三维电子显微镜工具彻底改变了我们对生物学中细胞结构和分子复合物的理解。这里,我们描述了研究两种单细胞寄生虫-布鲁氏锥虫和墨西哥利什曼原虫的鞭毛超微结构和生物发生的方法。我们描述了用于扫描电子显微镜细胞电子断层扫描的这些寄生虫的制备方法,和串行块面扫描电子显微镜(SBFSEM)。这些寄生虫具有高度有序的细胞形状和形态,具有内部细胞骨架结构和细胞器的明确定位。我们展示了如何将这些知识用于解剖两种寄生虫中的细胞周期,并从新的T.brucei中鉴定旧鞭毛。最后,我们展示了SBFSEM三维模型用于分析单个全细胞,证明了该技术在未来突变细胞系研究中的巨大潜力。
Three-dimensional electron microscopy tools have revolutionized our understanding of cell structure and molecular complexes in biology. Here, we describe methods for studying flagellar ultrastructure and biogenesis in two unicellular parasites-Trypanosoma brucei and Leishmania mexicana. We describe methods for the preparation of these parasites for scanning electron microscopy cellular electron tomography, and serial block face scanning electron microscopy (SBFSEM). These parasites have a highly ordered cell shape and form, with a defined positioning of internal cytoskeletal structures and organelles. We show how knowledge of these can be used to dissect cell cycles in both parasites and identify the old
flagellum from the new in T. brucei. Finally, we demonstrate the use of SBFSEM three-dimensional models for analysis of individual whole cells, demonstrating the excellent potential this technique has for future studies of mutant cell lines.