We appreciate assistance from members of the fly community, particularly Dr. Denise Montell, Dr. Lynn Cooley, and Dr. Pernille Rorth, for reagents. We thank Flybase, the Bloomington Drosophila Stock Center, and Developmental Studies Hybridoma Bank for information and providing fly stocks and antibodies, respectively. LM is supported by the Department of Education&#160;Grant, Graduate Assistance in the Areas of National Need (GAANN) training fellowship (P200A120017) and by a NIGMS Initiative for Maximizing Student Development Grant (2 R25-GM55036). A portion of the microscopy work was supported by NSF MRI grant DBI-0722569 and the Keith R. Porter Core Imaging Facility. Research was supported in part by a NSF CAREER Award (1054422) and a Basil O&#8217;Connor Starter Scholar Award from the March of Dimes, both awarded to MSG.

1.解剖卵巢管的<ol><li>转约15 2-4日龄雌性果蝇和少数的男性用加入活性干酵母新鲜飞食品小瓶中。使用棉作为插件的小瓶，并添加几滴水到棉花，以保持湿度较高。 </li><li>在25℃的培养箱中14-16小时处小瓶最大化阶段8-10卵室的数目。 注:取决于温度和所需的阶段孵化时间会有所不同。 </li><li>制备夹层媒体，0.1M KPO 4和NP40的解决方案，根据需要，为第二天（见材料）。 </li><li>麻醉使用CO 2的苍蝇，和解剖显微镜下放置。吸管几滴夹层媒体到玻璃中的凹陷滑动每个空腔。 </li><li>持在每手钳在一个大约30°角，以该表顶部和东方1雌蝇的翅膀下。用你的非惯用手，拿起女性使用镊子，在一个抓她nterior腹部，靠近胸部，并淹没她进入了抑郁症的幻灯片（ 图1A插图）解剖媒体。 </li><li>与其它对镊子，把握外骨骼在腹部的腹后和刺穿。抢卵巢在后端，然后将它们拉出来腹部并放入新鲜培养基上的凹陷滑动的另一侧。 （ 图1A小图）。避免拉开肠道。丢弃的尸体。 </li><li>与其他雌性果蝇重复，直到所有的卵巢解剖。丢弃的男性。 </li><li>用一对镊子，保持在后端卵巢（靠近最大卵腔），并用另一只手用一对镊子夹住的最前蛋室（germarium）之一。 （ 图1A）。 </li><li>缓慢而稳步推进，拉动了卵巢管链出卵巢鞘。继续解剖出卵巢管单独直到所有所需的卵室为Removed。 </li><li>重复步骤1.7-1.8为所有解剖卵巢。 </li><li>完成后，用塑料移液管转移到卵巢管一管0.6毫升。 </li><li>让卵巢管沉降到试管底部，然后进行定影和染色步骤。 </li></ol>卵巢管2.免疫荧光（IF）染色<ol><li>小心地从卵巢管用吸管取出术媒体。一定不要删除任何卵巢管和蛋室。留出大约50微升卵巢管和解剖媒体。 </li><li>加入50微升16％多聚甲醛的150微升0.1M的KPO 4缓冲在管，并添加解决鸡蛋室，以解决这些问题。孵育而摇动10分钟。注意:多聚甲醛是有毒的。 </li><li>除去固定液和用0.5ml NP40洗涤缓冲液冲洗两次。 </li><li> 15每个在RT（分钟）洗两次。 </li><li>参考标准IF协议15后续步骤。 <ol><li>简单地说，固定后，加在适当的稀释度的一抗孵育O / N在4℃。在NP40几次洗涤，然后加入第二抗体（1:200稀释度）。 </li><li>在NP40洗几次，然后加入DAPI（1:1000）进行10分钟，然后重复洗涤。一旦中频协议完成后，在PBS中加入200微升50％甘油的蛋腔室和让坐在在RT搅拌2小时。 </li><li>除去溶液，用在PBS中的70％的甘油代替，并盖上箔。将样品在4ᵒC直到准备安装。同时，继续执行步骤3。 </li></ol></li></ol>甘油果冻幻灯片3.事前准备<ol><li>测量至少4甘油果冻用抹刀毫升，并填补了玻璃细胞培养管中途大关。熔体甘油果冻在水浴中于55℃进行30分钟。 </li><li>倒入一小滴甘油，约300微升，从原液到两个显微镜载玻片。使用组织，传播甘油薄拉揭掉在每个幻灯片。这提供了一个基础，可以很容易地去除甘油果冻步骤5.6。 </li><li>切尖掀起了过滤1000微升枪头。使用剪切枪头1000-1500微升的温暖甘油果冻慢慢转移到每个显微镜载玻片。请注意，以防止泡沫。 </li><li>让甘油果冻滑梯坐5分钟RT设置。 </li><li>将在60毫米×15毫米的培养皿每个甘油果冻幻灯片，并盖上保鲜膜。发生在4°CO / N。可选，存储甘油果冻幻灯片长达5天4℃。 </li></ol> 4.安装蛋钱伯斯<ol><li>使用样品从步骤2.5，吸管若干3微升的跨越一个甘油果冻滑70％甘油蛋室滴。继续移液3微升滴，直到所有的鸡蛋厅在这张幻灯片。确保每一滴含有至少十蛋室。同时，热甘油果冻的培养管在55℃水浴。 </li><li>将鸡蛋室甘油果冻滑解剖显微镜下。调整倍率以使只有一滴卵腔中的视图（ 图1B）的字段。 </li><li>用30号针头的勺子，拿起所需的阶段卵室。必要时，独立所需卵子从卵巢管链用针作为一个刀腔。 </li><li>以避免碎片可以与成像干扰，蛋室传送到第二甘油胶冻滑动，具有面向左（ 图1E -1）前。重复，直到至少有7蛋室排成一列（ 图1C）。 </li><li>直到所有所需的卵室被转移到第二甘油果冻滑形成七个新栏目。 </li><li>撕下一小块组织和扭曲的。用这个来轻轻触摸每一个从蛋室吸收多余的PBS甘油。小心不要删除蛋室。 </li><li>切断尖端的过滤200微升枪头。使用此转移甘油果冻覆盖蛋室的所有列的150微升。 注意:需要以覆盖列甘油胶冻的量是基于卵腔的阶段和列的数目。量可以进行调节。 </li><li>让装蛋室坐5分钟，在室温直到甘油胶冻的顶层完全固化（ 图1D）。 </li><li>装的鸡蛋在商会60毫米×15毫米的培养皿的地方滑动，并用保鲜膜覆盖。储存在4°CO / N，使甘油果冻层凝固在一起（的地方在黑暗中，如果有关注漂白）。 </li></ol> 5.准备蛋钱伯斯的成像<ol><li>放置装蛋室幻灯片解剖显微镜下。调整倍率以使蛋室只有一列是在视场。 </li><li>采用45°角的微型手术刀，切straigHT线沿右侧的蛋室（接近卵腔的后侧）的第一列。 （ 图1D-E） </li><li>接着，切断上述第一蛋室的顶部和下面的最后蛋室在列。在这一点上的卵腔的列应三边裁剪。 </li><li>使用microknife，沿着列的左侧切片，尽可能接近到的蛋腔（ 图1E -3）的前侧。 </li><li>吸取100微升冷1X PBT过切列。 </li><li>使用圆边刮刀除去多余的甘油胶冻周围的蛋室并丢弃切列的三侧，留在幻灯片的其余部分。 </li><li>将在蛋室前侧了晋级的锅铲和列分开初级甘油果冻块。 </li><li>准备样品或者反转（5.8.1）或直立（5.8.2）显微镜。 <ol><li>对于倒置显微镜:转让吨蛋室，他列与蛋室前侧朝下盖玻片。直到甘油果冻中的所有列已被删除，并安装重复步骤5.2-5.8。添加一对夫妇的50％PBS-甘油滴上的鸡蛋室每个块，以防止其干燥。像鸡蛋室直接盖玻片。 </li><li>对于正置显微镜:蛋室的柱转移到与前侧朝上载玻片。直到甘油果冻中的所有列已被删除，并安装在显微镜载玻片重复步骤5.2-5.9。添加一对夫妇的50％PBS-甘油滴上蛋商会和盖砖每块有＃1个半盖玻片。 </li></ol></li></ol>

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The authors have no competing financial interests.

在这里，我们描述的方法来安装和图像小，从终端的角度上制定的蛋室。成像蛋室常见的技术横向的意见进行了优化，主要允许内 - 外滤泡细胞的精确的可视化，当用荧光抗体染色。在观看多个焦平面使用的Z-堆栈或三维重建助剂，但仍有不足之处为亚细胞的椭圆卵腔室（ 图2D）的极点的分辨率。虽然这可以部分地用诸如超分辨率成像的最新的显微技术来克服，这些方法是昂贵的并且不总是可用的。我们已经适应直立成像协议果蝇胚胎24,25用于小得多卵腔室。还有，在方法的几个关键的修改以考虑的卵腔的小得多的尺寸相比胚胎。所描述的技术的一个关键组成部分是物理组合通道ATION的个体腔室，这是不需要的胚胎。另一个变化是采用了第二甘油果冻滑动安装（4.4）中。卵室的规模及其附件通过柄细胞与其他鸡蛋室需要处理的针和引到灭亡甘油果冻。因此，蛋室必须传送用针到一个新的甘油胶冻滑动安装（4.5）。直立成像允许通过横向或水平的意见模糊不清的领域可视化，露出约卵子发育过程中的组织机构的新信息。这导致对图案形成事件的新见解，我们提出，该方法可以用来探索卵子发生的其它方面。 我们已经确定在成功安装直立的几个关键步骤。我们发现，是否有必要完全从护套剥离时除去卵巢管链，以及所需的蛋室必须从ovariol切Ë链。未能从卵巢管免费鸡蛋室，很难拿起正确的阶段，卵室（步骤4.3）。我们建议与少量甘油胶冻由等分入管的工作的时间。不要加热甘油果冻超过两次，因为反复加热改变其一致性，防止其凝固正常（3.1）。使用过滤嘴，以防止吸果冻进入移液器。允许甘油胶冻到在4℃下固化在载玻片至少8小时。缩短这一步使得安装蛋室难（3.6）。以确保吸收所有的PBS-甘油从装蛋室（4.6）是重要的。如果过多的液体存在时，它可以防止甘油胶冻的顶层粘附到底层。在这种情况下，卵腔室可以浮起并改变位置。为了获得最佳的成像效果，甘油果冻块必须削减接近蛋室前侧的身体可能（5.4）。在这个太多甘油果冻侧降低的图像质量由于试样和盖玻片之间的距离增加。 有固定样本中甘油果冻的一些注意事项。首先，它增加了样品和物镜之间的距离。这个距离在高放大倍率，特别是在使用的油的目标，可以建立困难聚焦在试样上，这可能会导致图像质量差。鉴于此，这是非常重要的，以能开出的甘油胶冻块非常接近卵腔室。虽然甘油果冻是光学透明的，但它确实defract一些轻。另一个限制是，抗体的信号可能由于物镜的工作距离和甘油胶冻的厚度的组合降低。增加这两个初级和/或次级抗体的浓度有时会有助于减少这个问题。尽管通过抗体变化，我们通常使用二次蚂蚁的两倍浓度取得了较好的图像ibody在直立成像相比在标准横向成像的使用量。我们知道没有替代甘油果冻安装蛋室直立。其它材料如琼脂糖和聚丙烯酰胺进行尝试，但都defracted光更加严重，引起的清晰度损失而成像。因此，这种安装方法是最好的，目前可用于标准显微镜的使用。 虽然我们专注于边界小区规格和运动的开始阶段，不同阶段的蛋室可以在这个协议，只有一些小的调整使用。最重要的是，针的规格可以改变操纵不同大小和发育阶段的卵腔室。对于阶段7-10，一个地下30针效果很好，因为鸡蛋室一拍即合的斜面。对于早期蛋室的较大规格针头（较小斜面）应使用并且类似一个小计（大斜角）应该用于购买ST中年蛋室。在任何一个旧的发展阶段将是太大，此计（4.3）。还蛋室的任何一方可以使用该技术通过安排的卵腔的甘油胶冻块具有朝向目标感兴趣区域进行成像。 安装在甘油胶冻支持小样本必须在多个上下文中有用的潜力。这种策略可以用于可视化的不同类型的小的，固定来自其它生物体组织，特别是当它是不够的，研究样本仅有一个视角。一旦这一技术被掌握，它可以用来查看卵腔室中的任何期望的角度，这取决于如何卵腔定位在果冻。这种方法允许创建细胞组织的一个更好的三维理解独到的见解。蛋室与免疫荧光抗体染色结合直立成像允许精确检测蛋白质s和细胞 - 细胞接触标准安装条件下，将是不可能的。我们计划在映射允许从上皮边界小区集群的聚结和分离的细胞 - 细胞相互作用使用此方法。此时，直立成像技术不能与活样品使用，但我们也正在努力克服这一障碍。我们预计这种方法也适用的果蝇卵子发生等方面来研究，或者可以适应图像等小型组织的新角度。

研究果蝇提供了巨大的见解的各种现象的遗传调控。特别是，出现了对蛋发展广泛的研究，因为卵子发生提供了一种易于处理的方式来调查许多不同的发育过程，包括组织图案化，细胞极性的变化，细胞周期的开关，和翻译调控1,2,3,4。卵子发生过程中一个重要的事件形态是一组细胞称为边缘细胞（5审查）的规格，收购蠕动，和迁移。因为细胞迁移是动物形态的一个主要特点，并且因为这个过程的基因调节是非常保守，在果蝇确定的机制很可能是在其他情况下非常重要的。因此，我们正在调查边境细胞迁移的分子控制。对于我们的研究，我们已经开发出一种新的方法来观察开发鸡蛋的前极，其中，边缘细胞产生，检查他们详细介绍了如何发展。 内卵巢，卵腔室中的不同发育阶段沿链称为卵巢管，其包封在薄鞘（ 图1A）存在。每个鸡蛋室将继续形成一个鸡蛋。在卵子发生，几种不同类型的细胞必须协调发展的方式。 D.在果蝇卵室由16个种系细胞，包括1卵母细胞，由单层滤泡上皮6包围。出现在上皮的前部和后部磁极少数特殊细胞，称为极性细胞。 6-8边缘细胞起源于卵室的前上皮细胞，诱导细胞极性5,7。在中期卵子发生（9级），边界细胞从他们的邻居和分离的滋养细胞之间迁移的卵室5,7的后到达卵母细胞。这个动作必须完成而边缘细胞停留在周边两个非能动极性细胞集群，使之成为一个类型的集体细胞迁移。边界小区集群的成功迁移确保蛋壳的珠孔，这是必要的施肥适当发展。 前极性细胞指示边境细胞命运通过激活的信号传导级联。极性细胞分泌细胞因子，不成对（UPD），其结合跨膜受体，无圆顶（DOME），对卵母细胞发育的8,9级8中相邻的毛囊细胞。 UPD的结合导致的Janus酪氨酸激酶（JAK）磷酸化转录（STAT）8,10,11的信号转导和激活。 STAT然后移动到细胞核来激活转录。慢边缘细胞（SLBO）是一种转录因子是STAT的直接转录目标，也需要边境细胞迁移12。蛋室外侧观点表明吨帽子STAT活性调节横跨前壁上皮8,11,13-阿松香三烯梯度。最接近极性细胞毛囊细胞具有活化的STAT的最高水平，因而它们变得边缘细胞和侵入相邻的种系组织。 理解边界细胞是如何内指定，并从上皮脱落，我们需要观察如何组织的组织结构。如果我们把鸡蛋室来自前上的角度来看，我们希望在周边极地细胞的毛囊细胞STAT活动的径向对称。一个端视图还更精确地之前和脱离比不同的焦平面相比较的细胞中表现出的膜蛋白和细胞间的接口的差异。因为蛋室是长方形和彼此附接由柄细胞，它们定居到载玻片横向，从而难以观察前架构。因此，有关的细胞在卵腔的磁极多的信息具有被推断，从横向的看法。虽然一些信息可以通过光的部分，光散射，光漂白算法的3-D重建，并在Z轴分辨率较差限制得到使该信息少在没有像超分辨率显微镜14昂贵的技术详细和可靠。其他种类的部分的基于成像（例如，电子显微镜或薄样切片机的切片）需要大量的操作的组织，包括脱水，增加了对工件的可能性。因此，我们开发了一种新的方法来形象D.果蝇卵室，而直立。这种方法已经被证明非常有用在阐明如何游动细胞注定（见代表性的成果和曼宁等人，正在审查 ），并且很可能是更广泛的有价值的卵子等方面的研究。

<table><tbody><tr><td>0.1 M Potassium phosphate Buffer (KPO&lt;sub&gt;4&lt;/sub&gt; Buffer)</td><td></td><td></td><td>Add 3.1 g of NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;H&lt;sub&gt;2&lt;/sub&gt;O and 10.9 g of Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (anhydrous) to distilled H&lt;sub&gt;2&lt;/sub&gt;O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 month at 4&amp;deg;C.</td></tr><tr><td>16% Paraformaldehyde aqueous solution, EM grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td>methanol-free to preserve GFP fluorescence</td></tr><tr><td>30G x 1/2 inch needle&amp;nbsp;</td><td>VWR</td><td>BD305106</td><td>Regular bevel</td></tr><tr><td>Active Dry Yeast</td><td>Genesee Scientific</td><td>62-103</td><td>To fatten female flies</td></tr><tr><td>Bovine Serum Albumin</td><td>PAA-cell culture company</td><td>A15-701</td><td>Used in NP40 Wash Buffer</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11295-10</td><td>Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection</td></tr><tr><td>DAPI (4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride)</td><td>Sigma Aldrich</td><td>D9542</td><td>5 mg/ml stock solution&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16140-071</td><td>Added to supplement dissection medium (10%)&amp;nbsp;</td></tr><tr><td>Glass culture tube</td><td>VWR</td><td>47729-576</td><td>14 ml</td></tr><tr><td>Glass Depression Slides</td><td>VWR</td><td>470019-020</td><td>1.2 mm Thick, Double Cavity</td></tr><tr><td>Glycerin Jelly&amp;nbsp;</td><td>Electron Microsocpy Sciences</td><td>17998-10</td><td>Mounting media</td></tr><tr><td>Glycerol</td><td>IBI Scientific</td><td>IB15760</td><td>70% in PBS</td></tr><tr><td>IGEPAL CA-630</td><td>Sigma Aldrich</td><td>I3021-500ML</td><td>(interchangable for Nonidet P-40)&amp;nbsp; Used in NP40 Wash Buffer&amp;nbsp;</td></tr><tr><td>Leica Fluorescent Stereoscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Leica SP5 Confocal Microscope</td><td>Leica Microsystems</td><td></td><td>40x/0.55NA dry objective&amp;nbsp;</td></tr><tr><td>Micro spatula&amp;nbsp;</td><td>VWR</td><td>82027-518</td><td>Stainless steel</td></tr><tr><td>Microscope Slide</td><td>VWR</td><td>16004-368</td><td>75x25x1 mm</td></tr><tr><td>NP40 Wash Buffer</td><td></td><td></td><td>50 mM TRIS-HCl, 150 mM NaCl, 0.5% Ipegal, 1 mg/ml BSA, and 0.02% sodium azide</td></tr><tr><td>Penicillin-Streptomycin-Glutamine</td><td>Life Technologies</td><td>10378-016</td><td>Added to supplement dissection medium (0.6X)</td></tr><tr><td>Petridish- Polysterine, sterile</td><td>VWR</td><td>82050-548</td><td>60 W x 15 H mm</td></tr><tr><td>Phosphate Buffer Saline</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td>10 packs of Powder</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma Aldrich</td><td>P3786-500G</td><td>Used in 0.1 M KPO&lt;sub&gt;4&lt;/sub&gt; Buffer&amp;nbsp;</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P9791-500G</td><td>Used in 0.1 M KPO&lt;sub&gt;4&lt;/sub&gt; Buffer&amp;nbsp;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Schneider&amp;rsquo;s Insect Medium</td><td>Life Technologies</td><td>&lt;br/&gt; 11720018</td><td>With L-glutamine and sodium bicarbonate</td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>S2002-25G</td><td>Used in fluorescent antibody staining</td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>S3014-500G</td><td>Used in NP40 Wash Buffer</td></tr><tr><td>TRIS-HCl</td><td>IBI Scientific</td><td>IB70144</td><td>1 M TRIS-HCl, pH 7.4</td></tr><tr><td>Volocity 3D Image Analysis Software</td><td>PerkinElmer</td><td></td><td>For processing confocal Z-stacks</td></tr></tbody></table>

upright imaging of drosophila egg chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

直立成像方法使我们能够看到细胞中的前滤泡上皮的直接的组织在阶段8，一种一般标记毛囊细胞命运，眼中的缺席（EYA）蛋白质，以及核DNA的DAPI标记，表明即使表达横跨细胞的这个领域，并表明，所有的细胞可被视为具有类似染色强度（ 图2B"）。蛋白响应于细胞因子UPD调节，但是，显示出可变图案和表达水平。极地细胞释放UPD顶部之前，这个阶段卵子发育16,17的，所以我们预计STAT被激活径向对极地细胞，有时这种情况。不过，通常来说，我们观察到STAT的下游目标，包括SLBO，有更复杂的表达模式。例如，GFP报告为SLBO表达出现在大多数，但不是所有的，细胞包围的极性细胞（ 图2B，和看到曼荷兰国际集团等审查人 ）。我们注意到，在E-钙粘蛋白的表达/本地化（ 图2B&#39;）轻微差异，这可能反映了变化的附着力，但更多的工作，将需要量化和解释了这一结果。这些细微之处检测不到通过分阶段卵腔的横向三维重建（ 图2C-D）。在毛囊细胞的顶 - 基底轴的差异也可以用竖直方法区别开来。我们使用了记者线为环管蛋白Visgun（VSG），其是存在于血浆膜18（ 图3A）的顶端区域。在毛囊细胞的顶部，检测直立成像证实VSG表达，靠近极细胞（ 图3B）的最窄部分。 我们还发现，所述直立成像方法工作得很好，以查看前上皮在阶段9卵腔室。在这个devel的opmental阶段，边界细胞凝聚一起绕极性细胞（ 图4A）。我们可以观察到使用终端上的成像（ 图4B-C），这些压实集群。如在阶段8中，我们发现EYA表达在所有的上皮细胞（未示出）相似的水平，而slbo -GFP以及激活，核的STAT标志着能动边界细胞（ 图4B和4C）。 FAS3蛋白的定位指示的极性细胞极性细胞接口（ 图4B）。此外，在这两个阶段8和9，我们可以看到大护士细胞，用DAPI或鬼笔环肽染色的皮质肌动蛋白（未示出），这表明该方法也将在生殖细胞或生殖细胞的研究中有用的指示-follicle细胞相互作用。 <img alt="图1" src="/files/ftp_upload/52636/52636fig1.jpg" /> 果蝇卵室的图1.直立安装秒。 （A）果蝇卵巢表明卵室，套，和卵巢管。描示出了用于从卵巢鞘拉动卵巢管慢慢钳子的位置。插图显示解剖飞的定位。雌蝇的腹面朝上。 A-前，P-后，安装蛋室（BD）程序。（B）小降蛋商会在70％甘油PBS，在解剖立体显微镜观察。黄色箭头表示卵巢管链;黄色箭头表示个别蛋室。红色箭头表示甘油的PBS液滴的边缘。布置在薄层的固化甘油胶冻蛋室（C）的列。黄色箭头表示个别蛋室。（D）第二薄层融化甘油果冻覆盖卵室列嵌入他们。黄色虚线表示的地方应该削减O / N孵育4ᵒC后进行。红色箭头指示甘油胶冻的第二层的边缘。红色的数字表示切割蛋室列（水平顶部和底部切口未示出）的顺序。前是向左。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52636/52636fig1large.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/52636/52636fig2.jpg" /> 图2.成像的早期阶段8卵腔的前上皮。 （A）阶段8 慢速边缘细胞（slbo）报告基因蛋室的横向视图（基因型:slbo -Gal4，UAS-mCD8-GFP）19,20，用DAPI染色和初级抗体指示或slbo&gt; GFP。犰狳（ARM）是β连环蛋白的飞同源物。前（A）是左侧，后（P）的权利。（BB"）最终的意见鸡蛋茶的MBER。阶段8 slbo-基因记者卵室Z堆叠图像三维重建垂直安装，沾上一抗所示。红色星号表示极性细胞。 DAPI标记的核。（B&#39;）的 slbo&gt;的GFP表达仅在推定边缘细胞（SLBO）。 E-钙粘蛋白（E-CAD）是定位于膜和富含边缘细胞粘附分子。（B&#39;）的眼睛缺席（EYA）被发现均匀表达于所有毛囊细胞除外极性细胞的细胞核。（C）的一个slbo -reporter级8卵室用DAPI染色（蓝色）和抗GFP（SLBO，绿色），臂（红色）的标准的横向图，而FAS3（白色）。插图示出了轴:X轴是向下（绿色箭头），Y轴是朝向左边的（红色箭头），并且Z轴是朝向观察者（蓝色）（D）中 ，为了比较，以新的技术。 ，此面板显示了端视CREA泰德由三维重建，具有Volocity软件处理，使用Z堆叠在C插图中所示的蛋室的横向图像显示转向轴:X轴是向下（绿色箭头），Z轴是现在向左（蓝色箭头），并且Y轴是朝向观察者（红色）。各个单元被模糊，由于在Z轴分辨率低;因此，在B所示的新方法代表了显著成像改善。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52636/52636fig2large.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/52636/52636fig3.jpg" /> 图3.在阶段8卵腔的滤泡上皮侧向和前结构域比较蛋白的表达。 （A）visgun（VSG）阶段8卵室- GFP记者的横向视图重建&lt;SUP&gt; 21-23，染色针对GFP或ARM（白色）的抗体。 DAPI标记在蓝色的细胞核和大的护士细胞核是显而易见的。 VSG定位于毛囊细胞的环运河（绿色）。（BB&#39;），最终在意见Z堆栈阶段8 VSG-GFP记者卵室的图像的一个鸡蛋chamber.Three三维重建，沾满初级抗体所指示的。 SLBO蛋白（红色）在两极和边境细胞核检测。 ARM标志着卵泡细胞膜和富集边缘细胞; DAPI标签细胞核。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52636/52636fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52636/52636fig4.jpg" /> 图4.成像早期9蛋室。 （A）的早期阶段9 SL的侧面观博-reporter卵室。边界细胞（绿色）内的前上皮（白色箭头）已聚集。 BC）最终的意见的蛋室。三维重建从早期阶段9 slbo-基因报告蛋室Z堆叠图像的，使用针对该蛋白的一级抗体所指示或GFP染色。（B）的 slbo&gt;的GFP表达于边缘细胞（绿色） ，而Fasciclin 3（FAS3，白色）的高度局部化的两个极性的细胞之间的膜; DAPI标记的核（蓝色）。品红星号表示极性的细胞。（C）的两个核（激活）STAT和slbo&gt; GFP表达被发现只在边缘细胞，而E-钙粘蛋白（E-CAD）的马克毛囊细胞的细胞膜，和DAPI表示细胞核。红色星号表示极性细胞。染色结果示单独为slbo&gt; GFP（C&#39;）和STAT（C&#39;） </stroNG&gt;。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52636/52636fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Upright Imaging of Drosophila Egg Chambers at JoVE.com

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

直立成像<em&gt;果蝇</em&gt;蛋钱伯斯

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

upright-imaging-of-drosophila-egg-chambers

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Upright Imaging of Drosophila Egg Chambers

7.6K 次观看.  University of Maryland Baltimore County. The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

视频: Upright Imaging of Drosophila Egg Chambers

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

在成像钙<em&gt;果蝇</em&gt;在蛋激活

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

为介观荧光体层摄影术在体内成像的发展果蝇</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

生物学

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

使用微计算断层扫描的 果蝇黑色素加斯特 全动物成像

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

使用实时成像可视化氧化损伤对果蝇卵室的影响

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

果蝇卵母细胞的核迁移

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

直立成像果蝇胚胎

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

用 CaspaseTracker 生物传感器检测 Anastasis体内

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

医学

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

直立成像

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实时成像

在成像钙

为介观荧光体层摄影术在体内成像的发展果蝇

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

使用微计算断层扫描的果蝇黑色素加斯特全动物成像

使用实时成像可视化氧化损伤对果蝇卵室的影响

果蝇卵母细胞的核迁移

直立成像果蝇胚胎

用 CaspaseTracker 生物传感器检测 Anastasis体内

果蝇蛹腹部免疫

直立成像

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使用微计算断层扫描的 果蝇黑色素加斯特 全动物成像

使用实时成像可视化氧化损伤对果蝇卵室的影响

果蝇卵母细胞的核迁移

直立成像果蝇胚胎

用 CaspaseTracker 生物传感器检测 Anastasis体内

果蝇蛹腹部免疫

使用微计算断层扫描的果蝇黑色素加斯特全动物成像