，直到所有的BSA溶解。 </li><li>冷冻前，填补了自动化的移动设备（Leica公司AFS）用液氮冷冻取代</li><li>设置AFS项目:-90°C 5-30小时*，5°C /小时至-60°C，-60°C 2小时（或选择"暂停"选项，如果您使用的是徕卡AFS 2）， 5℃/小时至-30℃和-30℃下72小时。如果AFS机器能够有五个以上步骤，在-30°C步骤应替换为"暂停"选项设置为0小时，和另外两个步骤应该加入:10°C /小时至-20° C及于-20℃下24小时如果机器是有限的5个步骤，成立了一个不同的内存插槽中的其他程序如下:10°C /小时至-20°C和24 hr在-20℃。 -60°C步骤开始在清晨的时间可以调整。 </li><li>准备1％四氧化锇原液混合0.1克四氧化锇晶体（EMS，RT19134）与10毫升无水丙酮（EMS，＃RT10016）。准备固定剂在如下的50毫升锥形管。首先，添加milliQ水中1毫升，并然后溶解在0.02克高锰酸钾（EMS，RT20200）。加入19毫升丙酮，拌匀。最后，加入20μl的1％锇股票溶液进入管。丙酮是一种自由基清除剂11，从而防止塑料的聚合，但使用丙酮作为冻结替代介质是必要的保存形态，因为其与脂类的相互作用。塑料渗透之前用乙醇将被取代的丙酮。等分试样1ml至每个离心管和保持冻结存储管在液氮中的固色剂。 </li><li>填充的试样用20％BSA或细菌载体。双方20％BSA和细菌作为冷冻保护剂。的试样载体的类型的选择取决于在试样上。对于C。线虫 ，使用100微米。 </li><li>试样放置在载体中，并使用高压冷冻冻结。 </li><li>冻结后，在液氮下，转移到含有固色剂的冷冻管中的试样。确保样本下的液体停留在任何时候都。 </li><li>重复1.5） - 1.7），直到所有的样本被冻结。 </li><li>将所有的冷冻管到AFS单位，并继续执行程序。 </li></ol> 2。冷冻替代<ol><li>当温度达到-60℃，放置的小瓶含有20毫升95％的丙酮到AFS。 </li><li>当温度达到-50℃，在2小时期间更换的固定剂，用95％的丙酮的六倍。 </li><li>将含有20毫升的0.1％醋酸双氧铀（Polysciences的，＃21447-25）的小瓶中，在进入腔室的95％丙酮和预冷却至-50℃。 </li><li>在洗涤步骤结束时，〜1毫升醋酸双氧铀溶液添加到每个管形瓶中。取消暂停的程序，如果必要的。 </li><li>当温度达到-30℃，用95％乙醇代替醋酸双氧铀的六倍，超过2小时的期间。 </li><li>在此期间，进行混合，10毫升22.3毫升GMA甲基丙烯酸正丁酯，milliQ水中1毫升，和0.2克过氧化苯甲酰，97％乙二醇的甲基丙烯酸甲酯树脂（GMA，SPI设备/结构探针，公司，＃02630-AA）由（催化剂）。将它存储在玻璃小瓶（EMS，＃72632），通过用95％乙醇混合，制备30％的GMA。 不要存储GMA媒体的塑料管中，因为与长期贮存在聚乙烯基塑料聚合的质量劣化。 GMA解决方案预冷到-30°C。 传统的环氧树脂，例如的Araldite和EPON不能在协议中使用，因为它们是无水的和酸性其中淬灭荧光蛋白。丙烯酸类树脂，如LR白角容忍少量的水，并且可以保留的荧光蛋白质，但它的酸性pH值的倾向，淬灭的荧光团12。 GMA容忍，事实上需要，少量的水和碱性（PH8）12。不幸的是，GMA是有点脆。此外，GMA不交叉链接类似传统的环氧树脂并用纸巾。切片质量中GMA原因不一致的这些特点，尤其是当切片厚度小于80纳米。 的GMA虽然保质期是一年左右，GMA应在购买后3个月内，以确保其质量。 </li></ol> 3。渗透和聚合步骤3.1-3.3进行冷冻取代用于在相同的离心管中。渗透和聚合在-30℃下进行，在AFS保留的荧光蛋白质。 <ol><li>准备渗透介质通过GMA原液混合，用95％的乙醇。孵育标本为3-5小时，在30％的GMA。 </li><li>孵育的标本中70％GMA 4-6小时。 </li><li>孵育标本中97％GMA过夜。 </li><li>第二天，新鲜的97％GMA。 </li><li>将样品到一个嵌入的模具（EBSciences，＃TC）。准备的磁盘ACLAR膜（EMS，＃50425-10）通过使用一个3/8"DISC冲床（特德培拉公司制），并放置在底部的BEEM胶囊磁盘。 </li><li>交易所97％的GMA三次超过6小时，在-30°C。 </li><li>第三次交换后，添加引发剂N，N-二甲基 - 对 - 甲苯胺（Sigma-Aldrich公司，＃D9912）的浓度为1.5微升/ 1毫升GMA GMA，并应用此解决方案，每个试样模具。立即定位在AFS嵌入模具中的试样。 需要注意的是过氧化苯甲酰是催化剂和被本所有浸润步骤期间使搅拌是没有必要的。 ，直到它被暴露于化学引发剂，N，N， - 二甲基 - 对甲苯胺，过氧化苯甲酰不聚合塑料。引发剂激活POLymerization立即将聚合树脂在1小时之内，即使在组织。然而，组织辍学，可能会导致深部组织由于不完整的聚合。此外，GMA的不交联的试样以及块，所以分离试样从冷冻保护剂，通过利用细菌的矩阵。 如果外执行的AFS聚合，嵌入模具必须在一个的ACLAR磁盘阻止暴露于氧气覆盖。 GMA不聚合时完全暴露在氧气中。 </li><li>允许的塑料治愈过夜，即使它聚合1小时内。 </li><li>存储氮充有气体的真空袋（Ziploc密闭）在冷冻（-20℃），直到试样在荧光蛋白的进一步的处理，以便不暴露于氧气。 </li></ol> 4。切片<ol><li>剖分GMA包埋标本，可以进行的方式类似于EPON​​包埋的标本。应该采取格外小心n不润湿分隔表面，因为GMA是非常亲水性和色带将被驱动到水浴中，当它们存在两侧润湿。 </li><li>收集的部分（50-80纳米）的玻璃盖玻片上的色带如果TIRF显微镜用于本地化。否则，使用透射电子显微镜为一个网格。应设置的切割速度at1.6 mm / s或更高。否则，色带可能无法形成。 </li><li>在-20°C的存储部分，如果不立即成像。这两个荧光基团保护UV光铝箔覆盖部分持有人。 </li></ol> 5。 PALM成像<ol><li>根据制造商的建议，成立了PALM显微镜。 EMCCD相机的温度应设置为-70℃或以下。 </li><li>应用金颗粒（＃790122-010 - 2x的浓缩，微球-nanospheres.com）在溶液中（约50微升），将被成像的样品。封面上的允许的解决方案，坐30秒唇，而下一个黑色封面的情况下。 </li><li>通过吹入到盖玻片的边缘和吸收具有kimwipe卸下金溶液。 </li><li>将盖玻片成圆形盖玻片持有人。如果需要，适用于真空油脂保持在盖玻片边缘。 </li><li>盖玻片底面应用浸油，直接下方的样品。 </li><li>在舞台上的显微镜，将样品架插入插槽。要特别小心不要接触到目标。 </li><li>调整的持有人，因此很紧，中心部分以上的目标。 </li><li>找到的部分，用10倍的物镜。 </li><li>切换到100倍的物镜。 </li><li>重点在试样上。 </li><li>找到感兴趣的区域，通过观察荧光信号的强度。使用488nm的激光，并在"通道"菜单中的强度增加至10％。重点领域最明亮的荧光。请注意，此步骤是没有必要的，如果区域Of利息可以识别的眼睛在明亮的领域。 </li><li>至561纳米的激光切换和增加强度为100％，以漂白剂的背景的自发荧光。漂白剂的样品〜2分钟。 </li><li>如果焦点在漂白，允许5分钟之前经过调整的重点和捕捉图像。此暂停允许温度稳定。如果PALM范围配备孵化室，设定温度为20℃和等待〜2小时，以在腔室中的温度稳定。 </li><li>漂白完成时，激活的405 nm激光，将其设置为最低强度。 </li><li>开始收集在每秒20帧的图像。我们通常会收集5000-6000的帧每秒实验，但根据实验目的，应调整帧的数量。例如，如果在感兴趣的区域中的所有的蛋白质必须被成像，帧序号应增加。 </li><li>如果信号稀疏或模糊的，慢慢增加本身的405nm激光的强度。 </li><li>在采集过程中，一定要仔细必要的调整旋钮，保持样品的焦点。 </li><li>当图像进行搜集，的PALM分析应该进行。对于tdEos，我们筛选出荧光长于500毫秒的信号，因为这些信号可能是由于自体荧光。 </li></ol> 6。扫描电镜成像<ol><li> SEM成像之前，染色为4分钟，用2.5％乙酸双氧铀（在水中）的部分。彻底过滤milliQ水中洗掉的醋酸铀。 </li><li>碳涂层的部分干燥后，使用碳盖玻片溅射直到盖玻片变得相当暗。应用碳导电带的一端被接地，使电子的盖玻片的表面上积聚的盖玻片的边缘处，而另一端上的金属存根。 </li><li>安装试样的扫描电镜室（FEI新星纳米）。 </li><li>将VCD检测。 </lI&gt; <li>关闭腔和泵用高真空环境。 </li><li>一旦抽真空，打开柱阀，使电子束被施加到试样。 </li><li>执行例行的电子束对准。 </li><li>找到试样。 </li><li>一旦调整对焦，链接试样阶段，至5毫米以下的极片的阶段。 </li><li>以低倍率的试样（约5000倍）的图像。 </li><li>进入该地区的利益，并获得高倍率变焦（50,000 x）的图像。 </li><li>移动到下一个部分，并重复步骤6.10）和6.11），直到所有的部分成像。 </li></ol> 7。对齐的PALM和EM图片<ol><li>打开Photoshop和收购的SEM图像。 </li><li>创建一个新的窗口尺寸为5,000 x 5,000像素，300像素/英寸分辨率。 </li><li>低倍SEM图像复制到新窗口中（ 图1A）。 </li><li>因此，它填补了缩放图像整个窗口使用变换操作（命令+ T为Mac）。 </li><li>复制更高的放大倍率SEM图像，将其换算为必要。 </li><li>通过从图像的下拉菜单栏选择"图像旋转，水平翻转和TIRF图像。 </li><li>复制并粘贴TIRF图像的总和（PALM）到一个新的层。 </li><li>使用的变换操作，缩放的图像，然后旋转，要匹配的金颗粒（在图1A中的白色箭头）与相应的结构在SEM观察（ 图1B）的荧光。 </li><li> PALM图像复制到一个新层，适用相同的转换（ 图1C）。可以提取高放大倍率的图像，从图像（ 图2）。 </li><li>介绍，复制转化PALM的图像到一个新的层。通过使用"色彩范围"，在"选择"下拉菜单中选择PALM信号，但不包括后台。确保SELECT背景像素作为参考像素，并打开上的"反转"选项。 </li><li>切所需的PALM的信号，并将其粘贴到一个新的层。 </li><li>应用的透明度，背景层，将它设置为10％。这使得进行可视化的PALM信号通过使背景透明而不会损害它们的强度（ 图2D）。 </li></ol> 8。代表性的成果组蛋白具有标记tdEos可以被稳定地表达在线虫C.线虫 ，和转基因动物，使用上面描述的协议处理。 PALM和电子显微镜照片，是从相同的部分（ 图1）获取。对齐图像的总和。TIRF图像，总结在整个时间过程中的荧光，被覆盖上的电子显微镜照片。金纳米粒子出现在荧光和电子显微镜照片，可以用来对齐两个图像使用&#39;互感器ORM"功能，在Photoshop中（ 图1A和B）。然后，相同的"变换"值被施加到对PALM图像（ 图1C）。在此放大倍数，结构的细节可以不加以区分，所以我们放大到的显微镜照片（ 图2）的上端部附近的区域。在高放大倍率的图像，可以观察到细胞内的详细信息，如一个核，一个核仁，核孔，内质网。此外，组蛋白分子标记的专门定位于细胞核的核仁，符合市场预期。 PALM和电子显微镜的相关性，从而使蛋白定位在最高分辨率。 五个方面的问题可以妥协的图像的质量。首先，冰晶损伤扭曲的超微结构（ 图3A，B）。名次标本中的冷冻保护，如细菌，从而降低冰晶的传播，可以减少这种损害。然而，还必须通过电子显微镜和筛选标本，抛弃那些与冷冻文物。二，GMA并没有交叉链接到组织如环氧树脂，从而标本往往挣脱从周围的塑料，拉伸，收缩，甚至掉出来的部分（ 图3C，D）。夹层的样品离的细菌或其它冷冻保护嵌入前提供更大的粘合性的塑料的试样（ 图3C）。同样地，如脂滴在肠道中的结构往往解离的组织中，由于交联的情况下（ 图3E）。第三，不完整的聚合塑料组织拉伸或折叠的原因（ 图3F），样品中的氧的存在下，也阻碍了GMA聚合。四，GMA切片质量差的结果往往是不一致的形态（ 图3G和H）。 GMA部分被切掉为70nm或更厚，且在约1.6毫米/秒的速度减少切片的工件。五，背景自发荧光的盖玻片上的灰尘或部分是不可避免的。自体荧光灰尘可以被最小化，通过使用预先清洁的盖玻片和如描述在协议中，避免灰尘污染从kimwipes和滤纸。 PALM分析程序可以编辑出信号从荧光长于从荧光蛋白（ 图2中A和B）的典型信号的检体或塑料。这样的文物，因此，最终的图像。 <img alt="图1" src="/files/ftp_upload/3995/3995fig1.jpg" fo:content-width="5in" fo:src="/files/ftp_upload/3995/3995fig1highres.jpg" /> 图1。校准利用黄金纳米粒子的荧光和电子显微镜。 （A）在低倍率从C 的横截面的电子显微镜照片线虫表达组蛋白tdEos标记:: HIS-11。白方rrows指示100纳米电子致密的黄金纳米粒子的应用PALM成像作为基准标记前。 （B）的黄金珠曝光时〜580 nm的光的荧光和创建的荧光图像中的基准标记。的总和。TIRF图像对齐到的基准标记的位置的基础上的电子显微镜照片。 TIRF图像的总和代表所有的光子相机检测到在实验时间过程。请注意，出现亮点上的左上角（白色箭头）从金颗粒簇。 （C）A PALM图像，然后添加到电子显微镜和旋转和翻译的基础上确定的值TIRF图像在（B）的总和的对齐方式。 <a href="http://www.jove.com/files/ftp_upload/3995/3995fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/3995/3995fig2.jpg" fo:content-width="5in" fo:src="/files/ftp_upload/3995/3995fig2highres.jpg" /><b转/&gt; 图2。相关纳米FEM组蛋白融合蛋白。（A）的总和的TIRF形象的tdEos :: HIS-11薄款（70纳米）收购。 （b）相应PALM图像的tdEos :: HIS-11。自体荧光（白色箭头）持续时间超过500毫秒的PALM程序被过滤掉了。 （C）的电子显微镜照片的核从相同的部分获得。 （D）的相关性的PALM图像和电子显微镜照片tdEos :: HIS-11。荧光被紧密地定位在细胞核染色质。 <img alt="图3" src="/files/ftp_upload/3995/3995fig3.jpg" /> 图3。存在的问题与纳米有限元分析。 （A）电子显微镜的C.线虫全身肌肉无冰晶体的损害。 （B）的电子显微镜照片的身体肌肉用冰晶体损伤。而不是离散的横截面，肌动蛋白和肌球蛋白丝被合并到聚集由于冰晶的形成。 （C，D）低倍率的电子显微镜，示出在从周围介质中的蠕虫的解离。中的标本，其周围由冷冻保护细菌（D）时相比，试样者塑料（C）包围的部分是更加扭曲。细菌冷冻保护被解剖在gallette应远离塑料包埋固定样本才。请注意，在右侧的（D）是动物的倾斜切片，并因此，该形状是不是由于组织的失真。 （E）的电子显微镜照片肠，辍学的组织（黑色箭头）。 （F）的神经环的电子显微镜照片，示出折叠的部分由于不完全聚合的塑料（黑色箭头）。 （G，H）的电子显微照片来自同一标本的神经元，切片在不同的日期。一天（G）的组织的保护是一流的，但这样的形态模糊的不一致的切片质量（H）。 <a href="http://www.jove.com/files/ftp_upload/3995/3995fig3large.jpg" target="_blank">点击这里给v</a>IEW较大的数字。

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在这里，我们将介绍如何保持荧光蛋白塑料，本地化荧光蛋白的部分，并用电子显微镜图像的超微结构。蛋白质本地化使用PALM显微镜纳米级分辨率的衍射极限以下。为了适应这一协议特定的标本，下面的参数应该被视为:荧光，量化和对齐方式。 荧光蛋白或有机荧光团的选择依赖于应用程序和模型系统。我们已经测试了各种不同的荧光蛋白，其中包括EGFP，YFP，黄水晶，mEosFP，mEos2，tdEos，魔橙，PA-mCherry，Dendra和12。保存从各荧光团的荧光是相似的，这表明所有的荧光蛋白质使用所描述的方法，可以被保存。我们选择tdEos，因为它表达C.线虫 ，蛋白质保持功能融合到tdEos时，并且，因为它PALM显微镜的光激活的特点是最佳的。然而，已经聚集或失败的表达tdEos偶尔观察到12。 根据不同的应用，不同的荧光团可能更适合。在许多情况下，这是没有必要使用光激活的荧光蛋白。简单相关荧光电子显微镜不需要光激活的荧光蛋白质。 GFP或有机染料，可以使用图像的荧光来自标签的蛋白的部分上方的衍射极限。例如，一种可以对图像中的神经纤维，用荧光显微镜轴突和关联的电子显微镜照片中的一个特定的轴突通过成像在荧光显微镜的荧光的荧光信号与。其他超分辨率技术，如受激发射损耗显微镜（STED）12，接地的状态耗尽显微镜其次由单个分子的回报（GSDIM）13，和结构照明显微镜（SIM）14，不需要光活化荧光蛋白。此外，超分辨率成像技术，使用有机染料9,15,16或荧光探针17的固有属性是容易适用。 PALM，可量化的，因为每个分子中的荧光分子的数目，被分离空间和时间。然而，量化可能会产生误导的原因有四:氧化，计数不足，超量，和过度表达。首先，荧光蛋白的一小部分可变性或样本处理期间5,12氧化。虽然〜90％的荧光信号被保存在我们的协议通过固定和嵌入，可能会发生的荧光蛋白质的氧化后的试验片已切片和表面暴露于氧。其次，光活化的蛋白的活化是随机的，从而多个分子可以是在一个给定的衍射受限点8激活。从多个分子荧光将显示为一个点，从而蛋白质的总数将被低估。第三，类似的问题可能会导致超量。于掌心，每个荧光蛋白本地化，然后单击"删除"漂白。然而，荧光蛋白可以返回从黑暗的状态，没有永久漂白18。然后，这样的分子将被计算多次。第四，标签的蛋白作为转基因的表达，并经常呈现在多个副本，这可能会导致过度。因此可以使用从PALM，量化估计，但不能准确地确定在一个给定的位置的分子数目。 对准的PALM的电子显微镜照片的图像，也可以是具有挑战性的，因为在光学和电子显微镜，由电子束引起的失真的分辨率差。金颗粒作为紧密禄alized在电子显微镜下的基准标记。然而，fluoresecence从金颗粒不被光激活的，并显示为一个大的衍射限制的现货。因此，放置在一个电子显微镜，荧光图像，是一种估计。扭曲也可以与塑料部分而产生的电子的相互作用。丙烯酸树脂，如GMA是不太稳定的电子束下，和塑料的尺寸可以改变。在这些情况下，对准的荧光超微结构可能需要的非线性变换的基准标记。

<table border="1"><tbody><tr><td>的试剂或设备的名称</td><td>公司</td><td>目录号</td><td>评论</td></tr><tr><td>高压冷冻机</td><td> ABRA </td><td> HPM 010 </td><td>徕卡或高倍视野-01从Wohlwend EMPACT HPM和100也可以被使用。 </td></tr><tr><td>自动冷冻置换单元的</td><td>徕卡</td><td> AFS 2 </td><td> AFS 1也可以被使用。 </td></tr><tr><td>蔡司PALM </td><td>蔡司</td><td> ELYRA P.1 </td><td>也卖尼康和Vutara的商业PALM显微镜。 </td></tr><tr><td>扫描型电子显微镜</td><td> FEI </td><td>新星纳米</td><td>可以使用其他高分辨率SEM显微镜。 </td></tr><tr><td>丙酮</td><td> EMS </td><td> RT10016 </td><td></td></tr><tr><td>乙醇</td><td> Sigma-Aldrich公司</td><td> 459844-1L </td><td></td></tr><tr><td>锇吨etroxide </td><td> EMS </td><td> RT19134 </td><td></td></tr><tr><td>高锰酸钾</td><td> EMS </td><td> RT20200 </td><td></td></tr><tr><td>从牛血清白蛋白</td><td> Sigma-Aldrich公司</td><td> A3059-50G </td><td></td></tr><tr><td>醋酸铀</td><td> Polysciences的</td><td> 21447-25 </td><td>该公司的醋酸铀pH值略高。 </td></tr><tr><td>乙二醇甲基丙烯酸酯（GMA）的</td><td> SPI </td><td> 02630-AA </td><td>低酸，TEM级。 </td></tr><tr><td> N，N-二甲基 - 对 - 甲苯胺</td><td> Sigma-Aldrich公司</td><td> D9912 </td><td></td></tr><tr><td>冷冻瓶</td><td>的Nalgene </td><td> 5000-0020 </td><td></td></tr><tr><td>玻璃小瓶</td><td> EMS </td><td> 72632 </td><td></td></tr><tr><td> ACLAR薄膜</td><td> EMS </td><td> 50425-10 </td><td></td></tr><tr><td>乙EEM胶囊</td><td> EBSciences </td><td> TC </td><td>聚丙烯</td></tr><tr><td> 3/8"DISC拳</td><td>特德·佩拉</td><td> 54741 </td><td></td></tr><tr><td>黄金纳米粒子</td><td>微球的nanospheres.com </td><td> 790122-010 </td><td>申请浓缩液的2倍</td></tr></tbody></table>

nano-fem: protein localization using photo-activated localization microscopy and electron microscopy

映射蛋白质的分布，对于理解蛋白质在细胞的功能是必不可少的。荧光显微镜被广泛用于蛋白质的定位，但在荧光图像中往往是不存在的细胞内的上下文。免疫电子显微镜，在另一方面，可以本地化的蛋白质，但该技术是有限的缺乏兼容的抗体，形态和保存差，因为大多数抗原不暴露在试样表面。相关的方法可以获取荧光图像，首先从整个细胞，无论是从免疫荧光基因标记的蛋白质。然后将样品固定和嵌入的电子显微镜和图像相关的1-3。然而，低分辨率的荧光图像和所述基准标记的缺乏排除蛋白的精确定位。 或者后可以做保持在塑料中的检体，荧光成像。在这种方法中，该块是切片，荧光图像和相同的部分的电子显微镜照片相关4-7。然而，在相关的图像的光的衍射极限的掩盖的单个分子的位置，和荧光常常延伸超出该单元格的边界。 本地化的蛋白质在纳米尺度成像相同的部分使用光激活定位显微镜（PALM）和电子显微镜，纳米分辨荧光电子显微镜（纳米-FEM）的设计。 PALM克服衍射极限，由成像单个荧光蛋白和随后各荧光点的质心8-10映射。 我们概述了纳米有限元技术的五个步骤。首先，将试料固定并嵌入使用条件，保持标签蛋白的荧光。其次，在树脂块划分成超薄段（70-80纳米），被安装盖玻璃上。第三，在这些部分使用蔡司PALM显微镜荧光成像。第四，电子致密的结构，在这些相同的部分，使用扫描型电子显微镜成像。第五，荧光和电子显微镜照片是使用金颗粒作为基准标记对准。总之，荧光标记蛋白的亚细胞定位，可以在大约一周的纳米级分辨率的决定。

Watch this Scientific Journal Video about Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy at JoVE.com

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

本地化荧光标记的蛋白质在电子显微镜下，我们描述了一种方法。荧光是第一次使用光激活定位显微技术在超薄切片本地化。然后，这些图像被对齐到相同的部分的电子显微镜照片。

纳米有限元分析:蛋白质定位的光激活定位显微镜和电子显微镜

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

nano-fem-protein-localization-using-photo-activated-localization

Research

JoVE Journal

Biology

16.0K 次观看.  University of Utah. 本地化荧光标记的蛋白质在电子显微镜下，我们描述了一种方法。荧光是第一次使用光激活定位显微技术在超薄切片本地化。然后，这些图像被对齐到相同的部分的电子显微镜照片。

视频: Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

科研

生物学

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

蛋白表达和共定位使用复用免疫组织化学染色和多光谱成像定量

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

三维超F-微丝高分辨率显微镜通过干涉光敏定位显微镜（iPALM）

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

活体哺乳动物细胞器中单膜蛋白的多色定位显微术

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

使用前 Vivo 共聚焦显微镜可视化淋巴节点结构和细胞定位

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

利用单分子定位显微镜分析人类 iPSC 衍生肌细胞中的α-Actinin 网络

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...