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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rho+GTPase-activating+proteins+RGA-3+and+RGA-4+are+required+to+set+the+initial+size+of+PAR+domains+in+Caenorhabditis+elegans+one-cell+embryos.">The Rho GTPase-activating proteins RGA-3 and RGA-4 are required to set the initial size of PAR domains in Caenorhabditis elegans one-cell embryos.</a> Proc. Natl. Acad. Sci. U. S. A. 104 (38), 14976-14981 (2007).</li><li>Schmutz, C., Stevens, J., Spang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+the+novel+RhoGAP+proteins+RGA-3+and+RGA-4+in+the+germ+line+and+in+the+early+embryo+of+C.+elegans.">Functions of the novel RhoGAP proteins RGA-3 and RGA-4 in the germ line and in the early embryo of C. elegans.</a> Development. 134 (19), 3495-3505 (2007).</li><li>Naganathan, S. R., F&#252;rthauer, S., Nishikawa, M., J&#252;licher, F., Grill, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+torque+generation+by+the+actomyosin+cell+cortex+drives+left-right+symmetry+breaking.">Active torque generation by the actomyosin cell cortex drives left-right symmetry breaking.</a> Elife. 3, e04165 (2014).</li><li>Kaletta, T., Schnabel, H., Schnabel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binary+specification+of+the+embryonic+lineage+in+Caenorhabditis+elegans.">Binary specification of the embryonic lineage in Caenorhabditis elegans.</a> Nature. 390 (6657), 294-298 (1997).</li><li>Schnabel, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cell+sorting+in+the+C.+elegans+embryo+defines+a+new+mechanism+for+pattern+formation.">Global cell sorting in the C. elegans embryo defines a new mechanism for pattern formation.</a> Dev. Biol. 294 (2), 418-431 (2006).</li><li>Murray, J. I., Bao, Z., Boyle, T. J., Waterston, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lineaging+of+fluorescently-labeled+Caenorhabditis+elegans+embryos+with+StarryNite+and+AceTree.">The lineaging of fluorescently-labeled Caenorhabditis elegans embryos with StarryNite and AceTree.</a> Nat. Protoc. 1 (3), 1468-1476 (2006).</li><li>Giurumescu, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+semi-automated+analysis+of+morphogenesis+with+single-cell+resolution+in+complex+embryos.">Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos.</a> Development. 139 (22), 4271-4279 (2012).</li><li>Dejima, K., Kang, S., Mitani, S., Cosman, P. C., Chisholm, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syndecan+defines+precise+spindle+orientation+by+modulating+Wnt+signaling+in+C.+elegans.">Syndecan defines precise spindle orientation by modulating Wnt signaling in C. elegans.</a> Development. 141 (22), 4354-4365 (2014).</li><li>Schnabel, R., Hutter, H., Moerman, D., Schnabel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+normal+embryogenesis+in+Caenorhabditis+elegans+using+a+4D+microscope:+variability+of+development+and+regional+specification.">Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of development and regional specification.</a> Dev Biol. 184 (2), 234-265 (1997).</li></ol>

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...

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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Stereo microscope</td>
			<td style="width:71px;">Motic/VWR</td>
			<td style="width:119px;">OT4005S</td>
			<td style="width:167px;">Stereo microscope for dissection and mounting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polybead Polystyrene Microspheres,&#160;</td>
			<td rowspan="2" style="width:71px;">Polysciences</td>
			<td rowspan="2" style="width:119px;">18329</td>
			<td rowspan="2" style="width:167px;">Embryo mounting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">20 &#181;m</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polybead Polystyrene Microspheres,&#160;</td>
			<td rowspan="2" style="width:71px;">Polysciences</td>
			<td rowspan="2" style="width:119px;">876</td>
			<td rowspan="2" style="width:167px;">Adult animal mounting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">0.1 &#181;m</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Microscope slides</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">631-0902</td>
			<td style="width:167px;">Adult animal mounting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Cover glass 18x18 mm</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">631-1331</td>
			<td style="width:167px;">Embryo/adult mounting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Cover glass 24x60 mm</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">631-1339</td>
			<td style="width:167px;">Embryo mounting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Scalpel</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">233-5455</td>
			<td style="width:167px;">Embryo dissection</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Silicone tubing</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">228-1501</td>
			<td style="width:167px;">Tubing for mouth pipette</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">30 mm PTFE membrane filter</td>
			<td style="width:71px;">NeoLab</td>
			<td style="width:119px;">Jul-01</td>
			<td style="width:167px;">Filter for mouth pipette</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Capillary tubes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">621-0003</td>
			<td style="width:167px;">Pipette tip for mouth pipette</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Vaseline</td>
			<td style="width:71px;">Roth</td>
			<td style="width:119px;">E746.1</td>
			<td style="width:167px;">Embryo/adult mounting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Agar</td>
			<td style="width:71px;">Roth</td>
			<td style="width:119px;">5210.5</td>
			<td style="width:167px;">Adult animal mounting</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Potassium-di-hydrogenphosphate</td>
			<td style="width:71px;">Roth</td>
			<td style="width:119px;">P018.2</td>
			<td style="width:167px;">M9 buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Di-sodium- hydrogenphosphate</td>
			<td style="width:71px;">Roth</td>
			<td style="width:119px;">P030.2</td>
			<td style="width:167px;">M9 buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium chloride</td>
			<td style="width:71px;">Roth</td>
			<td style="width:119px;">3957.1</td>
			<td style="width:167px;">M9 buffer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">VisiScope Spinning Disc Confocal System</td>
			<td style="width:71px;">Visitron Systems</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">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（?...

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

Universidad de Cartagena UDEC

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

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

使用微机电系统（MEMS）开发诊断工具

科研

生物学

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

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 ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using "Freeze-Cracking"

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用"冻结开裂"

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

开源高内涵分析利用自动荧光寿命成像显微镜

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

可视化和跟踪活果蝇黑色素细胞卵室的内源性 mRNA

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

测量在C.elegan的RAN肽毒性

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

跟踪和量化的发育过程

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