Three-dimensional images of enzymes at work in cells? This is the promise of a new advance in microscopy called fluorescence lifetime imaging microscopy (FLIM). Philippe Bastiaens and Anthony Squire at the Imperial Cancer Research Fund in London have published several papers this year describing this technique, in which the mean fluorescence lifetime of a chromophore is measured at each spatially resolvable element of a microscope image. The practical result is nanometer-scale resolution for quantifying protein activity and intracellular location. (For details on the microscopy, see Squire and Bastiaens, 1999. For a discussion of applications in cell biology, see Bastiaens and Squire, 1999.)

In conjunction with Peter Parker and his colleagues (also at the Imperial Cancer Research Fund), Bastiaens and Squire have used FLIM to describe the activation state and intracellular location of protein kinase C-α (PKC-α) in situ (Science March 26). They accomplished this by first identifying an autophosphorylation site on PKC-α that signals activation of the enzyme. The authors then used FLIM to measure the fluorescence resonance energy transfer (FRET) that resulted from the binding of fluorescently tagged antibodies at this site. In this manner, they were able to image PKC-α activation in live and fixed cultured cells, as well as in breast cell tumor pathology specimens.—Hakon Heimer

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Further Reading

Papers

  1. . Three dimensional image restoration in fluorescence lifetime imaging microscopy. J Microsc. 1999 Jan;193(Pt 1):36-49. PubMed.
  2. . Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol. 1999 Feb;9(2):48-52. PubMed.

Primary Papers

  1. . Imaging protein kinase Calpha activation in cells. Science. 1999 Mar 26;283(5410):2085-9. PubMed.