Mohler William A, Isaacson Ariel B
Cold Spring Harb Protoc. 2010 Mar;2010(3):pdb.top71. doi: 10.1101/pdb.top71.
Embryos are remarkable for their combination of pluripotency, three-dimensionality, and swiftness of subcellular and developmental rearrangements. Embryogenesis in the nematode Caenorhabditis elegans is uniquely suited among model systems to high-resolution dynamic imaging. Within a single high-magnification, high-numerical aperture (NA) microscope field, at submicrometer resolution, it is possible to observe several entire animals taking form. The full approximately 14-h course of embryonic cleavage and morphogenesis of this transparent, free-living worm is essentially invariant. Observing specific fluorescently labeled components during embryonic development promises to reveal the roles of organelles and molecules in an extremely diverse and reproducible set of contexts. The C. elegans community has created a growing collection of hundreds of transgenic strains expressing green fluorescent protein (GFP)-labeled versions of distinct endogenously expressed genes. The task of correlating the resulting expression and localization patterns in space and time is simultaneously alluring and technically demanding. This article describes the use of four-dimensional (4D) laser-scanning microscopy and subsequent data processing to record, portray, analyze, and compare the expression of fluorescently tagged gene products during development of the nematode embryo.
胚胎因其多能性、三维结构以及亚细胞和发育重排的迅速性而引人注目。线虫秀丽隐杆线虫的胚胎发生在模型系统中特别适合进行高分辨率动态成像。在单个高倍、高数值孔径（NA）显微镜视野内，以亚微米分辨率，可以观察到几只完整动物的形成过程。这种透明的自由生活蠕虫的胚胎分裂和形态发生的整个约14小时过程基本不变。在胚胎发育过程中观察特定的荧光标记成分，有望揭示细胞器和分子在极其多样且可重复的环境中的作用。秀丽隐杆线虫研究群体已经创建了越来越多的数百个转基因品系，这些品系表达绿色荧光蛋白（GFP）标记的不同内源性表达基因的版本。将由此产生的表达和定位模式在空间和时间上进行关联的任务既诱人又在技术上具有挑战性。本文描述了使用四维（4D）激光扫描显微镜及后续数据处理，来记录、描绘、分析和比较线虫胚胎发育过程中荧光标记基因产物的表达情况。
