Total internal reflection fluorescence (TIRF) microscopy of Chlamydomonas flagella

Methods Cell Biol. 2009;93:157-77. doi: 10.1016/S0091-679X(08)93009-0. Epub 2009 Dec 4.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Axoneme / ultrastructure
  • Biological Transport / physiology
  • Chlamydomonas reinhardtii / cytology*
  • Chlamydomonas reinhardtii / metabolism
  • Flagella / metabolism
  • Flagella / ultrastructure*
  • Fluorescence Recovery After Photobleaching / methods
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods*
  • Molecular Motor Proteins / metabolism
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Molecular Motor Proteins
  • Protozoan Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins