The authors have nothing to disclose.

1.编写C的线虫胚胎时间推移显微镜使用微球<ol><li>通过使用蠕虫拾取（铂丝）成一滴M9的缓冲区传送妊娠成虫和由第一剧烈搅拌，然后用眼睛睫毛安装到牙齿拾取/枪头将每个蠕虫的M9相邻滴擦去菌或用尖锐的玻璃毛细管。 </li><li>稀释包含20微米直径的聚苯乙烯珠10倍于M9缓冲液（1升M9缓冲液含有3克KH 2 PO 4，6克的 Na 2 HPO 4，5克NaCl的分散性，和，高压灭菌（20分钟，120°后C）中，添加额外的1毫升的1M 硫酸镁溶液）。 </li><li>做一个口吸管从任何类型的薄玻璃毛细管（ 例如，熔化用来拉注射针点毛细管或毛细管），大致0.5米长的一块橡胶，硅树脂或类似材料管（最好是透明的）中，200微升枪头，200μl的PCR管的盖子，疏水性过滤器为小的注射器（例如，一个0.2-0.45微米孔径和15-50毫米直径的尼龙，聚四氟乙烯或类似材料过滤器），和一个1000微升枪头。这种类型的嘴吸管确保没有生物材料可以与实验者接触得到。 </li><li>通过首先插入200μl的移液管尖的尖端插入管子组装吸管和用PCR管具有中央穿孔，所用的毛细管的直径相匹配的盖将其插入。一个非常尖锥子或制剂的针可以用于产生穿孔。 </li><li>画一条细细的毛管，将其插入穿孔。插入过滤器进入软管的另一端，并插入一个1000微升枪头作为口形件（ 图1）。 </li><li>通过切割蠕虫横向在外阴用干净的刀片或手术刀解剖妊娠成人。 注:通常情况下，很多的胚胎将通过电子邮件切割后xpulsed。然而，如果需要早期胚胎，他们有时必须从使用眼睛睫毛或尖端针胎体释放。 </li><li>吸取一滴稀释的聚苯乙烯珠分散体（〜4微升）的到24x60毫米的玻璃盖和转移胚胎到使用口吸管这种下降。 </li><li>小心将一个小的（ 例如，18×18或20×20毫米）覆盖的玻璃上滴顶部，确保降已经全面展开。 注:降也应该足够小不要触碰小护罩玻璃的边缘。 </li><li>密封安装液化凡士林。添加一滴浸油的24x60毫米盖玻片一侧，进入到"时间推移显微镜"部分。 </li></ol> 2.准备成人C的线虫动物使用纳米珠定时显微镜注:一般情况下，该协议用于安装成年动物是从文献的协议的适配。 18。与此协议，它是能够进行成年动物的长期时间推移共聚焦显微镜。 <ol><li>准备一个1.5％（重量/体积）的M9的缓冲区琼脂糖溶液，煮沸（1分钟，100℃水浴或微波）。使用较高浓度的琼脂糖不推荐，因为这可能会导致肠和/或性腺的部分挤压。 </li><li>坚持下来2-3层胶带上每两个显微镜载玻片。将幻灯片无两幻灯片用胶带的胶带。 </li><li>使用移液管头，并放置一滴在中间滑动件的中心的热琼脂糖溶液。迅速放置另一个滑动到压降，以便它支持在每一侧的带层。这将创造约10毫米直径的圆扁琼脂垫。 </li><li>放一滴100nm的珠子溶液（未稀释，这是2.6％（重量/体积））送到垫上。传输1-10清洁成虫进入下降了蠕虫挑。与18×18毫米的玻璃盖和凡士林密封在普罗特解释ocol 1。 </li></ol> 3.定时显微镜<ol><li>将一滴浸油到显微镜载玻片或玻璃盖三明治。使用明在低倍率（5倍或10倍镜头）定位样本。 注:限制暴露于蓝光尽可能多地（特别是线虫的胚胎）， 比如，不要用落射荧光显微镜发现/聚焦样本，但使用明来代替。 </li><li>切换到更高的放大倍率（40倍或60倍的目标），把样品放入对焦（无论是中心平面或样品的顶部/底部的飞机，这取决于采集软件）。 </li><li>设置采样间隔30飞机在1微米的间距记录每1分钟。万一一个自动化XY-或者XYZ阶段是可用的，多点可以被记录在一个区段。切换到荧光成像，并再次检查的重点。 注意:尽量避免使用高激光功率，而使用稍微更长的曝光时间在非常低的强度。测量获得的图像立刻，无论是在图像采集程序或ImageJ的以避免饱和，其中作物的动态范围，亦指过度的辐射暴露。 </li><li>开始录制时间推移系列。 注:要排除分析文物，开始样品扫描的长（3-10分钟）的时间间隔，并逐渐增加时间采样到所需的时间间隔。执行统计分析时发育时间以及结束点在两种情况下应该是相似的。另外，通常有必要评估光毒性和存活率。因此，恢复从安装标本长期时间推移显微镜后，检查他们是否继续开发/直播。 </li></ol> 4.重建与IMOD三维细胞形状模型注:IMOD软件可以从IMOD网页:http://bio3d.colorado.edu/imod/。安装软件的描述ð那里。如果使用的是Windows操作系统，Unix的工具，必须安装，也可发现作为一个整体上的IMOD的网页。此外，存在对IMOD网页（http://bio3d.colorado.edu/imod/doc/3dmodguide.html）一个极好编译3dmod可用介绍。 <ol><li>打开Unix外壳，键入"IMOD"。在IMOD窗口将打开。选择与原始数据（理想的层叠TIF或相似），从该生成的三维模型的文件。 </li><li>通过使用信息窗口（向上和向下的滚动工具）定位在ZAP窗口的对象的底部和顶部。 </li><li>开始追踪细胞轮廓。重要的是，确保每个细胞是一个对象。首先每个单元的顶部平面，并使用该信息窗口中创建第一轮廓在那里。 Z中向下滚动，并为每个Z平面中的新轮廓。 </li><li>根据需要在3D建模做同样的尽可能多的细胞。不要忘了一个新的小区启动时创建新​​的对象。 ñOTE:有许多不同的方式进行轮廓跟踪和一些有用的插件。请访问IMOD网页和在线教程了解详细信息。在这里所示的情况下，默认设置已被使用。 </li><li>打开"模型视图"窗口，检查对象及其轮廓。 </li><li>在"模型视图"工具栏上选择"编辑" - "对象"。相应的编辑窗口将打开。 </li><li>为了模拟细胞填充和封闭的对象，选择"网格"为"绘制数据类型"和"填充"作为"绘画风格"。在可滚动选择点击"啮合"在窗口左下角。请在选择该选项的"帽子"在右下角。检查尽可能多的对象必须点击"网全部"。该方案将产生从轮廓的烟囱固体物质。的z采样因子可以通过改变"Z尺度"，在"查看"窗口被调整。 </li><li>移动/旋转3D objecT（"编辑" - "查看"），并保存快照或动画（"文件" - "快TIFF文件为..."或"电影/蒙太奇"）。 </li></ol> 5.跟踪荧光标记的结构与Endrov 注意:为了跟踪细胞分裂残余，使用的菌株具有核或质膜标记小区识别（例如，H2B，PH-PLC-d1）和一个细胞分裂残余标记（例如，NMY-2，ZEN-4 ）。核或质膜标记是很重要的，以确定哪些细胞继承了细胞分裂残余。 <ol><li>快速装载和易用性，原始数据必须被转换为TIFF文件（例如，在ImageJ的）。接下来，打开Endrov并加载文件，选择从文件菜单中的"加载文件"选项进行分析。点击工具栏上的"2D查看器"图标。 </li><li>通过点击"适合范围"图标（蓝色图标右下角调整直方图在Endrov屏幕）。 "文件名" - - "Imageset" - 进入"数据"将XYZ分辨率的"通道" - "设置XYZ分辨率"。类型为X，Y和Z， 例如，5，5，1值如果X和Y轴分辨率为0.2微米，样品Z.每1微米</li><li>对于标注的核，移动到感兴趣的平面，然后点击"2D查看器"。从下拉菜单中选择"天堂"，然后选择"新建血统"。 </li><li>通过点击并拖动的核心，它可以被标记。要命名的核心，右击并选择从下拉菜单中选择"重命名粒子"。以增加或减少该圆的直径，拖动上标记圆圈的左侧显示的箭头图标。为了纪念核的中心平面，点击右边的图标。当使用质膜标记，大球体可以得出，将覆盖大部分细胞。 </li><li>要继续在时间和飞机，选择"框架"和"Z"选择底部的工具栏。后进入下一个时间点，调节其相应标​​记细胞核的圆的位置或大小。重复该步骤直至跟踪的细胞分裂。 </li><li>当有一个细胞分裂，标记子细胞核和切换到"天堂观众"。这可以通过点击顶部工具栏上的"Windows"图标来选择。马克同时所有三个核，进入"天堂"选项顶部的工具栏并选择"关联父"。 </li><li>当细胞跟踪完成后，切换到信道标记的细胞分裂残用于跟踪。要切换频道，可点击该文件名工具栏的左下角。细胞分裂残余（或任何其他结构）可以如上述的核进行跟踪。 </li><li>当关闭后返回到软件，就必须去"2D查看器" - "天堂" - "编辑"，并点击将要编辑的谱系号。 </LI&gt; </ol> 6.与PIVlab分析皮层肌动球蛋白流注:PIVlab是一个免费的软件:http://pivlab.blogspot.de/;它可以从MATLAB命令行环境中键入PIVlab_GUI调用。当与PIVlab工作，建议保存图像系列在一个单独的文件夹中。 <ol><li>开始从"文件菜单"一个新的会话。通过点击"载入图像"按钮加载图像文件。 </li><li>选择排序风格1-2，2-3，...并导航到包含所需图像序列的目录。选择图像，然后点击"添加"按钮，导入。 </li><li>进入"分析设置"，然后选择"排除（投资回报率，面具）"。另外，按"Ctrl + E"。使用按钮"画掩模（秒）当前帧"失活的图像/秒（即，胚外区）的不希望的部分。 </li><li>选择"PIV设置"FROM的"分析设置"菜单。另外，按"Ctrl + S"。选择"FFT窗口变形"为所需的PIV算法。 注意:快速傅立叶变换（FFT）的降低了计算成本，但是只建议的信号是远高于噪声和如果所研究的粒子的位移不超过一半所分析的通区域（见6.5）。 </li><li>输入以下值三遍，这是审问，后续图像之间的强度互相关的领域:通过1:讯问犯罪嫌疑人64像素面积与32通2步:讯问犯罪嫌疑人32像素的面积与16的步骤选择从窗口变形的"线性"下拉菜单。选择高斯2 * 3点法的亚像素位移估计。 </li><li>选择"分析"！从分析菜单，并使用按钮"分析所有帧"开始进行分析。 </li><li>对于后处理从选择"向验证"";后处理"菜单和设置标准偏差（标准偏差）滤波器阈值7，并适用于所有的帧。 </li><li>选择"导出参数/修改数据"，从"暗算"菜单。从下拉菜单中选择"矢量[像素/帧]"。调整向量和高通滤波器的平滑度，并适用于所有帧。 </li><li>要计算所有帧的平均载体，设置"使用的框架"，以1:结束，然后点击"计算平均向量"按钮。 </li></ol>

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Research in the lab of CP is funded by the Deutsche Forschungsgemeinschaft (EXC 115; FOR 1756) and the LOEWE Research Cluster Ubiquitin Networks. CP is further supported by the European Union Framework Program 7 (Marie Curie Actions Project 326632).

通过对象跟踪的发展，特别是核跟踪，已经能够阐明C.中央构图机制线虫胚胎发育1,23,24。扩展这一战略提供更高的吞吐量，它最近已经可以发现更多的构图规则，并提出一种方法如何演绎构图规则从头 10。对于许多突变体，但是，精确的构图缺陷，还是一个未知数。此处描述的方法的工具，可有助于其澄清。重要的是，现在已经很清楚，近年来，虽然许多其他模型系统不遵循像C.一个不变的发展线虫 ，目标跟踪和量化的跟踪数据的分析是确定发展机制和疾病的机制的一个重要工具。 此处示出的方法特别适合于这种情况:要么太有挑战性实现更复杂和/或自身产生的软件解决方案，或简单地太昂贵实现商业软件工具，分别。重要的是，这里所讨论的工具使研究者加载和独立地在其上仪器他们已经记录分析的任何数据。此外，如果专有的图像采集软件的使用，ImageJ的提供生物格式插件，可以在专有格式读取数据，然后将它们保存在标准格式（JPEG，TIFF）。 在依赖于时间的经过显微镜所有协议的一个关键步骤是选择条件权获得足够的对比度显微镜图像，并且，在同一时间，以减少辐射照射到试样。这在很大程度上取决于所用显微镜的类型和它被一般建议使用诸如旋转盘或单个平面照明显微镜快速扫描技术。具体地，对于PIV皮质动力学，旧版本点扫描的显微镜可能太慢，以允许以足够的分辨率和速度来执行有效的分析捕获皮质信号。因此，总是关键仔细评估辐射暴露和测试样品的可行性。另外，虽然这里讨论的协议使用micobeads装入胚，但也可以使用琼脂糖垫，这只是需要更多一点的时间来准备，但是更便宜，并与一些经验同样可重复的。然而，应该指出的是琼脂糖垫和纳米珠的组合为成虫的长期固定化的优良的方法，因为它避免使用任何麻痹剂，例如，左旋咪唑 。 应当指出的是，这里讨论的工具也有其局限性，因为它们不执行完全自动数据分析。然而，对于像细胞分裂残余的结构，它通常有一定难度，以确定一个分子标记物只发tains这种结构，这对于可靠的跟踪是至关重要的。因此，一个重要的未来的挑战在于，包括像机器学习和我们在这里讨论的易于使用和开放存取软件适应性检测/分割算法的特点。其中的一些特征可以在其他软件模块可以发现，在可与重复跟踪处理更长的时间间隔特定的软件，或者可以比较大集的跟踪数据 （如，StarryNite / AceTree 25或Nucleitracker4D 26，其目的是为了用组蛋白融合蛋白或纱锭27轨核，或西米的BioCell 28）。这些工具可以是更复杂的，当瞄准结构比其它核的跟踪来实现。尽管应跟踪的特定结构，这些程序通常是当大量的图像数据将被分析非常有用。重要的是，上述的软件模件之间的兼容性上课。 当与其他模型系统的工作（例如，飞胚胎，哺乳动物培养细胞等），这里提出的方法也非常适合作为上述追踪直向同源结构。更重要的是，独立地被调查的模型系统中，不同的亚细胞结构，例如，中心体，核仁，微粒，囊泡等可以容易地跟踪，仅采集参数必须被调整，以确保对象磁道完全捕获。

用荧光蛋白质，基因组工程，光学显微镜，和计算机软，硬件的稳步提高，现在有可能在一个前所未有的时空分辨率来记录的多模式生物发展。这使研究人员询问了无法事先解决问题或重新已知的发育过程，以寻找忽视的方面。这一进展已经引发了定量发育生物学，其目的是改变定性的，非正式的模型转化为定量模型被彻底的测量和统计分析领域。 跟踪细胞和亚细胞结构使得有可能导出胚胎发育，神经系统的活动，或细胞分裂1-12的定量模型。到了C的早期发育过程中追踪细胞分裂残部线虫胚胎，我们可以最近发现，他们遵循一个立体类型化的道路，构成了重要的偏光因素13,14。 在这里，协议提出，使定量发育生物学的方法对非专业人士访问。重点在于三个直接的，免费的工具，是可实现的，有访问标准共聚焦显微镜和计算机的任何实验室。这些包括一个协议，以产生三维细胞形状，一个协议来跟踪细胞分裂残余，和协议来定量描述皮质肌动球蛋白动力学。线虫C.线虫用作示例性情况下，然而，这里讨论的方法和工具适于多种其他生物模型 ，例如，培养的细胞，组织外植体，组织体或球状体，其它胚胎等问题一般情况下，一些这里显示的分析也可以用流行的开源工具的ImageJ（http://imagej.nih.gov/ij/docs/index执行。HTML;或斐济，"电池列入"版本的ImageJ，http://fiji.sc/Fiji），这些很多可用的插件不同的定量分析。然而，节目这里讨论的目的，以解决特定的问题。 首先，IMOD，图像处理，建模和显示套件可以被用于从电子或光镜15连续切片三维重建。 IMOD还包括从任何方位观看3D数据的工具。其次，Endrov，设计成执行图像分析网络或一个扩展插件架构的基础上轨道（等等）的，数据处理，和注释，用ImageJ的插件兼容性16的Java程序。它包含超过140图像处理操作，并在模型和原始数据分别显示一个可扩展的用户界面。它的源代码可以在https://github.com/mahogny/Endrov找到。第三，PIVlab，A M专家组组成工具数字粒子图像测速，它允许用户以定量和定性分析粒子流场17。使用这种方案需要包括图像处理工具箱（http://mathworks.com）MATLAB的许可证。 PIVlab是被设计来定量描述的流程的程序。它计算的速度分布，规模，涡度，散度，或图像对或系列中的剪切。对于这一点，它互相关的图像对的图像（称为"遍"在协议部分）的小区域来导出最可能的粒子的位移。此交叉相关产生，可以在使用直接互相关或快速傅立叶变换（FFT），分别在空间域或频域中进行分析的相关矩阵。 此处所使用的设备是倒置显微镜配备有尼普科夫（&#39;纺丝&#39;）盘，一个EMCCD，488和561 nm的标准固激光器和20X空气或40倍或60倍的计划/复消色差透镜油或水浸泡的目标。但是，它也可以与其他成像方式执行时间推移成像，例如，点- ，线-或片扫描基于激光显微镜，多光子显微术，以及落射荧光显微镜并结合反卷积或结构化照明。使用尼普科夫盘系统的优点是非常快的图像的获取，特别是如果一个流模式（连续运动和扫描在z方向的对象）是可用的。此外，为了提高分辨率，在EMCCD前面1.5倍放大扩展器都可以使用。

<table><tbody><tr><td>Stereo microscope</td><td>Motic/VWR</td><td>OT4005S</td><td>Stereo microscope for dissection and mounting</td></tr><tr><td>Polybead Polystyrene Microspheres,&amp;nbsp;</td><td>Polysciences</td><td>18329</td><td>Embryo mounting</td></tr><tr><td>20 &amp;micro;m</td><td></td><td></td><td></td></tr><tr><td>Polybead Polystyrene Microspheres,&amp;nbsp;</td><td>Polysciences</td><td>876</td><td>Adult animal mounting</td></tr><tr><td>0.1 &amp;micro;m</td><td></td><td></td><td></td></tr><tr><td>Microscope slides</td><td>VWR</td><td>631-0902</td><td>Adult animal mounting</td></tr><tr><td>Cover glass 18x18 mm</td><td>VWR</td><td>631-1331</td><td>Embryo/adult mounting</td></tr><tr><td>Cover glass 24x60 mm</td><td>VWR</td><td>631-1339</td><td>Embryo mounting</td></tr><tr><td>Scalpel</td><td>VWR</td><td>233-5455</td><td>Embryo dissection</td></tr><tr><td>Silicone tubing</td><td>VWR</td><td>228-1501</td><td>Tubing for mouth pipette</td></tr><tr><td>30 mm PTFE membrane filter</td><td>NeoLab</td><td>Jul-01</td><td>Filter for mouth pipette</td></tr><tr><td>Capillary tubes</td><td>VWR</td><td>621-0003</td><td>Pipette tip for mouth pipette</td></tr><tr><td>Vaseline</td><td>Roth</td><td>E746.1</td><td>Embryo/adult mounting</td></tr><tr><td>Agar</td><td>Roth</td><td>5210.5</td><td>Adult animal mounting</td></tr><tr><td>Potassium-di-hydrogenphosphate</td><td>Roth</td><td>P018.2</td><td>M9 buffer</td></tr><tr><td>Di-sodium- hydrogenphosphate</td><td>Roth</td><td>P030.2</td><td>M9 buffer</td></tr><tr><td>Sodium chloride</td><td>Roth</td><td>3957.1</td><td>M9 buffer</td></tr><tr><td>VisiScope Spinning Disc Confocal System</td><td>Visitron Systems</td><td>n/a</td><td>Confocal microscopy</td></tr></tbody></table>

tracking and quantifying developmental processes in c. elegans using open-source tools

定量捕捉发育过程是非常重要的衍生机理模型和关键识别和描述突变表型。在这里，协议提出了准备胚胎和成人C.线虫动物的短期和长期的时间推移显微镜和用于跟踪发育过程和定量的方法。提出的方法都是基于C.线虫菌株可从线虫遗传学中心和开源软件，可以在任何实验室可以轻松实现使用的显微系统的独立。一个三维细胞形状模型的使用建模软件IMOD甲重建，使用多功能图像分析程序Endrov，和皮质收缩流的使用PIVlab分析荧光标记的亚细胞结构的手动跟踪（时间分辨数字粒子图像测速显示了MATLAB工具）。它是讨论如何将这些方法也可以部署吨Ø定量捕捉不同的模型 ，例如，细胞跟踪和谱系等发育过程跟踪，跟踪囊泡流动。

通过使用协议2，3和4中，在野生型C.性腺延时成像线虫的成人进行（株OD58（UNC-119（ED3）III; ltIs38 [PAA1;馅饼-1 :: GFP :: PH（PLC1delta1）+ UNC-119（+）]），从一个种系的启动子表达的膜标记物）。着眼于性腺的转弯，从显微镜数据（图2）产生的生殖细胞的3D模型。此模型允许的变化来分析细胞大小而细胞中转形成远端到近端臂，揭示了脊柱的组织和个体细胞对脊柱触点的大小（也见的http://www.wormatlas。组织/雌雄同体/生殖％20LINE / Germframeset.html）。 接着，协议1，3和5被用于执行C的长期时间推移显微镜线虫的胚胎（株CHP52这是应变RW10226的横（UNC-119（ED3）III; itIs37 [馅饼-1P :: mCherry :: H2B ::饼1 3&#39;UTR + UNC-119（+）]; stIs10226 [他-72启动HIS-24 :: mCherry翻译融合让-858 3&#39;UTR + UNC-119（+）]）和应变LP162（NMY-2（CP13 [NMY-2 :: GFP + LoxP位]）一）。在该菌株中，有可能按照两个核（通过mCherry组蛋白融合蛋白），并通过基因组修饰非肌肉肌球蛋白II基因座，其中一个的GFP已经集成在帧通过CRISPR / Cas9技术19细胞分裂残余（ ）同时采用双色时间推移显微镜（图3时，左图）。通过lineaging两种结构在Endrov中获得的模型显示了细胞分裂残继承13,14前面描述的千篇一律的模式。而且，从lineaging数据，磁迹的每个细胞和细胞分裂残余（图3，右小图）和细胞分裂残余辉时间（基于NMY -2- GFP -信号），以及相关的细胞分割时序可以得到（&lt;STRONG&gt;图4所示为只在ABA血统）。当使用等离子体的膜标记物，此外，还可以观察到的细胞分裂残内化的时间点。 最后，通过使用协议1，3，和6中，短期的时间推移显微镜具有高时间分辨率的皮层非肌球蛋白II（NMY-2 GFP）（记录的z堆叠之间间隔5秒）被执行。这里的重点通过比较野生型和胚胎的RNAi贫为的Rho GTP酶激活蛋白的RGA-3 20,21之间的这种流动位于皮质偏振流动动力学的差异。类似于最近的观察22，流动在野生型胚胎主要是沿胚胎的长轴（图5，左小图），而流在RGA-3的RNAi胚胎是正交于该轴。这是显而易见的，从覆盖连续时间点或从PIV analys是（图5，下图）。 观察这些差异，选择采样间隔是很重要的，这样皮质的流动粒子可以明确地对数据进行准确的解释来解决。更小的时间间隔（≤5秒）的建议，以避免大的位移和失踪载体。询问窗口应足够大，以容纳要分析的颗粒的尺寸（皮质颗粒）。相邻的询问窗口之间的重叠允许减小向量间隔，从而增加在网格向量的数目。 <img alt="图1" src="/files/ftp_upload/53469/53469fig1.jpg" /> 图1.装配有口吸左图 :组装一张嘴吸带过滤器所需的零件。右图 ​​:组装吸管<a href="https://www.jove.com/files/ftp_upload/53469/53469fig1large.jpg" target="_blank">请</a>点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53469/53469fig2.jpg" /> 性腺转弯区域的图2的三维模型左上:从时间推移显微镜数据的单个时间点的三维投影。顶部中间和右边:在显微镜数据的三维模型。下图:细胞，穗轴联系人详细信息<a href="https://www.jove.com/files/ftp_upload/53469/53469fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53469/53469fig3.jpg" /> 图3.跟踪细胞核和细胞分裂的残余左:从时间推移显微镜数据3D预测。从跟踪这两个核（彩色球）和细胞分裂的残余（灰色球）获得的模型创建的快照:中东。右:快照从细胞分裂残跟踪。对于残存的路径也显示， <a href="https://www.jove.com/files/ftp_upload/53469/53469fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53469/53469fig4.jpg" /> 在如图4细胞和细胞分裂残系的亚系阿坝州，宗族，从有代表性的胚胎。细胞分裂的标志是正方形。每个刻度为1分钟。细胞分裂残部被评为出现从在此期间，残余产生师的女儿后， <a href="https://www.jove.com/files/ftp_upload/53469/53469fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53469/53469fig5.jpg" /><br/&gt; 皮层收缩流的PIV顶图5.分析:从时间推移显微镜描绘开始时（第一行），并且用于计算与PIVlab矢量场的时间窗的结束（第二行）的3D投影剧照。板中的第三行表示时间窗的所有时间点的叠加。下图:由PIVlab获得向量场<a href="https://www.jove.com/files/ftp_upload/53469/53469fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="电影1" src="/files/ftp_upload/53469/53469movie1.jpg" /> <a href="https://www.jove.com/files/ftp_upload/53469/movie_3dmod_annotated.mp4">视频1</a> -关于图性腺转弯区域2，一种三维模型的视频开始通过用来分割细胞平面的整个堆叠滚动。叠层的事后一个三维最大突出<html>示出，随后由3D segementation模型的x和y轴（有和没有被分割性腺的穗轴）周围旋转。 <img alt="电影2" src="/files/ftp_upload/53469/53469movie2.jpg" /> <a href="https://www.jove.com/files/ftp_upload/53469/Max_projection_Endrov_data_annotated_for_movie.mp4">视频2</a> -相关图3.跟踪细胞核和细胞分裂的残余左 :3D预测从时间推移显微镜的数据。中东:从跟踪这两个核（彩色球）和细胞分裂的残余（灰色球）获得的模型。右:细胞分裂残余和他们的轨迹。比例尺= 10微米。 <img alt="电影3" src="/files/ftp_upload/53469/53469movie3.jpg" />_for_movie_with_PIV_data.mp4"&gt;视频3 -与图皮质收缩流动5.分析与PIV用于计算与PIVlab比例尺矢量场具有代表性的野生型和RGA-3 RNA干扰胚胎的三维最大投影定时录音。 = 10微米。

Watch this Scientific Journal Video about Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools at JoVE.com

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

跟踪和量化的发育过程<em&gt;℃。线虫</em&gt;使用开源工具

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

tracking-quantifying-developmental-processes-c-elegans-using-open

Nanyang Technological University

Research

JoVE Journal

Biology

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

8.8K 次观看.  Goethe University. Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

视频: Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

基于微流体Electrotaxis的，为按需定量分析<em&gt;线虫</em&gt;&#39;LOCOMOTION的

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

科研

生物工程

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

线虫跟踪和行为测量

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

神经科学

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

发育生物学

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

线粒体疾病脑 膜炎基因 模型中量化动物活动的实验方法比较分析

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

行为学

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

生物学

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

的自动分析<em&gt;℃。线虫</em&gt;游泳行为使用天书软件

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

利用广域跟踪平台对大型线虫种群进行自动行为分析

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

免疫与感染

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

跟踪和量化的发育过程

摘要

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成像 C。线虫胚胎使用啶显微镜和开放源码软件

的自动分析

利用广域跟踪平台对大型线虫种群进行自动行为分析

基本的

跟踪和量化的发育过程

摘要

探索更多视频

此视频中的章节

基于微流体Electrotaxis的，为按需定量分析

线虫跟踪和行为测量

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

线粒体疾病脑 膜炎基因 模型中量化动物活动的实验方法比较分析

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

琼脂糖微室的长期钙成像

成像 C。线虫胚胎使用啶显微镜和开放源码软件

的自动分析

利用广域跟踪平台对大型线虫种群进行自动行为分析

基本的

线粒体疾病脑膜炎基因模型中量化动物活动的实验方法比较分析