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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.

Original publication

DOI

10.1016/bs.mcb.2014.12.011

Type

Journal article

Journal

Methods Cell Biol

Publication Date

2015

Volume

127

Pages

509 - 542

Keywords

Axoneme, Basal body, Electron tomography, Flagellum, Leishmania, SBFSEM, SEM, Trypanosoma, Animals, Cell Cycle, Cell Movement, DNA, Protozoan, Electron Microscope Tomography, Flagella, Imaging, Three-Dimensional, Leishmania mexicana, Microscopy, Electron, Scanning, Trypanosoma brucei brucei, Tsetse Flies