跟踪和量化的发育过程<em>℃。线虫</em>使用开源工具
Summary
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.
Abstract
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。
Introduction
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（'纺丝'）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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors have nothing to disclose.
Materials
| Stereo microscope | Motic/VWR | OT4005S | Stereo microscope for dissection and mounting |
| Polybead Polystyrene Microspheres, | Polysciences | 18329 | Embryo mounting |
| 20 µm | |||
| Polybead Polystyrene Microspheres, | Polysciences | 876 | Adult animal mounting |
| 0.1 µm | |||
| Microscope slides | VWR | 631-0902 | Adult animal mounting |
| Cover glass 18×18 mm | VWR | 631-1331 | Embryo/adult mounting |
| Cover glass 24×60 mm | VWR | 631-1339 | Embryo mounting |
| Scalpel | VWR | 233-5455 | Embryo dissection |
| Silicone tubing | VWR | 228-1501 | Tubing for mouth pipette |
| 30 mm PTFE membrane filter | NeoLab | Jul-01 | Filter for mouth pipette |
| Capillary tubes | VWR | 621-0003 | Pipette tip for mouth pipette |
| Vaseline | Roth | E746.1 | Embryo/adult mounting |
| Agar | Roth | 5210.5 | Adult animal mounting |
| Potassium-di-hydrogenphosphate | Roth | P018.2 | M9 buffer |
| Di-sodium- hydrogenphosphate | Roth | P030.2 | M9 buffer |
| Sodium chloride | Roth | 3957.1 | M9 buffer |
| VisiScope Spinning Disc Confocal System | Visitron Systems | n/a | Confocal microscopy |
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