我们感谢托马斯·贝茨的批判性阅读和改善手稿。我们也感谢克里斯蒂娜·艾伯特和SabrinaStötzer鱼类维护。

所有动物实验均根据"实验动物护理的原则"以及关于动物保护的当前版本的德国法进行。 1.反义吗啉的制备（MO） <ol><li>要准备3毫米MO原液，加100微升灭菌，超纯水的冻干MO（在玻璃小瓶300纳摩尔）。完全溶解，加热储备溶液至65℃5分钟。密封用封口膜的小瓶中并在室温下储存（RT）下将MO原液。不冷却在MO原液，因为这可能会导致在MO的关联与药瓶墙壁。 </li><li>通过稀释的MO原液用无菌，超纯水制备1mM的MO工作溶液，并添加0.5％酚红溶液，以0.05％的终浓度。 <ol><li>要获得10微升MO工作液中，加入3.3微升MO原液和1微升0.5％酚红溶液5.7微升水。是前准备一批新的工作溶液加热MO原液至65℃5分钟。 </li></ol></li><li>在使用之前，离心机将MO工作溶液，以防止针堵塞。例如，旋转10微升的MO原液以15,000 xg离心5分钟（RT）并取下8微升上清液用于注射。 注:随着时间的推移11吗啉的解决方案，可能会出现活性损失。为了排除此，可以按照制造商提供的协议测试通过UV光谱法的解决方案，在吗啉降低的有效性尚未观察到在存储的长时间的使用wt1a的情况下，（3毫工作液储存超过一个长年）。 </li></ol> 2.设置接合对受精卵的收集<ol><li>之前显微注射下午，建立5对的Tg（wt1b:GFP）线路，每在装有可拆卸的筛子以分离繁殖容器夜间男性和女性。 注:要确保有足够的鸡蛋，可为成功注入实验中，作为接合对鱼应该是预先筛选和评价为良好"生产者"。 </li><li>第二天早上，开灯后，将男性和女性在一起，防止鱼类吃了他们的鸡蛋筛以上。 </li><li>观看产卵。一旦被放置的蛋可以在4细胞期之前被注入的量再次达到从女性（约50），单独的男性。用巴氏吸管转移将鸡蛋打入培养皿装满胚胎水用于第一轮注射。为了让鸡蛋的额外回合，把一对交配在一起，分开几次。 </li></ol> 3.显微注射准备工作注:提前执行步骤3.1和3.2。 <ol><li>要准备，注射（注射菜）期间保持在原位胚胎菜插入GL在40-45°角的屁股滑入94毫米的培养皿充满液体，透明，纯净，食品级硅。取出幻灯片当硅硬化。产生在硅层的槽。 注:另外，用1.5-3％琼脂糖代替硅和菜储存在4℃。 </li><li>产生从用针拔出器的玻璃毛细管注射针。 <ol><li>使用含有内部细丝毛细血管。 注:灯丝将有助于回填（见3.3）后向针尖的MO解决运输。 </li><li>在针拉马建立适当的设置。良好的针注射到斑马鱼应该有长而薄的技巧12。产生具有大约10毫米的长度，并在其最末端的直径为1.5-2微米的提示。例如，使用一个水平针车夫，设置如下:热= 330，拉= 0，速度= 40，时间= 100。 </li><li>检查针的下一个质量解剖显微镜和梳理那些短或过长提示。 注:针短提示趋向而这种非常长的和薄的提示弯曲触摸绒毛膜时损坏胚胎和不穿透它。 </li></ol></li><li>通过使用微加载尖端回填2微升的MO工作溶液的针，并将其插入到注射装置的针保持器。 </li><li>作为针在前端封闭，产生由捏背面用解剖显微镜下急剧镊子针的最末端的开口。另外也可以使用显微注射设备尖端轻轻推到坚硬的表面（ 如小金属块或镊子的背面）。 </li><li>校准通过注入在MO工作液入放置在刻度矿物油的液滴的注射体积。调节喷射的持续时间用于产生75微米的水滴直径以获得约200复注射体积。 </li></ol><p类="jove_title"&gt; 4。显微注射过程<ol><li>安排沿着具有朝向从在针来（朝向槽的45°角）的方向上的植物极显微注射培养皿的槽1-细胞期的胚胎。 </li><li>穿孔绒毛膜和与针蛋黄和注入1mM的MO工作溶液（0.2皮摩尔）200复成1-蛋黄至2-细胞胚胎。 </li><li>使用巴斯德吸管具有宽开口，收集所有的注射的胚胎在培养皿中填充有胚胎水中，并在28.5℃培养它们。 </li><li>当天下午，通过使用巴斯德吸管用广口取出死胚和替换胎水。 </li></ol> 5.准备胚胎的体内成像<ol><li>第二天早晨，用含有0.003％N-苯基硫脲（PTU）抑制黑化胚胎水取代胚胎水。原肠胚形成，因为它INTERF结束前不要使用PTUERES与早期胚胎发育。 注意:PTU是毒性和致畸。在任何时候都戴上手套，避免吸入以及直接接触皮肤。 </li><li> Dechorionate在所需发育阶段解剖显微镜下用尖锐的镊子的胚胎。抓住绒毛膜一个镊子，并试图抓住绒毛膜的另一侧与第二镊子。小心拉在相反方向上镊子而不破坏胚胎。 </li><li>通过用含有PTU（0.003％）和0.02％3-氨基苯甲酸乙酯甲磺酸盐（三卡因）胚胎水代替包含PTU胚胎水（0.003％）麻醉的胚胎。 </li><li>琼脂糖嵌入在一个玻璃罩底μ-菜与一个50微米搬迁电网低熔点胚胎。适当的嵌入需要一些练习。 <ol><li>制备0.7％琼​​脂糖的1ml等份在1.5ml反应管，并把它们设定为36℃的加热块上。使用巴斯德吸管具有广泛的开放TRAnsfer胚胎μ-菜。删除使用巴斯德吸管采用超薄尖多余胚胎的水和覆盖与回火琼脂糖胚胎。 </li><li>而琼脂糖仍然液体，定向在所希望的位二凝胶加载提示胚胎，关于感兴趣的结构以及对重定位网格的距离。放置的兴趣尽可能接近玻璃底部（这里:背侧向下）的结构（此处为前肾），并在紧挨着的网格。 </li><li>为获得最佳的图像质量通过放置的胚胎尽可能接近底部的感兴趣结构最小化的胚胎和玻璃底之间的琼脂糖层。除此之外，定位在靠近附近向电网胚胎但照顾该网格将不与感兴趣的结构叠加。嵌入3-4胚胎在不同的μ-菜每使用定位最好的之一。 </li><li>当琼脂糖很难足以容纳胚胎等待2-3分钟，并覆盖嵌入胚胎用含有0.003％PTU和0.02％三卡因胚胎水。倒出多余的胚胎的水或通过使用巴氏吸管超薄尖端移除。在锁定位置，以防止蒸发关闭μ-培养皿的盖子。 注:避免气泡的发生在μ-菜的，因为它们会损害图像记录。 </li></ol></li></ol> 6.显微镜注:使用配备通过结构照明的光学切片显微镜。图像记录包含以下模块软件:多通道，Z-Stack的，时间序列，软件自动对焦和扩展对焦。 <ol><li> Z-栈收购 <ol><li>反转μ-菜。玻璃底现在朝向物镜和μ-培养皿用作成像室。 </li><li>切换滑块到网格位置和使能在软件控制结构照明模式。 </li><li>校准通过下面的焦点校准向导的指示进行检查电网重点选择的通道（S）与试样（嵌入式胚胎）。 </li><li>激活的Z-Stack采集模式，并将其设置为中心的模式。聚焦到试样的中间，并设置为通过点击中心 Z-堆栈的中心平面。通过定义围绕中心位置的范围内的z堆栈的尺寸。 注:对象结构不应被第一和Z堆叠的最后面以外急剧看到。 </li><li>调整光部分之间的间距。为了达到最佳的z分辨率点击最佳按钮。基于该目标的景深的软件将设置以下奈奎斯特绕圈（50％重叠）建议的距离。 注意:如果瞄准较小的文件大小和结构许可，手动设置间距较大尺寸。设置非推荐较小的步骤只会导致更大的文件大小不增加以后人的信息。 </li></ol></li><li> 时间推移成像 <ol><li>打开时间序列工具，并设置时间序列实验的时间间隔。例如记录Z堆栈图像在一段5小时每隔30分钟。 </li><li>随着时间的推移修正焦点漂移，建立一个自动聚焦战略。检查聚焦战略对话中，从下拉列表中选择软件自动对焦并选择TL-明场通道作为参考通道。 </li><li>先从自动对焦每个时间点。为了保证优化的时间框架内一个可靠的搜索范围，设定一个固定的200微米的相对的搜索范围。这个范围是完全在扫描最大步长（采样参​​数）（覆盖范围参数）基本品质（Quality参数）。 </li><li>以提高自动聚焦精度，激活的兴趣（聚焦ROI），并位置的对焦区域内的计量所述周围的感兴趣结构的实时取景窗口焦点投资回报率。 注:如果系统缺乏软件自动对焦模块延时录制过程中自动更新中心z位置，暂停试验在给定的时间和手动调焦​​，并继续实验。 </li><li>以时间序列记录期间补偿潜在的xy漂移，定位印迹格（的成像室）的一个特定点到软件的十字线并且通过使屏幕截图保存的定位。 </li><li>开始的时间系列实验。的时间序列区间结束之前，暂停实验和，如果需要的话，定位根据截图重新调整。与实验继续。 </li></ol></li></ol> 7.图像处理<ol><li>切换到处理选项卡，选择对焦（EDF）在方法对话框中的扩展深度。由于光学切片被收购，应用最大投影算法对图像系列。 注:这里的算法将投影BRightest或从堆的复合2D图像中的第一步骤中的最暗的像素，并最终使用具有最高方差为每个时间点的结果的图像。 </li><li>使用影片导出方法了EDF图像系列创建一个时间的推移电影（ 例如 AVI或移动文件）。 </li></ol>

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作者迈克尔·格拉夫是卡尔·蔡司显微镜有限公司产生本条法器的员工。

斑马鱼已成为脊椎动物发育和建模人类疾病的研究流行的模式生物。因为斑马鱼胚胎保持透明，如大脑和心脏显影器官可以使用标准制备显微镜观察到。以转基因品系的优势与器官特异性荧光使荧光显微镜下活胎内的整个开发过程中的不同阶段器官的评估。与标准荧光显微镜成像整个器官的结构细节一个限制是信号从上方和下方的聚焦平面的物体的影响。外的焦点该光不仅导致降低图像的对比度和分辨率，它也可能会掩盖感兴趣13,14重要结构。几种技术如激光扫描confocal-，旋转盘confocal-或多光子显微镜已开发，以减少外的焦点的信息，并由此人口增加Ë图像对比度以及轴向分辨率。的替代方法，以获得光学切片是被激光和扫描自由结构照明显微镜，宽基于场的照明技术，该技术是简单的，定期显微镜15来实现。此处呈现的结果表明，光学切片，由结构照明来实现，在解剖显微镜来实现，并与扩展聚焦图像重建相结合，使得能够正常结构细节的可视化，并在斑马鱼干扰肾发展。特别是，在wt1a morphants GFP阳性细胞被分散在不同的聚焦深度，并与失焦模糊仅在一个平面的图像会低估表型的三维的复杂性。 跟随器官组织，重排或中断的动态，时间推移的荧光显微镜是一种强大尽管复杂的技术。在延时成像轻微的振动或轻微的温度波动引起的x，y或z方向上漂移。这是为了获得稳定的结果需要额外的设备（环境室，抗震动表）。该方法采用解剖显微镜的能力与录制时间推移图像，而无需额外的设备电动对焦驱动。保持稳定的聚焦，自动聚焦策略建立和用于x的校正，y轴不稳定印迹到成像室的底部的重定位网格。 胚胎的适当嵌入是在协议中的一个关键步骤。需要一些实践以放置感兴趣尽可能接近到玻璃底和中紧挨给电网的结构，而没有与它重叠。 该方法的主要优点是，它是对发育过程如在活生长和迁移的直接观察实惠和简单的工具胚胎在几个小时。此外，解剖显​​微镜特定收益如观察和扩展工作距离大视场更大的方便样本，包括整个器官的检查。然而，一些限制必须牢记。实验的暂停来检查和重新调整的定位使得费时和缺乏可靠的温度控制的改变"标准"发育时间，这是在28.5℃的斑马鱼16定义为小时受精后的过程。另一个潜在的问题是成象的受限制的深度到动物，尤其是当感兴趣结构位于胚胎或幼虫的内部。这样的结构是斑马鱼pronephric肾小球，这是位于该体节和卵黄囊之间。成像肾小球限制深度被发现是大约200微米，以后发展5天达到该距离。相反，荧光结构，其为1-ocated接近动物的表面能够被成像为一个较长的时间。例如肝细胞成像访问至少要等到9 DPF。进一步的限制是，在琼脂糖和三卡因治疗胚胎或幼虫的长期固定诱导发育迟缓和心脏水肿，分别。因为这可能与正常发展干扰，建议时间推移记录的持续时间限制到感兴趣的一定期间。以确保在感兴趣的成像结构正常发展是有帮助的比较（在末端）与该动物的整体外观保持相同的实验条件下，但没有嵌入和麻醉。 记录光学部分的Z堆叠在一段5小时和聚焦图像的扩展深度，随后计算每30分钟提供有关正常的早期事件的空间和时间信息和干扰肾发展其诱导的BŸ吗啉介导wt1a的击倒。先前执行啉拦截实验已经表明，Wt1a满足在前肾形成的早期和必不可少的作用。在肾标记17,18和转基因wt1b就业原位杂交:在固定的时间用于成像前肾发展的GFP线指向9,10-揭示这wt1a缺乏导致肾小球破坏形态和失败的足规范。相对于这些静态的方法，定时录音，直接可视化控制胚胎早期nephrogenesis和GFP阳性细胞从pronephric区域客场wt1a morphants迁移的动态。随着时间的推移跟踪错误路由的细胞的可能性允许的更详细的表型分析。 在一般情况下，在这里提出的方法提供了一种廉价和易于使用的替代复杂的，并且较少可访问本身tups通常用于延时成像如激光扫描显微镜（装备有环境舱和抗振动台）或光片的显微镜。所描述的程序来解决漂移问题，也可以应用到没有光学切片的选项，如荧光立体显微镜上执行设置时间推移的图像。 该技术不仅适用于研究肾发展，但也可以适用于监控其他器官，如心脏或肝脏的正常和有缺陷的形态。此外，该方法可以用来观察在胚胎和成年模式生物的各种更多的动态过程，如伤口​​愈合或再生。

以下原肠胚形成，器官是个体的生命周期的下一阶段。它涉及到重排，互动和经常细胞的迁移，以产生组织和器官。误差在器官的发育所依据的过程往往导致可立即或也表现出来以后的生活中的疾病。因此，了解器官已经发育生物学和生物医学研究的重大努力。为了能够调查特定器官的发育，它具有即使是通过胚胎的体壁可见。这使得能够在开发过程中发生的空间和时间变化的记录。也为了分析参与器官发生因素的重要性，它需要容易操纵。 一个生物体是非常适合于体内器官的调查是斑马鱼。其transpar与使用的荧光转基因系在组合胚胎发育过程中ency允许蛋白定位和表达动力学的观察，以及内部器官1的可视化。这提供了有关相比，哺乳动物模型，其器官都无法进入微观分析器官发育的实时调查的独特优势。此外，不同的工具可用来操纵斑马鱼的胚胎发育。旁的突变系的产生，反义吗啉代寡核苷酸（MO）可以用来敲除所涉及的某些器官的发育特定基因的活性。无论是材料订单目标剪接位点或翻译起始密码子（AUG），从而与mRNA前体或翻译的拼接干涉。 斑马鱼的胚胎肾中，前肾，是一种解剖学上简单而有价值的模型来研究肾脏发育和根的功能与肾脏疾病2上课。它仅由两个被称为肾功能单元。每个肾单位，其中包括血液过滤发生3肾小球的。进一步组件是短颈部区域以及分割小管分泌和溶质的重吸收和管道，在泄殖腔4中结束。不管其简单组合物，组织和不同细胞类型的斑马鱼前肾的非常相似的哺乳动物肾5,6。 一个因素，这是极其参与肾发展，由肾母细胞肿瘤抑制基因WT1 7编码。斑马鱼具有两个旁系称为wt1a和wt1b以重叠但不相同的模式的前肾8的发育过程中表达。通过使用GFP标记前肾结构转基因斑马鱼，它已被证明的wt1a &lt;即完整击倒/ em&gt;的或特定剪接形式导致9,10分别胚胎肾严重或轻微畸形。 这里所描述的方法允许在斑马鱼胚胎正常和受损nephrogenesis的时间推移分析通过使用配备用于经由结构照明光学切片荧光解剖显微镜。一般光学切片允许收购其中只包含焦点对准信息图像。外的焦点可通过各种方法，如数学算法（ 例如去卷积），光学设计（ 例如共聚焦激光扫描显微镜）或两者的组合（ 例如 ，结构照明）来避免信息。 诱导肾发育缺陷，我们使用的反义MO针对注入的转基因斑马鱼线wt1a（TG（wt1b:GFP））。这条线表示在肾小球，颈部和前的GFP荧光细管9,10的一部分。相比于更复杂的设置时间推移的录音与光学切片，如激光扫描或光片的显微镜，变焦显微镜是易于处理和更便宜。此外，结构照明设备可以很容易地与传统的荧光设备（ 如荧光灯，过滤器），并提供特定的解剖显微镜等优势，如延长工作距离和大视场相结合。带漂移问题解决了没有使用通常需要稳定业绩的补充设备（环境试验箱，抗震动表）。为了校正成立自动聚焦策略和与压印搬迁网格成像室中利用重新调整之后在x或y方向上的漂移的定位焦点的漂移。 所提出的方法也可以应用到显微镜没有光学切片的选项，如荧光立体声米icroscopes并提供更复杂的设备通常用于发育组织和器官的动态时移记录的替代品。

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Control Morpholino</td><td>Gene tools, LLC</td><td>Standard control oligo</td><td>Prepared&amp;nbsp;control&amp;nbsp;oligo</td></tr><tr><td>&lt;em&gt;wt1a&lt;/em&gt; Morpholinos&amp;nbsp;</td><td>Gene tools, LLC</td><td>customized</td><td>Designted to target the first splice donor site, sequence as published in Ref. &lt;sup&gt;9&lt;/sup&gt;</td></tr><tr><td>0.5% Phenol red solution</td><td>Sigma</td><td>P0290</td><td>Injection tracer</td></tr><tr><td>peqGOLD universal agarose</td><td>peqlab</td><td>35-1020</td><td>To prepare microinjection dish</td></tr><tr><td>Glass capillaries&amp;nbsp; (GC100F-10)</td><td>Hugo Sachs Elektronik</td><td>30-0019</td><td>To generate injection needles&amp;nbsp;</td></tr><tr><td>Microloader tips</td><td>Eppendorf</td><td>5242956003</td><td>To load the microinjection needle</td></tr><tr><td>Dumont forceps&amp;nbsp;</td><td>Fine Sience Tools</td><td>91150-20</td><td>To generate an opening at the needle tip&amp;nbsp;</td></tr><tr><td>Embryo water (0.3x Danieaus&amp;acute; solution)</td><td></td><td></td><td>1x: 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO&lt;sub&gt;4&lt;/sub&gt;,&amp;nbsp; 0.6 Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;, 5 mM HEPES, pH: 7,5; Do not add methylene blue!</td></tr><tr><td>Graticule 5 mm/0.05 mm</td><td>Science Services</td><td>PY68039-02</td><td>For calculation of injection amount</td></tr><tr><td>Pasteur pipette 137 mm, 4.8 ml</td><td>Roth</td><td>E305.1</td><td>To collect eggs, to transfer embryos and to remove dead embryos</td></tr><tr><td>Pasteur pipette with ultra-thin tip 157 mm, 3.5 ml</td><td>Roth</td><td>E304.1</td><td>To remove excess of water&amp;nbsp;</td></tr><tr><td>N-Phenylthiourea (PTU)</td><td>Sigma</td><td>P7629</td><td>For suppression of melanization</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td><td>Sigma</td><td>E10521</td><td>To anethesize zebrafish</td></tr><tr><td>Low melting agarose</td><td>Biozym</td><td>850111</td><td>For embedding&amp;nbsp;</td></tr><tr><td>&amp;micro;-dish&lt;sup&gt; 35 mm, high&lt;/sup&gt; Glass Bottom Grid-50</td><td>ibidi</td><td>81148</td><td>Imaging chamber</td></tr><tr><td>Gel loader tips</td><td>Hartenstein</td><td>GS05</td><td>To orient the embryo for proper embedding</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micropipette puller (model P-97)</td><td>Sutter Instrument</td><td></td><td>To generate injection needles&amp;nbsp;</td></tr><tr><td>Micromanipulator</td><td>Saur Laborbedarf</td><td></td><td>For microinjection</td></tr><tr><td>Thermomixer copact</td><td>Eppendorf</td><td></td><td>Thermoblock for tempering the low melting agarose</td></tr><tr><td>SZ61 (Stereomicroscope)</td><td>Olympus</td><td></td><td>For injection control</td></tr><tr><td>Axio Zoom. V16</td><td>Zeiss</td><td></td><td>For embedding and imaging</td></tr><tr><td>ApoTome.2 slider</td><td>Zeiss</td><td></td><td>For optical sectioning</td></tr><tr><td>AxioCam MRm</td><td>Zeiss</td><td></td><td>For image acquisition&amp;nbsp;</td></tr><tr><td>ZEN 2012</td><td>Zeiss</td><td></td><td>Imaging software</td></tr></tbody></table>

analysis of zebrafish kidney development with time-lapse imaging using a dissecting microscope equipped for optical sectioning

为了了解器官，需要记录组织发育过程中出现的空间和时间的改变。这里所描述的方法允许在斑马鱼胚胎正常和受损肾发展的时间推移分析使用装备结构照明和z堆栈采集荧光解剖显微镜。为了显现nephrogenesis，转基因斑马鱼（TG（wt1b:GFP））用于与荧光标记的肾脏结构。肾缺陷是由对Wilms肿瘤基因wt1a，已知可用于肾发展的关键因素的反义吗啉代寡核苷酸的注射触发。 实验装置的优点是用在X，Y或Z方向上没有附加设备重新调整的动作简单的策略的变焦显微镜的组合。规避是受温度变化和机械振动引起的焦距漂移，自动聚焦策略应用，而不是利用通常需要环境室。为了重新调整由于XY-漂移位置的变化，采用了与印迹搬迁改造成像室。 相比于更复杂的设置为与光学切片的时间推移记录如共焦激光扫描或光片的显微镜，变焦显微镜是容易处理。此外，它提供了具体的解剖显微镜效益，如高景深和扩展工作距离。 研究这里提出器官的方法，也可以用荧光立体显微镜不能光学切片的使用。虽然不限于高通量的，这种技术提供了更为复杂的设备通常用于发育组织和器官的动态时移记录的替代品。

时间推移显微镜是一种强大的技术在一段较长的时间来观看动态生物过程。时间推移成像过程中经常发生的问题是，在X，Y或Z方向的移动，被称为漂移，这是由温度变化和机械振动引起的。这里介绍的方法实现定时录制在解剖显微镜的斑马鱼胚胎或幼虫的荧光标记的结构不受环境室和抗振动台。 为XY-漂移的补偿，将试样包埋在成像室带有印迹重定位网格（ 图1）。相关的软件工具的十字线网格的一个特定点的位置被用于检体返回到预调整的位置（ 图2A）。所呈现的结果是通过使用放大显微镜获得为经由结构照明（ 图2B）的光学切片的集成滑块。对于连续焦点校正，自动对焦的战略正式成立。在这种策略中，软件自动对焦使用基于各时间点之前最大对比度找到的焦点。这使定义焦平面随后被设置为Z堆栈购书中心的计划。为了最大限度地减少在样品中光毒性，透射光明信道被用作参考信道，因为它在自动对焦步骤（ 图2C）可以足够短的曝光时间。 已申请在转基因斑马鱼线的控制和wt1a morphant胚胎（TG（wt1b:GFP））肾脏发育的比较分析的方法。在早期nephrogenesis此行显示在中间的中胚层绿色荧光，被肾脏祖细胞从9,10和后来出现在成形管和肾原基（ 图3）。 隔时图像记录表明nephrogenesis在控制啉注射的胚胎相比是未注射的那些未改变的（结果未显示），并如下早期斑马鱼肾发展3所描述的步骤。在20 HPF显影pronephric管是可见的，在其前部的提示，细胞球形聚集，代表成形肾原基可以被检测出来。在接下来的时间，小管和肾原基生长和稍后的原基开始在中线（ 图4，视频2）融合。相比之下，nephrogenesis严重的wt1a morphant胚胎破坏。尽管GFP阳性的管状结构是在20高倍视野可见的，它们显得更弥漫和欠发达国家。此外，已形成​​没有合适的肾原基。最显着的区别为t他在这个时间点控制胚胎，但是，是在显影前肾（ 图4，视频2）以外的大量的荧光细胞的外观。随后，这些细胞离开pronephric字段和腹侧迁移（ 视频2）。 在控制胚胎和转基因wt1b线wt1a morphants肾脏发育都通过不同的固定时间点9,10拍摄图像以前相比。与此相反的静态方法，定时录音允许按照正常nephrogenesis而造成wt1a枯竭肾脏祖细胞的错打的动态。 <img alt="图1" src="/files/ftp_upload/53921/53921fig1.jpg" /> 图1:市售商会与搬迁网格嵌入式胚胎的示意图 。这道菜有四宫格，每个细分为50μm的重复距离，印迹到玻璃盖玻片底部。要成像的胚胎被嵌入倒挂，在靠近观察肾方结构，但没有与电网覆盖。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53921/53921fig1large.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/53921/53921fig2.jpg" /> 图2:调整对漂移的补偿时间推移成像和结构照明的网格投影搬迁电网的不同位置被带进图像中心（黄色十字标记），并作为一个参考点以后的XY对准在小变化的情况下，。红色矩形表示感兴趣区域（ROI）的自动对焦策略（A）使用的区域。光学切片WA■通过结构照明获得。该图像示出了在正确的校准（B）的后焦平面伸出的格子结构。对于自动对焦策略（红色矩形）的投资回报率被设定为涵盖独特的胚胎结构的高对比度（在这里个体节），允许可靠的自动对焦到感兴趣（C）的结构。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53921/53921fig2large.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/53921/53921fig3.jpg" /> 图3:转基因 wt1b:GFP胚胎绿色荧光在发展中肾背（A）或横向（B）传输和荧光图像叠加显示有前左侧。 NP，肾原基; PT，pronephric肾小管;一般般螨。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53921/53921fig3large.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/53921/53921fig4.jpg" /> 图4:wt1a敲除 打乱胚胎肾发展代表，从时间推移录音对焦的图像扩展焦深。在控制啉注射胚胎，肾发展呈现出不断增长的肾小管和肾原基开始其在中线融合的正常进展。与此相反，wt1a morphants不能形成适当的肾原基和GFP阳性细胞的巨量是pronephric字段之外。 （NP，肾原基; PT，pronephric肾小管）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53921/53921fig4large.jpg" target="_blank">请点击此处查看该网络的放大版本</a>古尔。 <img alt="电影1" src="/files/ftp_upload/53921/53921movie1.jpg" /> <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53921/co-MO.mov" target="_blank">补充视频1.在控制啉注射胚胎正常肾发展延时录制。 （点击下载）。</a>视频显示管如何生长和肾原基开始融合。在20 HPF开始，图像30分钟间隔取一段5小时。 <img alt="电影2" src="/files/ftp_upload/53921/53921movie2.jpg" /> <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53921/wt1a-MO.mov" target="_blank">在wt1a morphant胚胎感到不安nephrogenesis的补充视频2.定时录制。 （点击下载）。</a>视频显示GFP阳性细胞的迁移出pronephric地区。在20 HPF开始，图像拍摄于30英里N个间隔了一段5小时。

Watch this Scientific Journal Video about Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning at JoVE.com

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

这里介绍的方法允许在斑马鱼胚胎器官发育的时间推移分析，通过使用荧光解剖能够执行光学切片和调整的简单的策略来纠正焦点和平面漂移的显微镜。

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

analysis-zebrafish-kidney-development-with-time-lapse-imaging-using

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

10.7K 次观看.  Leibniz Institute on Aging - Fritz Lipmann Institute (FLI). 这里介绍的方法允许在斑马鱼胚胎器官发育的时间推移分析，通过使用荧光解剖能够执行光学切片和调整的简单的策略来纠正焦点和平面漂移的显微镜。 

视频: Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

科研

发育生物学

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

斑马鱼发展长期成像的多功能安装方法

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

多囊肾一种可能的斑马鱼型号:击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

医学

Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, proliferation, and differentiation. Time-lapse imagining offers unique insight to the behavior of cells during eye development because it allows us to visualize oculogenesis in vivo. Zebrafish are an excellent model to visualize this process due to their highly conserved vertebrate eye and their ability to develop rapidly and externally while remaining optically transparent. Time-lapse imaging studies of zebrafish eye development are greatly facilitated by use of the transgenic zebrafish line Tg(rx3:GFP). In the developing forebrain, rx3:GFP expression marks the cells of the single eye field, and GFP continues to be expressed as the eye field evaginates to form an optic vesicle, which then invaginates to form an optic cup. High resolution time lapse imaging of rx3:GFP expression, therefore, allows us to track the eye primordium through time as it develops into the retina. Lightsheet microscopy is an ideal method to image ocular morphogenesis over time due to its ability to penetrate thicker samples for fluorescent imaging, minimize photobleaching and phototoxicity, and image at a high speed. Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software. The resulting dataset can provide valuable insights into the process of ocular morphogenesis, as well as perturbations to this process as a result of genetic mutation, exposure to pharmacological agents, or other experimental manipulations.

Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software.&#160;

使用rx3:GFP转基因斑马鱼通过光片显微镜可视化眼部形态发生

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, ...

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

生物学

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

成年斑马鱼肾脏的解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

斑马鱼光学杯形态形成的四维成像

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...