Chapter 28 Preparation of Cilia and Flagella for Thin-Section Transmission Electron Microscope Analysis

Abstract

Publisher Summary Flagellar images are composed of multiple overlapping structures. This is because the thickness of a standard “thin section” (50–60 nm) is much greater than the dimensions of many important axonemal components. Preparations that provide a high density of flagellar sections allow the investigator to find satisfactory images much quickly. Fixation of isolated axonemes yields a large number of specimens in a given section and allows tannic acid to be used as a mordant. Preparations of isolated flagella also yield a large number of specimens per section and allow observation of the flagellar matrix and membrane, although the matrix may obscure some axonemal structures. Fixation of flagella in situ can preserve information about developmental stages of flagellar growth and spatial relationships between the flagella and the underlying cells, and allows portions of the basal body/flagellum proximal to the abscission point to be studied. The conditions of fixation must also be decided. Novel combinations of fixation protocols and specimens are unpredictable, and so literature or a protocol that has worked for a similar specimen needs to be referred. The methods suggested in this chapter are based on those that have worked for Chlamydomonas and related species, but may be suitable for other specimens as well. The chapter discusses the sourcing of materials and the procedure involved—fixation of isolated axonemes, of isolated flagella, and of flagella in situ. Flagellar membranes are osmotically active until after postfixation with OsO 4 , so the fixation and wash solutions should have the appropriate tonicity. Usually, this has to be determined by trial and error. Fixative solutions that are hypotonic ensure penetration by the fixative, but can also result in swelling of the flagella and sometimes even cause the axoneme to curl up inside the flagellar membrane.