• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biophysjLink to Publisher's site
Biophys J. Oct 2001; 81(4): 2395–2402.
PMCID: PMC1301710

Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes.

Abstract

Green fluorescence protein (GFP)-based fluorescence resonance energy transfer (FRET) is increasingly used in investigation of inter- and intramolecular interactions in living cells. In this report, we present a modified method for FRET quantification in cultured cells using conventional fluorescence microscopy. To reliably measure FRET, three positive control constructs in which a cyan fluorescence protein and a yellow fluorescence protein were linked by peptides of 15, 24, or 37 amino acid residues were prepared. FRET was detected using a spectrofluorometer, a laser scanning confocal microscope, and an inverted fluorescence microscope. Three calculation methods for FRET quantification using fluorescence microscopes were compared. By normalization against expression levels of GFP fusion proteins, the modified method gave consistent FRET values that could be compared among different cells with varying protein expression levels. Whole-cell global analysis using this method allowed FRET measurement with high spatial resolutions. Using such a procedure, the interaction of synaptic proteins syntaxin and the synaptosomal associated protein of 25 kDa (SNAP-25) was examined in PC12 cells, which showed strong FRET on plasma membranes. These results demonstrate the effectiveness of the modified method for FRET measurement in live cell systems.

Full Text

The Full Text of this article is available as a PDF (833K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adams SR, Harootunian AT, Buechler YJ, Taylor SS, Tsien RY. Fluorescence ratio imaging of cyclic AMP in single cells. Nature. 1991 Feb 21;349(6311):694–697. [PubMed]
  • Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. [PubMed]
  • Chapman ER, Alexander K, Vorherr T, Carafoli E, Storm DR. Fluorescence energy transfer analysis of calmodulin-peptide complexes. Biochemistry. 1992 Dec 29;31(51):12819–12825. [PubMed]
  • Gordon GW, Berry G, Liang XH, Levine B, Herman B. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J. 1998 May;74(5):2702–2713. [PMC free article] [PubMed]
  • Heim R. Green fluorescent protein forms for energy transfer. Methods Enzymol. 1999;302:408–423. [PubMed]
  • Heim R, Tsien RY. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol. 1996 Feb 1;6(2):178–182. [PubMed]
  • Jacob JM, Zhou Q, Liu Y. Novel method for the labeling of distant neuromuscular junctions. J Neurosci Res. 2000 Jul 1;61(1):61–66. [PubMed]
  • Jovin TM, Arndt-Jovin DJ. Luminescence digital imaging microscopy. Annu Rev Biophys Biophys Chem. 1989;18:271–308. [PubMed]
  • Mahajan NP, Linder K, Berry G, Gordon GW, Heim R, Herman B. Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer. Nat Biotechnol. 1998 Jun;16(6):547–552. [PubMed]
  • Martin TF, Kowalchyk JA. Docked secretory vesicles undergo Ca2+-activated exocytosis in a cell-free system. J Biol Chem. 1997 May 30;272(22):14447–14453. [PubMed]
  • Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature. 1997 Aug 28;388(6645):882–887. [PubMed]
  • Ng T, Squire A, Hansra G, Bornancin F, Prevostel C, Hanby A, Harris W, Barnes D, Schmidt S, Mellor H, et al. Imaging protein kinase Calpha activation in cells. Science. 1999 Mar 26;283(5410):2085–2089. [PubMed]
  • Olwin BB, Keller CH, Storm DR. Interaction of a fluorescent N-dansylaziridine derivative of troponin I with calmodulin in the absence and presence of calcium. Biochemistry. 1982 Oct 26;21(22):5669–5675. [PubMed]
  • Pollok BA, Heim R. Using GFP in FRET-based applications. Trends Cell Biol. 1999 Feb;9(2):57–60. [PubMed]
  • Robinson LJ, Martin TF. Docking and fusion in neurosecretion. Curr Opin Cell Biol. 1998 Aug;10(4):483–492. [PubMed]
  • Stryer L. Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem. 1978;47:819–846. [PubMed]
  • Südhof TC. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature. 1995 Jun 22;375(6533):645–653. [PubMed]
  • Trón L, Szöllósi J, Damjanovich S, Helliwell SH, Arndt-Jovin DJ, Jovin TM. 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. 1984 May;45(5):939–946. [PMC free article] [PubMed]
  • Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67:509–544. [PubMed]
  • Uster PS, Pagano RE. Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells. J Cell Biol. 1986 Oct;103(4):1221–1234. [PMC free article] [PubMed]
  • Vanderklish PW, Krushel LA, Holst BH, Gally JA, Crossin KL, Edelman GM. Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer. Proc Natl Acad Sci U S A. 2000 Feb 29;97(5):2253–2258. [PMC free article] [PubMed]
  • Verveer PJ, Squire A, Bastiaens PI. Global analysis of fluorescence lifetime imaging microscopy data. Biophys J. 2000 Apr;78(4):2127–2137. [PMC free article] [PubMed]
  • Wolf DE, Winiski AP, Ting AE, Bocian KM, Pagano RE. Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer. Biochemistry. 1992 Mar 24;31(11):2865–2873. [PubMed]
  • Wu P, Brand L. Resonance energy transfer: methods and applications. Anal Biochem. 1994 Apr;218(1):1–13. [PubMed]
  • Xia Z, Zhou Q, Lin J, Liu Y. Stable SNARE complex prior to evoked synaptic vesicle fusion revealed by fluorescence resonance energy transfer. J Biol Chem. 2001 Jan 19;276(3):1766–1771. [PubMed]
  • Yang Y, Xia Z, Liu Y. SNAP-25 functional domains in SNARE core complex assembly and glutamate release of cerebellar granule cells. J Biol Chem. 2000 Sep 22;275(38):29482–29487. [PubMed]
  • Zhou Q, Xiao J, Liu Y. Participation of syntaxin 1A in membrane trafficking involving neurite elongation and membrane expansion. J Neurosci Res. 2000 Aug 1;61(3):321–328. [PubMed]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...