• 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. Dec 2002; 83(6): 3652–3664.
PMCID: PMC1302440

Fluorescence resonance energy transfer-based stoichiometry in living cells.


Imaging of fluorescence resonance energy transfer (FRET) between fluorescently labeled molecules can measure the timing and location of intermolecular interactions inside living cells. Present microscopic methods measure FRET in arbitrary units, and cannot discriminate FRET efficiency and the fractions of donor and acceptor in complex. Here we describe a stoichiometric method that uses three microscopic fluorescence images to measure FRET efficiency, the relative concentrations of donor and acceptor, and the fractions of donor and acceptor in complex in living cells. FRET stoichiometry derives from the concept that specific donor-acceptor complexes will give rise to a characteristic FRET efficiency, which, if measured, can allow stoichiometric discrimination of interacting components. A first equation determines FRET efficiency and the fraction of acceptor molecules in complex with donor. A second equation determines the fraction of donor molecules in complex by estimating the donor fluorescence lost due to energy transfer. This eliminates the need for acceptor photobleaching to determine total donor concentrations and allows for repeated measurements from the same cell. A third equation obtains the ratio of total acceptor to total donor molecules. The theory and method were confirmed by microscopic measurements of fluorescence from cyan fluorescent protein (CFP), citrine, and linked CFP-Citrine fusion protein, in solutions and inside cells. Together, the methods derived from these equations allow sensitive, rapid, and repeatable detection of donor-, acceptor-, and donor-acceptor complex stoichiometry at each pixel in an image. By accurately imaging molecular interactions, FRET stoichiometry opens new areas for quantitative study of intracellular molecular networks.

Full Text

The Full Text of this article is available as a PDF (654K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Elsliger MA, Wachter RM, Hanson GT, Kallio K, Remington SJ. Structural and spectral response of green fluorescent protein variants to changes in pH. Biochemistry. 1999 Apr 27;38(17):5296–5301. [PubMed]
  • Erickson MG, Alseikhan BA, Peterson BZ, Yue DT. Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells. Neuron. 2001 Sep 27;31(6):973–985. [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]
  • Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem. 2001 Aug 3;276(31):29188–29194. [PubMed]
  • Janetopoulos C, Jin T, Devreotes P. Receptor-mediated activation of heterotrimeric G-proteins in living cells. Science. 2001 Mar 23;291(5512):2408–2411. [PubMed]
  • Jayaraman S, Haggie P, Wachter RM, Remington SJ, Verkman AS. Mechanism and cellular applications of a green fluorescent protein-based halide sensor. J Biol Chem. 2000 Mar 3;275(9):6047–6050. [PubMed]
  • Kenworthy AK, Petranova N, Edidin M. High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. Mol Biol Cell. 2000 May;11(5):1645–1655. [PMC free article] [PubMed]
  • Kraynov VS, Chamberlain C, Bokoch GM, Schwartz MA, Slabaugh S, Hahn KM. Localized Rac activation dynamics visualized in living cells. Science. 2000 Oct 13;290(5490):333–337. [PubMed]
  • Miyawaki A, Griesbeck O, Heim R, Tsien RY. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2135–2140. [PMC free article] [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]
  • Sourjik Victor, Berg Howard C. Receptor sensitivity in bacterial chemotaxis. Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):123–127. [PMC free article] [PubMed]
  • Ting AY, Kain KH, Klemke RL, Tsien RY. Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):15003–15008. [PMC free article] [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]
  • Xia Z, Liu Y. Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys J. 2001 Oct;81(4):2395–2402. [PMC free article] [PubMed]
  • Zacharias David A, Violin Jonathan D, Newton Alexandra C, Tsien Roger Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science. 2002 May 3;296(5569):913–916. [PubMed]

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


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...