Takada Saeko, Cha Byeong J
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 55455, Minneapolis, MN, USA.
Methods Mol Biol. 2011;782:75-92. doi: 10.1007/978-1-61779-273-1_7.
Live-imaging of cells has been an excellent technique to provide us with highly accurate and valuable information about cell cycle checkpoint regulation and DNA damage responses. Early stage Drosophila embryos have several advantages to be studied by live-imaging. Fly embryos are much tougher than cultured cells and stand up to relatively rough manipulation, such as protein/chemical microinjection followed by time-lapse imaging. Cell cycles in the embryonic cleavage stage progress rapidly (9-20 min/cycle) and nuclear divisions are synchronous, allowing observation of multiple nuclei/cell cycles in a short period of time. Somatic precursor nuclei form a monolayer at the cortex of the embryo during the syncytial blastoderm stage (cell cycles 10-13). Thus the nuclei in this stage are particularly accessible by various microscopic techniques (Sullivan and Theurkauf, 1995, Curr. Opin. Cell Biol. 7, 18-22). Live-imaging of embryos complements the versatility of the Drosophila embryonic system, in which we can utilize various approaches, including genetics and biochemistry, to obtain comprehensive understanding of biological processes. In this chapter, we will describe basic methods of microinjection and live-imaging during early embryogenesis by differential interference contrast (DIC) or confocal microscopy, and the use of such methods to study cell cycle checkpoints.
细胞实时成像一直是一项出色的技术，能为我们提供有关细胞周期检查点调控和DNA损伤反应的高度准确且有价值的信息。早期果蝇胚胎具有一些适合通过实时成像进行研究的优势。果蝇胚胎比培养细胞更坚韧，能承受相对粗暴的操作，如蛋白质/化学物质显微注射，随后进行延时成像。胚胎卵裂期的细胞周期进展迅速（每个周期9 - 20分钟），且核分裂是同步的，这使得在短时间内就能观察到多个细胞核/细胞周期。在合胞体胚盘期（细胞周期10 - 13），体细胞前体细胞核在胚胎皮层形成单层。因此，这个阶段的细胞核特别适合用各种显微技术进行观察（Sullivan和Theurkauf，1995年，《细胞生物学当前观点》7卷，18 - 22页）。胚胎的实时成像补充了果蝇胚胎系统的多功能性，在该系统中，我们可以利用包括遗传学和生物化学在内的各种方法，全面了解生物学过程。在本章中，我们将描述通过微分干涉差（DIC）或共聚焦显微镜在早期胚胎发育过程中进行显微注射和实时成像的基本方法，以及利用这些方法研究细胞周期检查点。
