Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research News and Views
  • Published:

Spying on the hidden lives of proteins

Nature Biotechnology volume 17, pages 425–426 (1999)Cite this article

Fluorescence microscopy studies of protein localization in the cell have contributed enormously to our understanding of biological processes. Since the introduction of immunofluorescence techniques more than 50 years ago, however, most fluorescence microscopy procedures have changed very little. Rapid advances in technology have led to the development of many new imaging modalities, but most of these have found little general use in biomedical research and languish in the labs of the developers. In the March 26 issue of Science, Ng and colleagues1 show us how a combination of two advanced imaging methods, fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), may revolutionize the way immunofluorescence is performed. In their study, the authors use these fluorescence microscopy techniques to acquire a spatial map of the distribution of the activated form of the enzyme, protein kinase C α (PKCα) in intact cells.

In practice, however, the use of FRET microscopy is limited by problems of labeling specificity, probe concentration, and photobleaching2. These latter two limitations can be overcome by detecting FRET through its effect on the fluorescence lifetime of the donor fluorophore using FLIM. The average excited-state lifetime of a fluorescent molecule is fleetingly short, typically less than 10 ns, and is critically dependent on the local environment surrounding the probe3,4. The FLIM technique detects a spatial lifetime map of these nanosecond decay kinetics, and therefore can reveal localized variations in the probe environment, in addition to a simple picture of fluorophore localization4,5. Since FRET occurs during the excited-state lifetime of the donor molecule, it significantly affects the donor fluorescence lifetime. The change in lifetime due to the presence of an acceptor fluorophore in the donor's local environment can be detected by FLIM (Fig. 1). An important advantage of combining the FRET and FLIM imaging modalities is that only the donor emission lifetimes need to be monitored to detect interactions between the labeled proteins. The technique is blind to acceptor emission, and only those acceptor molecules close enough to receive donor excited-state energy are detected.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Detection of fluorescence resonance energy transfer (FRET) by fluorescence lifetime imaging microscopy (FLIM).

References

  1. Ng, T. et al. Imaging protein kinase Cα activation in cells. Science 283, 2085–2089 (1999).

    Article  CAS  Google Scholar 

  2. Day, R.N., Nordeen, S.K. & Wan, Y. Visualizing protein interactions in the nucleus of the living cell. Mol. Endocrinol. 13, 517–526 (1999).

    Article  CAS  Google Scholar 

  3. French, T. et al.. Fluorescence lifetime imaging techniques for microscopy. Methods Cell Biol. 56, 277–304 (1998).

    Article  CAS  Google Scholar 

  4. Bastiaens, P.H.I. & Squire, A. Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol. 9, 48–52 (1999).

    Article  CAS  Google Scholar 

  5. Gadella, T.W. Jr & Jovin, T.M. Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation. Cell Biol 129, 1543–1558 (1995).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. associate professor Departments of Medicine and Cell Biology, National Science Foundation Center for Biological Timing, University of Virginia, Charlottesville, 22908, VA

    Richard N. Day

  2. associate professor Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, 37232, TN

    David W. Piston

Authors
  1. Richard N. Day
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David W. Piston
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Day, R., Piston, D. Spying on the hidden lives of proteins. Nat Biotechnol 17, 425–426 (1999). https://doi.org/10.1038/8592

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/8592

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing