HIGH-RESOLUTION IMAGING OF FLAGELLA

In flagellated orciliated cells, motility is equated, with dynein-based ciliary or flagellar beating in addition to beating; the eukaryotic flagellum displays four motilities that are independent of flagellar dynein. These dynein- and beat-independent motilities continue to be studied in the biflagellate, unicellular, green alga, Chlamydomonas. The motilities studied by conventional light microscopy include the gliding of whole cells across surfaces by means of their flagella, the saltatory bidirectional movement of polystyrene beads attached to the flagellar membrane, and the movement (tipping) of glycoproteins from positions along the length of the flagellum to the flagellar tip during mating. Bidirectional movement of granule-like particles was observed along the length of the flagellum beneath the flagellar membrane. This motility has been termed intraflagellar transport. This chapter focuses on video-enhanced differential-interference-contrast (DIC) microscopy techniques used to study the beat-independent flagellar motilities of Chlamydomonas. The most important criterion in choosing a specimen for high-resolution imaging is obtaining immotile cilia/flagella. Excessive movement of cilia/flagella causes them to change focal planes, giving no opportunity to adjust the optics and video camera for maximum resolution and optimal contrast. The method of choice for obtaining an immotile specimen is biological. Advantage should be taken of naturally immotile cilia/flagella, or use organisms in which motility mutants exist. The second method for obtaining immotile specimens is by chemical modification of the medium. With Chlamydomonas, the addition of 1 mM ethylene glycol bis (P-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) to the medium or the omission Ca2+ inhibits gliding, leaving the cells attached to the coverslip. It is a method for completely immobilizing cells for the study of intraflagellar transport. Specimens may be also immobilized mechanically. The chapter discusses sample preparation, equipments and process of obtaining image. Each advance in light microscopy has made it easy to obtain new information about cilia and flagella. © 1995, Academic Press Inc.