Total internal reflection fluorescence (TIRF) microscopy of Chlamydomonas flagella.
The eukaryotic flagellum is host to a variety of dynamic behaviors, including flagellar beating, the motility of glycoproteins in the flagellar membrane, and intraflagellar transport (IFT), the bidirectional traffic of protein particles between the flagellar base and tip. IFT is of particular interest, as it plays integral roles in flagellar length control, cell signaling, development, and human disease. However, our ability to understand dynamic flagellar processes such as IFT is limited in large part by the fidelity with which we can image these behaviors in living cells. This chapter introduces the application of total internal reflection fluorescence (TIRF) microscopy to visualize the flagella of Chlamydomonas reinhardtii. The advantages and challenges of TIRF are discussed in comparison to confocal and differential interference contrast techniques. This chapter also reviews current IFT insights gleaned from TIRF microscopy of Chlamydomonas and provides an outlook on the future of the technique, with particular emphasis on combining TIRF with other emerging imaging technologies.
Engel
Benjamin D
BD
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94158, USA.
Lechtreck
Karl-Ferdinand
KF
Sakai
Tsuyoshi
T
Ikebe
Mitsuo
M
Witman
George B
GB
Marshall
Wallace F
WF
D000818:Animals; D054468:Axoneme / Q000648:ultrastructure; D001692:Biological Transport / Q000502:physiology; D016825:Chlamydomonas reinhardtii / Q000166:cytology* / Q000378:metabolism; D005407:Flagella / Q000378:metabolism / Q000648:ultrastructure*; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D020409:Molecular Motor Proteins / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D020409:Molecular Motor Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
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