Fluorescence Resonance Energy Transfer: Techniques for Measuring Molecular Conformation and Molecular Proximity

Michael Edidin1

1 Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.10
DOI:  10.1002/0471142735.im1810s52
Online Posting Date:  February, 2003
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


This overview unit focuses on the basics of fluorescence and of the FRET phenomenon, and on methods for detecting FRET and data interpretation. FRET is very versatile and there are more application of these basics than can be covered in a single overview. However, some examples are given of applications of various FRET techniques.

PDF or HTML at Wiley Online Library

Table of Contents

  • Basics of Fluorescence and FRET Mechanisms
  • Practical FRET Microscopy
  • Donor Dequenching After Acceptor Photobleaching
  • Donor Fluorescence Lifetime Imaging
  • Frequency Domain Lifetime Imaging
  • Analysis of FRET Data
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Adams, S.R., Campbell, R.E., Gross, L.A., Martin, B.R., Walkup, G.K., Yao, Y., Llopis, J., and Tsien, R.Y. 2002. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications. J. Am. Chem. Soc. 124:6063‐6076.
   Bastiaens, P.I.H. and Squire, A. 1999. Fluorescence lifetime imaging microscopy: Spatial resolution of biochemical processes in the cell. Trends Cell Biol. 9:48‐52.
   Beecham, J.M. 1992. Global analysis of biochemical and biophysical data. Methods Enzymol. 210:37‐54.
   Beecham, J.M., Knutson, J.R., Ross, J.B.A., Turner, B.W., and Brand, L. 1983. Global resolution of heterogeneous decay by phase/modulation fluorometry: Mixtures and proteins. Biochemistry 22:6054‐6058.
   Catipovic, B., Talluri, G., Oh, J., Wei, T., Su, X‐m., Johansen, T.E., Edidin, M., and Schneck, J.P. 1994. Analysis of the structure of empty and peptide‐loaded major histocompatibility complex molecules at the cell surface. J. Exp. Med. 180:1753‐1761.
   Clayton, A.H., Hanley, Q.S., Arndt‐Jovin, D.J., Subramaniam, V., and Jovin, T.M. 2002. Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM). Biophyis J. 83:1631‐1649.
   Clegg, R.M. 1996. Fluorescence resonance energy transfer spectroscopy and microscopy. In Fluorescence Imaging Spectroscopy and Microscopy. (X.F. Wang and B. Herman, eds.) pp. 179‐252. John Wiley & Sons, New York.
   Dewey, T.G. and Hammes, G.G. 1980. Calculation of fluorescence resonance energy transfer on surfaces. Biophys. J. 32:1023‐1035.
   Elangovan, M., Day, R.N., and Periamsamy, A. 2002. Nanosecond fluorescence resonance energy transfer‐fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell. J. Microscopy 205:3‐14.
   Gaietta, G., Deerinck, T.J., Adams, S.R., Bouwer, J., Tour, O., Laird, D.W., Sosinsky, G.E., Tsien, R.Y., and Ellisman, M.H. 2002. Multicolor and electron microscopic imaging of connexin trafficking. Science 296:503‐507.
   Gautier, I., Tramier, M., Durieux, C., Coppey, J., Pansu, R.B., Nicolas, J.C., Kemnitz, K., and Coppey‐Moisan, M. 2001. Homo‐FRET microscopy in living cells to measure monomer‐dimer transition of GFP‐tagged proteins. Biophys. J. 80:3000‐3008
   Gordon, G.W., Berry, G., Liang, X.H., Levine, B., and Herman, B. 1998. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74:2702‐2713
   Inoue, S., Shimomura, O., Goda, M., Shribak, M., and Tran, P.T. 2002. Fluorescence polarization of green fluorescence protein. Proc. Natl. Acad. Sci. U.S.A. 99:4272‐4277.
   Jovin, T.M. and Arndt‐Jovin, D.J. 1989. Luminescence digital imaging microscopy. Ann. Rev. Biophys. Biophys. Chem. 18:271‐308.
   Kenworthy, A.K. and Edidin, M. 1998. Distribution of a GPI‐anchored protein at the apical surface of MDCK cells examined at a resolution of <100 Å using imaging fluorescence resonance energy transfer. J. Cell Biol. 142:69‐84.
   Kenworthy, A.K. and Edidin, M. 1999. Imaging fluorescence resonance energy transfer as probe of membrane organization and molecular associations of GPI‐anchored proteins. Methods Mol. Biol. 116:37‐49.
   Kenworthy, A.K., Petranova, N., and Edidin, M. 2000. High resolution FRET microscopy of cholera toxin B‐subunit and GPI‐anchored proteins in cell plasma membranes. Mol. Biol. Cell 11:1645‐1655.
   Kuhn, H. 1987. Energy transfer mechanisms. In Biophysics (W. Hoppe, W. Lohmann, H. Markl, and H. Ziegler, eds.) pp. 279‐288. Springer‐Verlag, Berlin.
   Lakowicz, J.R. 1999. Principles of Fluorescence Spectroscopy. Kluwer Academic/Plenum, New York.
   Matko, J. and Edidin, M. 1997. Energy transfer methods for detecting molecular clusters on cell surfaces. Methods Enzymol. 278:444‐462.
   Matko, J., Bushkin, Y., Wei, T., and Edidin, M. 1994. Clustering of class I HLA molecules on the surfaces of activated and transformed human cells. J. Immunology 152:3353‐3360.
   Murakoshi, H.R., Iino, C., Yasui‐Oshima, T., Kobayashi, M., Murakami, S., Minoguchi, A., Yoshimura, and Kusumi, A. 2002. H‐ras activation and induction of localized signalling as studied by single molecule fluorescence energy transfer. Biophys J. 82:47A.
   Murphy, D.B. 2001. Fundamentals of Light Microscopy and Electronic Imaging. John Wiley & Sons, New York.
   Pentcheva, T. and Edidin, M. 2001. Clustering of peptide‐loaded MHC class I molecules for ER export imaged by fluorescence resonance energy transfer. J. Immunology 166:6625‐6632.
   Pentcheva, T., Spiliotis, E.T., and Edidin, M. 2002. Tapasin is retained in the endoplasmic reticulum by dynamic clustering and exclusion from endoplasmic reticulum exit sites. J. Immunol. 168:1538‐1541.
   Periasamy, A. (ed.) 2001. Methods in Cellular Imaging. American Physiological Society/Oxford University Press, New York.
   Rocheleau, J.V., Edidin, M., and Piston, D.W. 2002. Monitoring the membrane rotational mobility of the MHC class I protein using an intra‐sequence expressed GFP construct. Biophys. J. 82:627A.
   Runnels, L.W. and Scarlatam, S.F. 1995. Theory and application of fluorescence homotransfer to melittin oligomerization. Biophys. J. 69:1569‐1583.
   Selvin, P.R. 2000. The renaissance of fluorescence resonance energy transfer. Nat. Struct. Biol. 7:730‐734.
   Sharman, K.K., Demsas, J.N., Ashworth, H., and Periasamy, A. 1999. Error analysis of therapid lifetime determination (RLD) method for double exponential decays: Evaluating different window systems. Anal. Chem. 71:947‐952.
   Spiliotis, E.T., Pentcheva, T., and Edidin, M. 2002. Probing for membrane domains in the endoplasmic reticulum: Retention and degradation of unassembled MHC class I molecules. Mol. Biol. Cell 13:1566‐1582.
   Squire, A., Verveer, P.J., and Bastiaens, P.I.H. 2000. Multiple frequency fluorescence lifetime imaging microscopy. J. Microsc. 191:39‐51.
   Szollosi, J., Damjanovich, S., Mulhern, S.A., and Tron, L. 1987. Fluorescence energy transfer and membrane potential measurements monitor dynamic properties of cell membranes: A critical review. Prog. Biophys. Mol. Biol. 49:65‐87.
   Torigoe, C. and Metzger, H. 2001. Spontaneous phosphorylation of the receptor with high affinity for IgE in transfected fibroblasts. Biochemistry 40:4016‐4025.
   Triantafilou, K., Triantafilou, M., and Dedrick, R.L. 2002. A CD14‐independent LPS receptor cluster. Nat. Immunol. 2:338‐345.
   Triantafilou, K., Fradelizi, D., Wilson, K., and Triantafilou, M. 2001. GRP78, a coreceptor for coxsackievirus A9, interacts with major histocompatibility complex class I molecules which mediate virus internalization. J. Virol. 76:633‐643.
   Tron, L., Szollosi, J., Damjanovich, S., Helliwell, S.H., Arndt‐Jovin, D.J., and Jovin, T.M. 1984. Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative evaluation of the transfer efficiency on a cell‐by‐cell basis. Biophys. J. 45:939‐946.
   Varma, R. and Mayor, S. 1998. GPI‐anchored proteins are organized in submicron domains at the cell surface. Nature 394:798‐801.
   Verveer, P.J., Squire, A., and Bastiaens, P.I.H. 2000. Global analysis of fluorescence lifetime imaging microscopy data. Biophys. J. 78:2127‐2137.
   Verveer, P.J., Squire, A., and Bastiaens, P.I.H. 2001. Frequency‐domain fluorescence lifetime imaging microscopy: A window on the biochemical landscape of the cell. In Methods in Cellular Imaging. (A. Periasamy, ed.) pp. 273‐294. American Physiological Society/Oxford University Press, New York.
   Werner, T.C., Bunting, J.R., and Cathou, R.E. 1972. The shape of immunoglobulin G molecules in solution. Proc. Natl. Acad. Sci. U.S.A. 69:795‐799.
   Wolber, P.K. and Hudson, B.S. 1979. An analytic solution to the Forster energy transfer problem in two dimensions. Biophys. J. 28:197‐210.
   Wu, P. and Brand, L. 1994. Resonance energy transfer: Methods and applications. Anal. Biochem. 218:1‐13.
   Zacharias, D.A., Violin, J.D., Newton, A.C., and Tsien, R.Y. 2002. Partitioning of lipid‐modified monomeric GFPs into membrane microdomains of live cells. Science 296:913‐916.
   Zal, T., Zal, M.A., and Gascoigne, N.R. 2002. Inhibition of T cell receptor‐coreceptor interactions by antagonist ligands visualized by live FRET imaging of the T‐hybridoma immunological synapse. Immunity 16:521‐34.
PDF or HTML at Wiley Online Library