title: Scanning and three-dimensional electron microscopy methods for the study of Trypanosoma brucei and Leishmania mexicana flagella.
abstract: 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.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
authors.forename: Eva
authors.initials: E
authors.lastname: Gluenz
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
authors.forename: Richard John
authors.initials: RJ
authors.lastname: Wheeler
authors.affiliation: Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, UK.
authors.forename: Louise
authors.initials: L
authors.lastname: Hughes
authors.affiliation: Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, UK.
authors.forename: Sue
authors.initials: S
authors.lastname: Vaughan
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000235:genetics; D002465:Cell Movement / Q000502:physiology*; D016054:DNA, Protozoan / Q000235:genetics; D055032:Electron Microscope Tomography / Q000379:methods; D005407:Flagella / Q000502:physiology / Q000648:ultrastructure*; D021621:Imaging, Three-Dimensional / Q000379:methods; D007894:Leishmania mexicana / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D008855:Microscopy, Electron, Scanning / Q000379:methods; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D014370:Tsetse Flies / Q000469:parasitology
chemical_list: D016054:DNA, Protozoan
keywords: Axoneme; Basal body; Electron tomography; Flagellum; Leishmania; SBFSEM; SEM; Trypanosoma
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