Verbrugghe Koen J C, Chan Raymond C
Human Genetics, University of Michigan, USA.
J Vis Exp. 2011 Mar 24(49):2625. doi: 10.3791/2625.
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 samples(1,2). 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 lineage(3), thus providing an ideal experiment model for studying questions in cell biology(4,5)and development(6-9). C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis(10,11)) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis(12-15)). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters(16,17). These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo(18-21). 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.
细胞过程，如染色体组装、分离和胞质分裂，本质上是动态的。利用荧光标记的报告蛋白或微分干涉差（DIC）显微镜对活细胞进行延时成像，能够检查这些动态事件的时间进程，而这些进程通常是通过对固定样本的分析推断出来的(1,2)。此外，对细胞过程的发育调控进行研究需要在发育过程中的完整生物体上进行延时实验。秀丽隐杆线虫胚胎是透明的，具有快速、不变的发育程序以及已知的细胞谱系(3)，因此为研究细胞生物学(4,5)和发育(6 - 9)问题提供了理想的实验模型。秀丽隐杆线虫适合通过正向遗传学（基于随机诱变(10,11)）和反向遗传学来进行基因操作，以靶向特定基因（基于RNAi介导的干扰和靶向诱变(12 - 15)）。此外，可以很容易地创建转基因动物来表达荧光标记的蛋白质或报告基因(16,17)。这些特性相结合，使得在体内识别调控基本细胞和发育过程的遗传途径变得容易(18 - 21)。在本方案中，我们介绍了在复合落射荧光显微镜上使用DIC光学系统或GFP荧光对秀丽隐杆线虫胚胎进行实时成像的方法。我们展示了通常用于固定样本成像的现成显微镜也可以很容易地应用于延时分析，使用开源软件实现成像过程的自动化。
