Müller-Reichert T, Mäntler J, Srayko M, O'Toole E
Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany.
J Microsc. 2008 May;230(Pt 2):297-307. doi: 10.1111/j.1365-2818.2008.01985.x.
The early Caenorhabditis elegans embryo is currently a popular model system to study centrosome assembly, kinetochore organization, spindle formation, and cellular polarization. Here, we present and review methods for routine electron microscopy and 3D analysis of the early C. elegans embryo. The first method uses laser-induced chemical fixation to preserve the fine structure of isolated embryos. This approach takes advantage of time-resolved fixation to arrest development at specific stages. The second method uses high-pressure freezing of whole worms followed by freeze-substitution (HPF-FS) for ultrastructural analysis. This technique allows staging of developing early embryos within the worm uterus, and has the advantage of superior sample preservation required for high-resolution 3D reconstruction. The third method uses a correlative approach to stage isolated, single embryos by light microscopy followed by HPF-FS and electron tomography. This procedure combines the advantages of time-resolved fixation and superior ultrastructural preservation by high-pressure freezing and allows a higher throughput electron microscopic analysis. The advantages and disadvantages of these methods for different applications are discussed.
秀丽隐杆线虫早期胚胎目前是研究中心体组装、动粒组织、纺锤体形成和细胞极化的常用模型系统。在此，我们展示并综述了用于秀丽隐杆线虫早期胚胎常规电子显微镜和三维分析的方法。第一种方法使用激光诱导化学固定来保存分离胚胎的精细结构。这种方法利用时间分辨固定在特定阶段停止发育。第二种方法是对整条线虫进行高压冷冻，然后进行冷冻替代（HPF-FS）以进行超微结构分析。该技术能够对虫体子宫内发育中的早期胚胎进行分期，并且具有高分辨率三维重建所需的卓越样本保存优势。第三种方法采用一种相关方法，通过光学显微镜对分离的单个胚胎进行分期，随后进行HPF-FS和电子断层扫描。该程序结合了时间分辨固定的优势以及高压冷冻带来的卓越超微结构保存效果，并允许进行更高通量的电子显微镜分析。讨论了这些方法在不同应用中的优缺点。
