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Keywords:

  • fluorescence microscopy;
  • FRAP;
  • FRET;
  • fusion protein;
  • green fluorescent protein;
  • IFP

Summary

The intrinsically fluorescent proteins (IFPs), such as the green, cyan and yellow fluorescent proteins, have revolutionized how we can image the dynamics of cellular events. Intrinsically fluorescent proteins have been used as reporter genes to monitor transcriptional regulation, as targeted markers for organelles and subcellular structures, in fusion proteins to directly observe protein motility and dynamics, and in sensors designed to show changes in cellular environments ranging from pH to protein kinase activity. The IFPs hold tremendous potential to reveal the dynamic processes that underlie plant cell function; however, as with all technology there are artifacts and pitfalls inherent in their use. In this review, we highlight some of the practical issues in using IFPs for live cell imaging. These include choice of the appropriate IFP, dealing with autofluorescence, photobleaching and phototoxicity, and application of approaches such as fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM) and fluorescence recovery after photobleaching (FRAP) to gain high-resolution data about protein dynamics within the cell. We also discuss some of the more common artifacts associated with these fluorescence imaging approaches and suggest controls that should help both spot these problems and suggest their solutions.