这项工作是由一个赠款（R01GM098394）由美国国立卫生研究院和国家普通医学科学研究所以及发放1121176从美国国家科学基金会的支持。

1。现场睾丸的轨道A.准备<ol><li>制备PBS溶解8.0克氯化钠，0.2克的KCl，1.44克Na 2 HPO 4 0.24克KH 2 PO 4在800毫升的蒸馏水中，调整pH至7.4。使体积达到1升，并通过高压灭菌消毒。 </li><li>隔离睾丸如前所述27。 </li><li>制备DAPI染色缓冲液稀释1毫克/毫升储到1μg/ ml的PBS中。用铝箔保护管的光，并储存在-20°C。 </li><li>分离睾丸后，浸入试样中6微升的DAPI染色缓冲上10分钟，带正电荷的玻璃显微镜载片的上方。睾丸很好地粘附在市售的带正电荷的幻灯片，可以很容易操纵的标本。带正电荷的载玻片共价修饰以赋予静电正电荷到玻璃表面。类似的多聚赖氨酸涂层，这促进睾丸wi的相互作用TH滑的表面。 </li><li>通过用6微升PBS替换染色缓冲液洗两次过量的DAPI。用一张滤纸每次洗涤步骤后，以吸走的缓冲区。要小心，不小心每次洗涤过程中随缓冲取出睾丸。 </li><li>加入6微升PBS的标本。所用缓冲液的量应该为盖玻片的大小进行调整。在这个协议中提供的容积为18×18毫米盖玻片。可选的:最好是刺破睾丸附近的感兴趣的细胞类型的相对位置。这将确保有在挤压工序对这些细胞的最小压力，从而保持其结构的完整性。可以使用锋利，干净的刀片进行睾丸穿刺。 </li><li>小心地将盖玻片上的样品的上方。 </li><li>用透明指甲油，以确保缓冲区不从活体标本蒸发密封盖玻片的边缘，而IM老化。 </li><li>使用一个标记来指定睾丸为便于位置中定位所述样品上滑动，​​并继续成像。 </li><li>用6微升PBS替换缓冲区洗两次多余的DAPI染色缓冲液。用一张滤纸每次洗涤步骤后，以吸走的缓冲区。小心不小心每次洗过程中伴随着缓冲取出标本。 </li></ol> 2。睾丸固定轨道B.编制<ol><li>通过在PBS中稀释37％的甲醛原液中的1×PBS的3.7％甲醛的最终浓度制备修复缓冲</li><li>准备DAPI染色缓冲液稀释1毫克/毫升的DAPI股票为1微克/毫升的PBS。 </li><li>沉浸睾丸在PBS中的上带正电荷的玻璃显微镜载片的顶部上的6微升下降。 </li><li>手动定位以线性方式或以其他方式优选睾丸。 </li><li>用滤纸吸走的PBS中，并用6微升修复的替换缓冲液5分钟。 </li><li>用滤纸吸走修复缓冲液和浸入试样中6微升的DAPI染色缓冲液10分钟。 </li><li>小心地将一个18×18毫米的盖玻片上的样品的上方。 </li><li>用透明指甲油，以确保缓冲区不从标本而成像蒸发密封盖玻片的边缘。 </li><li>使用一个标记来指定睾丸为便于位置中定位所述样品上滑动，​​并继续成像。 </li></ol> 3。免疫染色睾丸的轨道。制备方法 盖玻片硅化:浸入多个18×18毫米的盖玻片在含有硅化溶液的小托盘，并根据通风柜后，于室温下放置1分钟。确保盖玻片充分暴露，并且不堆积在彼此的顶部。 <ol><li>将样品在PBS上的硅化盖玻片5微升下降。东方的睾丸的期望，用锋利的手术刀刺入睾丸。轻轻地放在一个带正电荷的玻璃显微镜载片在盖玻片上，使PBS成为盖玻片与载玻片之间均匀地分散。用一小片滤纸吸走盖玻片与载玻片之间的多余的缓冲区。由于缓冲区是由滤纸除去，增加的压力将挤压的睾丸。许多标本应同时准备，因为有些人可能会丢失或损坏了该协议的过程。玻璃雕刻机可用于标记的幻灯片，如果不同类型的标本将同时准备。 <ol><li>洗涤盖玻片3次，每次1分钟在水中，随后3次，每次1分钟70％的乙醇，并在水中1分钟的最终洗涤。允许硅化盖玻片风干在通风橱内。 </li></ol></li><li>丢弃滑入液氮，并允许样品冻结5-10分钟。 </li><li>从L中删除幻灯片采用大钳iquid氮气。用手术刀快速去除盖玻片。试样应保持在幻灯片上。要小心，不要沿滑道滑动盖玻片在此过程中涂抹标本。 </li><li>孵育在预冷的甲醇中的幻灯片的玻璃科普林氏染色缸在-20℃下15分钟。 </li><li>转移到载玻片含有预冷的丙酮在-20℃下进行30秒的玻璃科普林氏染色缸。 </li><li>为1分钟，在PBS中于室温下用玻璃科普林氏染色缸洗的幻灯片。 </li><li>通过补充PBS，0.1％的Triton-X100和1％牛血清白蛋白（BSA）的准备PBST-B。可以使用来自同一宿主物种作为第二抗体（来自步骤3.14）5％正常血清代替牛血清白蛋白，以减少非特异性二级抗体结合产生的背景噪声。 </li><li>孵育载玻片在PBST-B 10分钟，在玻璃科普林氏染色缸用于阻断非特异性位点。 </li><li>填写的井湿气室中的水。湿度的室内将在孵育步骤从样品抗体溶液的蒸发最小化。 </li><li>通过补充PBST-B与100微克/毫升RNA酶A。的RNaseA降解RNA的试样，并通过强烈吸收到玻璃表面作为一个附加的封闭剂制备的PBST-BR。 <ol><li>放置在试样的顶约1×1厘米片封口膜的均匀分布的抗体溶液和保护抗体溶液的蒸发。关闭该腔室的水分和孵育的样品中的第一抗体1小时，在室温下进行。 </li></ol></li><li>从PBST-B拆下幻灯片。用一张滤纸干周围的标本的载玻片的面积，使用小心不干燥样品本身。最后，放置在幻灯片中的湿气室与试样朝上</li><li>轻轻地加入100微升一抗稀释在PBST-BR在规范上imen（一般1:200是一个很好的起点浓度未知抗体）。 <ol><li>盖上样品与约1×1厘米的封口膜和关闭水分室。孵育样品在二次抗体1小时，在室温下进行。 </li></ol></li><li>打开防潮室，并使用镊子从幻灯片轻轻取出的封口膜。孵育玻片5分钟，在PBST中的玻璃科普林氏染色缸于室温下洗。共洗涤三次重复此过程。 </li><li>从PBST-B拆下的幻灯片，并将其放置在湿气室内与试样朝上。轻轻地添加100μl的第二抗体的稀释在PBST-BR对试样的顶部。 </li><li>打开防潮室，并使用镊子从幻灯片轻轻取出的封口膜。再次，在一个玻璃科普林氏染色缸随后3次，每次5分钟用PBS洗涤样品3次，每次5分钟在PBST中。 </li><li>使用的Kimwipe和滤纸<html>仔细擦干滑的表面，小心避免接触或干燥的标本。 </li><li>加入6微升安装媒体到标本，盖上干净盖玻片（不涂硅胶），并用指甲油密封边缘。 </li><li>使用一个标记来指定睾丸为便于位置中定位所述样品上滑动，​​并继续成像。 </li></ol> 4。影像注释成像可以使用一个直立或倒置常规光学显微镜或共聚焦显微镜来实现。重要的是，该显微镜配备有相位对比，特别是对活睾丸标本（轨迹A）成像。这个功能已经在共聚焦显微镜最近变得可用。 当睾丸打破自发（轨迹A），尖端（干细胞区域）通常保持完整并是容易识别的，提供了良好的标记，以初步定位和用于定向。 _content"&gt;中心粒是相当小的结构和图像，因此应使用63X或100X物镜，4-6X变焦倍率拍摄，并且至少512×512像素时，可能的分辨率。

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensory-defective,+male-sterile+unc+mutants+identify+a+novel+basal+body+protein+required+for+ciliogenesis+in+Drosophila.">Mechanosensory-defective, male-sterile unc mutants identify a novel basal body protein required for ciliogenesis in Drosophila.</a> Development. 131, 3411-3422 (2004).</li><li>Avidor-Reiss, T., Gopalakrishnan, J., Blachon, S., Polyanovsky, A., Schatten, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Centrosome: Cell and Molecular Mechanisms of Functions and Dysfunctions in Disease. , (2012).</li><li>Gopalakrishnan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+SAS-6+multimer+is+a+core+centriole+building+block.">Self-assembling SAS-6 multimer is a core centriole building block.</a> J Biol Chem. 285, 8759-8770 (2010).</li><li>Hassell, J., Hand, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+fixation+with+diimidoesters+as+an+alternative+to+aldehydes.+I.+Comparison+of+cross-linking+and+ultrastructure+obtained+with+dimethylsuberimidate+and+glutaraldehyde.">Tissue fixation with diimidoesters as an alternative to aldehydes. I. Comparison of cross-linking and ultrastructure obtained with dimethylsuberimidate and glutaraldehyde.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 22, 223-229 (1974).</li><li>Pisano, C., Bonaccorsi, S., Gatti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kl-3+loop+of+the+Y+chromosome+of+Drosophila+melanogaster+binds+a+tektin-like+protein.">The kl-3 loop of the Y chromosome of Drosophila melanogaster binds a tektin-like protein.</a> Genetics. 133, 569-579 (1993).</li></ol>

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研究中心体生物学中使用基因标记的中心体标志物蝇睾丸是用于评估在两个野生型和突变型的上下文中心体功能和活性的有效方法。特别是，跟踪A是适合于中心体异常，如畸形，misegregation，不稳定，或在努力寻找新的异常突变体长度的快速筛查。此外，在现场准备精子细胞的纤毛运动型保持解剖后约15分钟，并在通道A中使用活的睾丸也允许精子活力可以很容易地解决。由于精子运动纤毛的活性直接相关，中心体功能，测定可以被执行以确定对纤毛功能的各种中心体的突变的影响。跟踪B可用于特别是当统计数据是必需的，例如，用于计数每个细胞和或每囊肿细胞的数目中心粒的数目更具体的观测。轨道C是最useful对于需要染色的抗体详细的观察。实例包括特定的细胞类型，例如干细胞，不具有可用的标签，例如微管蛋白乙酰化，或者验证一个蛋白质中的突变的缺失或错误定位的蛋白质染色的标记。 当成像中心体和中心体有关的结构，使用基因标记的标记物，而不是抗体不仅实验更容易，而且提供了更可靠的和可重现的结果。因此，利用基因标记的中心体蛋白的是，需要一个大的数据集机械和定量分析的可靠方法。例如，使用基因标记的中心体标志物一直是中心粒长度的量化特别有价值。这样的分析表明，各突变可以根据在中心粒的长度的可变性可以分为两类。一类包括基因突变，陈GE中心粒的长度，但不影响标准偏差为1，而另一类别包括同时影响中心粒长度和长度为11的标准偏差的突变。这种定量的数据可以提供有用的洞察特定的中心体基因的功能。表现出与增加的标准偏差有缺陷的中心粒长度的中心体的突变体可能是由于中心粒结构的不稳定。然而，有缺陷的中心粒长度与正常标准错误可能表明该突变不会在结构上不稳定的中心粒和更可能是由于在调节机制控制中心粒的长度的变化。由于染色的不一致性，定量分析是很难与使用抗体标记孤单。 中心体是一个大型的，复杂的蛋白质结构和它的许多蛋白质​​仅在其内部找到。使用基因标记的中心体而标记的抗体允许一个统一标记的抗原决定簇可能是其它方法无法抗体标记的中心体的内部组件。例如，使用抗体BLD10的定位研究发现该蛋白在中心粒13的远端和近端富集，而BLD10-GFP显示了更均匀的分布1。但是，同样重要的是考虑一个特定的遗传标记的蛋白质的表达水平，因为这会影响蛋白质的分布。 SAS-4-GFP和SAS-6-GFP的定位在其内源性表达被限制在中心粒1,16,35的近端另一方面，SAS-4-GFP和SAS-6-GFP下的表达强大的泛素启动子沿的中心粒12,14的整个长度本地化。另一个重要的考虑是对蛋白质功能的遗传标记的效果。如果基因标记的蛋白质是功能分析可以工商业污水附加费特德通过引入转基因蛋白进入一个突变的背景和检查，如果转基因蛋白抢救突变表型。 可以使用各种化学固定剂来进行果蝇睾丸固定。在这里，我们描述了与两个甲醛（轨迹B）和甲醇 - 丙酮（课程C）固定。然而，无论是固定剂可以互换使用，并且由于各种固定剂可以化学破坏天然抗体的表位，适当的固定剂对免疫染色的选择必须通过实验确定。下面的固定剂和培养条件，通常采​​用:3.7％甲醛，5分钟，在室温下的甲醇，15分钟，在-20℃;丙酮，10分钟，在-20℃;甲醇，15分钟，于-20℃。接着用丙酮，30秒，在-20℃;乙醇，20分钟，于-20℃下虽然固定用丙酮，甲醇，乙醇和不需要对免疫染色，固定无线网络的扩展透化步骤第甲醛时，应遵循由1小时温育在PBST-B在室温下透化细胞膜，允许抗体进入细胞内的抗原决定簇。此外，某些固定剂更适合于特定的抗原。例如，甲醛的功能以及用于小蛋白的固定，而甲醇和丙酮是非常适合的大分子复合物36的固定。 果蝇睾丸的免疫染色也观察染色质结构和微管骨架29,37已如前所述。这里（课程C），程序已被优化，含有基因标记的中心体蛋白质结构的分析。我们提供这个程序来指导个人谁是经验不足在果蝇精巢工作的详细说明。此过程还包括修改通过使用s到改善睾丸，如保存和形态学iliconized盖玻片和带正电的显微镜玻片上。

果蝇睾丸是一个合适的器官系统来研究各种细胞和发育过程，并进行了审查，多年来1-9广泛。这篇稿子的重点是利用果蝇睾丸学习中心体，一个保守的细胞器。如在其它系统中，在有丝分裂，减数分裂，并ciliogenesis 10 果蝇睾丸功能的中心体。中心体是由一对被称为中心粒由称为pericentriolar材料（PCM）的复杂蛋白质网络包围的微管为基础的结构中的。中心粒对由高龄产妇中心粒和小女儿中心粒的。随着细胞的进行对有丝分裂，无论是中心粒分离，复制，并获得了大量的PCM，最终形成两种截然不同的中心体。包含原始母中心粒的中心体被称为母中心体和离心泵含原子中心粒osome被称为子中心体。 果蝇睾丸是理想的学习中心体生物学的分子基础，荧光显微镜用于各种原因。 <ol><li>大部分被在睾丸需要中心体生物学果蝇蛋白质是保守的真核生物中，这表明相关的人类和其它物种的中心体生物学见解可以通过研究该中心体在果蝇来获得睾丸1,11-14。 </li><li>在果蝇突变体进行分析提供了显著的优势，因为，不像许多其他车型，在中心体果蝇突变不是胚胎致死的，因此允许中心体功能的经典遗传分析。 果蝇的这种独特的功能，是由于产妇的贡献存在于胚胎开发板的的关键阶段仍然存在pment。1,11-14。因此，人们可以一）研究基因突变，完全消除中心体的形成和b）研究形成于胚胎早期，通过在突变情况下产妇的贡献产妇的贡献已经耗尽后，一个正常的中心体的命运（该方法的原理在11中描述）。 </li><li>可使用该标签的中心体基因编码的荧光标记功能转基因。很多这些线的使用蛋白质的自身启动子来驱动转基因的转录，以防止强烈过表达。这是特别重要的，因为蛋白质的过表达通常导致与中心体功能1,11的分析干涉工件。 </li><li>中心粒和中心体是唯一在整个长果蝇精子，从而使该中心体由成像快速，简单的分析。 </li><li>在精子发生和中心体b连续的步骤iology按时间顺序组织沿睾丸，开始于睾丸尖与精子干细胞的中心体并在具有降低的中心体的大小和活动中成熟的精子细胞（ 图1和2）睾丸的底部为止。这使得在精子发育的不同阶段容易识别中心体的功能和分析。 </li><li>睾丸很容易从雄性幼虫，蛹和成人27解剖。 </li><li>在精子发生，中心体，其中心粒继续通过多种成分，结构和功能状态的有丝分裂，减数分裂和纤毛的形成发挥作用。在这些过程中，中心体组装，复制，迁移，锚到细胞的特定部分，成熟，分裂，并创建一个纤毛。此外，成熟的精子细胞的中心粒产生了名为PCL 1一个中心粒前体。另外，在成熟精子的中心体经历了一个方法CALLED中心体减少，从而失去在PCM的许多组件和中心粒（ 图1和2）。因此，在本研究中睾丸允许一个处理中心体寿命的多个方面，如中心体滞留在干细胞，中心粒复制，中心粒的稳定性，中心粒伸长，中心粒的分离和离析，PCM招聘，ciliogenesis，PCL形成，中心体减少，并且其中包括星体微管成核。 </li><li>最后，中心体生物学研究是由果蝇的其他知名的特性，都使得它的最佳模式生物的生物学研究资助。这些包括繁殖时间短，易于遗传学，以及随机和位点定向诱变。 </li></ol>一起， 果蝇睾丸的上述特性提供了其中中心体可以通过简便，快速，和详细的成像进行研究的模型。所描述的技术在本文中已被应用到调查中心体生物学的许多方面，包括中心粒形成11，中心粒复制15，PCM招募16，中心体17调节，并ciliogenesis 18。这些技术也被应用到研究中心体在生物学的其他领域，如减数分裂第19，主轴组件20，而在非对称干细胞分裂21在许多其他中心体的活动。 成像在中心体开始与苍蝇获得表达基因标记的中心体蛋白和隔离从雄性幼虫，蛹或成蝇睾丸睾丸。这些苍蝇都可以从几个研究小组1,11,15,22-25。幼虫睾丸生精功能包含前减数分裂各阶段和分析突变是致死的蛹或成人时是有用的。然而，后期蛹或年轻的阿杜LT睾丸最健壮和含有精子发生的所有预处理和后减数分裂的阶段，从而使它们优选用于分析。由于精子细胞的数量会随着年龄的苍蝇，使用成年睾丸也适合研究中心体在老化26的上下文中。从成蝇睾丸分离法已如前所述27。 可以通过在此处（ 图3）提出了三个相关的轨道实现中心体和它们在果蝇睾丸功能成像。选择哪一个磁道是最合适的是依赖于问题的研究者正在处理的性质。 跟踪A涉及现场睾丸成像。它是最快速的三个磁道，但只能应用时试样不需要予以保留，并进行免疫染色时是不需要的。在课程A，睾丸安装（完好，刺穿或切割）在载玻片上和下一个盖玻片小心压扁以形成易于识别的细胞单层。然后将细胞同时使用相位对比显微镜和荧光显微镜观察。使用相衬是果蝇睾丸的分析特别重要，因为它表明，不与其他形式的透射照明的可见光，从而允许快速识别精子发育28,29的各阶段的细胞的信息。然而，轨道A有两个主要的缺点。首先，细胞的形态学完整性常常受到损害一旦睾丸破裂。第二，未定影的细胞有时移动样品内，使得在指定的区域内难以多个共焦层的成像。促进特定细胞类型的分析，睾丸可刺穿或切割，以直接的方式，睾丸破裂，使得下盖玻片最低限度地影响挤压感兴趣的细胞类型的形态。通道B涉及睾丸化学固定。这条赛道需要的时间，样品制备的中间量，并且具有固定的标本可以保存供以后分析的优势。此外，固定供应，使细胞结构更坚固，在成像过程中最大限度地减少样品的运动。然而，相衬显微术变得非常少翔实化学固定后，使精子发育难以识别的某些阶段。 轨道C是最耗时的，但有额外的好处，细胞结构是固定的免疫染色，允许蛋白质，是无法用适当的遗传学标记的可视化。有用于免疫染色中心体和中心体有关的结构在果蝇精巢提供许多抗体两种商业和来自不同研究组。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/ Equipment</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments/Description</td>
 </tr>
 <tr>
 <td>DAPI</td>
 <td>Invitrogen</td>
 <td>D1306</td>
 <td></td>
 </tr>
 <tr>
 <td>Positively Charged Slides</td>
 <td>AZER Scientific</td>
 <td>EMS200A+</td>
 <td></td>
 </tr>
 <tr>
 <td>Feather Microscalpel</td>
 <td>Electron Microscopy Sciences</td>
 <td>72045-30</td>
 <td></td>
 </tr>
 <tr>
 <td>37% Formaldehyde or Paraformaldehyde</td>
 <td>Fisher Scientific</td>
 <td>BP531-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate Buffered Saline (PBS)</td>
 <td>Boston Bioproducts</td>
 <td>BM-220</td>
 <td></td>
 </tr>
 <tr>
 <td>18x18 mm coverslips number 1.5</td>
 <td>VWR</td>
 <td>48366 205</td>
 <td></td>
 </tr>
 <tr>
 <td>Nail Polish</td>
 <td>Electron Microscopy Sciences</td>
 <td>72180</td>
 <td></td>
 </tr>
 <tr>
 <td>Sigmacote</td>
 <td>Sigma-Aldrich</td>
 <td>SL2-100 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>Fisher Scientific</td>
 <td>A412-4</td>
 <td></td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>Fisher Scientific</td>
 <td>A949-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Triton-X100</td>
 <td>Sigma-Aldrich</td>
 <td>T9284-100ML</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Jackson ImmunoResearch</td>
 <td>001-000-162</td>
 <td></td>
 </tr>
 <tr>
 <td>RNAse A</td>
 <td>5 prime</td>
 <td>2900142</td>
 <td></td>
 </tr>
 <tr>
 <td>Filter paper</td>
 <td>Whatman</td>
 <td>1001-055</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass engraver</td>
 <td>Dremel</td>
 <td>290-01</td>
 <td></td>
 </tr>
 <tr>
 <td>TCS SP5 confocal microscope</td>
 <td>Leica </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Mounting Media</td>
 <td>Electron Microscopy Sciences</td>
 <td>17985-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Immuno Stain Moisture Chamber, Black</td>
 <td>Electron Microscopy Sciences</td>
 <td>62010-37</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass Coplin Staining Jar, Screw Cap</td>
 <td>Electron Microscopy Sciences</td>
 <td>70315</td>
 <td></td>
 </tr>
 </tbody>
</table>

imaging centrosomes in fly testes

中心体是保守的基于微管的细胞器，其结构和功能的变化显着地在整个细胞周期和细胞分化。中心体是必不可少的有丝分裂过程中，以确定细胞分裂轴和间期成核纤毛。介导这些动态变化的蛋白质的身份仍只是部分已知的，并且许多已牵涉在这些过程中的蛋白质的功能仍是基本的。最近的研究表明， 果蝇精子发生过程提供了一个强大的系统，以确定中心体的功能和形成的关键新的蛋白质，以及深入了解已知的球员 ​​在中心体有关的过程的特定功能。 果蝇是一个既定的遗传模型有机体突变的地方在中心体的基因可以很容易地获得和容易地进行分析。此外，在光学显微镜的灵敏度和分辨率的最新进展和健壮的遗传标记的中心体标记的发展已经改变了使用果蝇睾丸作为一种简单方便的模型系统，研究中心体的能力。本文介绍了利用遗传标记的中心体标记为新的中心体突变体进行遗传筛选，并深入了解新发现的基因的具体功能。

中心体进行多重形态和功能转换在精子发生的过程。精子发生的这种特性使得睾丸一个有用的系统研究中心体生物学的各个方面。这样一个很容易观察到的过程是中心体的伸长率。在精原细胞中，中心粒标记ANA1-GFP标志着0.6微米长的中心粒（ 图4a）。精子发生过程这个中心粒拉长，并达到近成熟精子2.5微米的长度。因为在果蝇精子中心粒是唯一长，成像可以用来做出关于中心体伸长（ 图4b）的定量陈述。也可以在一个突变体背景进行中心粒长度的分析和各种突变体已经确定，改变中心粒的生长（ 图5）。例子总是早（ALY）和炮弹 （ 可以），基因突变，arres吨精子发生减数分裂前30的发作，但不阻止中心粒伸长。在这些突变体中，精母细胞成熟的中心粒生长至约〜2.4微米相比于对照细胞的中心粒其中最大可达1.8微米。 <img src="/files/ftp_upload/50938/50938fig1.jpg" alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50938/50938fig1highres.jpg"/> 图1。精子发生和中心体生物。 A）发展干细胞和精原细胞。来自于睾丸的顶端被发现干细胞的所有精子细胞起源。每个干细胞不对称分裂，形成一个干细胞，它继承了母亲中心体和一个精原细胞祖继承女儿中心体31。由于精原细胞的形式，因为通过2囊肿细胞，从而继续围绕着精母细胞和精子细胞在整个精子包围。精原细胞分裂4次，产生16精原细胞，每块包含两个中心粒B）精母细胞减数分裂和发展。在精母细胞的发展，中心粒复制一次生成分为2双每个细胞和每囊肿64中心粒4中心粒。早期精母细胞发育过程中，每个中心粒移动到质膜，码头到它，并伸长，以形成一个类似于初级纤毛18,32-34的结构。在成熟精母细胞，中心粒的长度达到〜1.8微米。中心体在减数分裂中发挥重要作用。在减数分裂I，那些仍然附着在朝向电池中心质膜移动，并在它们的远侧末端创建细胞膜内陷的中心粒对。围绕中心粒在PCM的增长，成核星体微管，并用共定位的锭杆。在过渡到减数分裂II中，中心粒一对分隔开，以便只有一个单一的中心粒是PRESENT AT每锭杆。C）精子形成。在减数分裂II的末端，早期圆形精子细胞具有连接到套叠质膜单个中心粒。大而圆的线粒体衍生发现附近的原子核，后来开始沿着增长的轴丝在后来圆形精子细胞伸长。在精子发生，轴丝的形式和伸长细胞质内。另外称为PCL（近端中心粒等）的新中心粒结构出现不久的预先存在的中心粒1。在精子发生过程结束时，细胞共享一个共同的囊肿彼此分离，成为完全成熟活动精子。生精细胞发育（B）及精子形成（C）的图描述只有一个单元格出16精母细胞和精子细胞64，分别为每囊肿，不描绘囊肿细胞。 <a href="https://www.jove.com/files/ftp_upload/50938/50938fig1large.jpg" target="_blank">点击这里查看大</a>R图。 <img src="/files/ftp_upload/50938/50938fig2.jpg" alt="图2" fo:content-width="4in" fo:src="/files/ftp_upload/50938/50938fig2highres.jpg"/> 图2。 果蝇睾丸。整体式安装睾丸表达的泛中心粒标记安娜-1-GFP的相衬和GFP荧光显微照片的叠加。对应于面板1-3 图1中不同的区域用红色虚线A的分离）干细胞和精原细胞，B），精母细胞减数分裂和，C）的精子形成。 <img src="/files/ftp_upload/50938/50938fig3.jpg" alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/50938/50938fig3highres.jpg"/> 图3。在果蝇睾丸跟踪迈向成像中心体。 图4。中心体的断裂伸长率在精子发生。 A）的每个阶段显示了相位对比图像（左），荧光图像（中），和中心体的放大的图像（右）。的相位对比图像演示基于细胞，细胞核，核仁和线粒体的位置和形态的特定细胞的阶段。荧光图像显示的DNA用DAPI染色（蓝色）。中心体图像显示中心体标记的Asterless-GFP（ASL，顶部图像）和ANA1-GFP（ANA1，底部图像）。白色圆圈突出显示在这两个相位对比图像和荧光图像的细胞膜。虚线白圈和灰色圆圈中的荧光图像突出的髓核和核仁的位置，分别。黄色箭头指向的Y染色体一些循环。红色虚线圆圈标记线粒体。阶段1-6指的是29和13-17级是 ​​指细胞阶段的描述32。第1阶段:初级精母细胞。确定为小细胞睾丸的尖端。第二期甲:极地生精细胞。通过对细胞核的一侧的线粒体帽的存在下确定。第3阶段:非极性生精细胞。确定它的相对较大的尺寸比初级精母细胞和缺乏线粒体帽。第4阶段:初级精母细胞。全部3 Y染色体循环:所有的外观标识。第五阶段:成熟的初级精母细胞。在精子发生产生最大的生精细胞，确定在核大小的进一步增加。第六阶段:减数分裂初级精母细胞。 Y染色体循环瓦解和细胞核消失。阶段13:洋葱舞台精子细胞。圆形细胞含有同样大小的细胞核和线粒体。阶段17:晚精子。确定为一个细长的精子，剩下一个bundl的一部分电子商务64精子与睾丸的基地附近发现的细胞核。，核比在精囊发现成熟精子细胞稍宽。比例尺，10微米和1微米。 B）图形描绘了用于每个阶段由Asterless-GFP（蓝色）和ANA1-GFP（绿色）测量的平均和标准偏差。在第6阶段，Asterless-GFP（蓝色）和ANA1-GFP（绿色）平均值和标准偏差重叠，并且只有Asterless-GFP（蓝色）是显而易见的。在开始阶段13，Asterless-GFP不装点整个中心粒和测量都是没有确定（ND）。 <img src="/files/ftp_upload/50938/50938fig5.jpg" alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/50938/50938fig5highres.jpg"/> 图5。异常长的中心粒在第6阶段即已精母细胞，并能突变体。整个睾丸和代表性的中心体拉的荧光板AC）光镜照片通过ANA1-GFP贝莱德。注意在ALY睾丸的异常形状并能突变体，从缺乏精子细胞，导致作为减数分裂停滞的结果。D）图形描绘了平均，标准偏差，和中心粒长度的野生型数据分配， 即已和可 。对于每个数据点样本数是在括号中。比例尺，1微米。

Watch this Scientific Journal Video about Imaging Centrosomes in Fly Testes at JoVE.com

Imaging Centrosomes in Fly Testes

中心体蛋白的果蝇中精子成像是一种功能强大的方法来识别中心体生物学重要的新蛋白质以及阐明已知的玩家在这个过程中的特定功能。

在飞睾丸成像中心体

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

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15.9K 次观看.  University of Toledo. 中心体蛋白的果蝇中精子成像是一种功能强大的方法来识别中心体生物学重要的新蛋白质以及阐明已知的玩家在这个过程中的特定功能。 

视频: Imaging Centrosomes in Fly Testes

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

Ferumoxides植入人类间质干细胞在软骨缺损的标记

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

科研

生物学

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

精子发生的细胞学分析:Live和固定制剂<em&gt;果蝇</em&gt;睾丸

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

全睾丸针浸泡超快速冷却法超低温保存斑马鱼细胞

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...

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 ...

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