European Biophysics Journal

, Volume 36, Issue 6, pp 669–674 | Cite as

Periodic acceptor excitation spectroscopy of single molecules

  • Sören DooseEmail author
  • Mike Heilemann
  • Xavier Michalet
  • Shimon Weiss
  • Achillefs N. KapanidisEmail author
Biophysics Letter


Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques.


Single-molecule fluorescence spectroscopy Alternating-laser excitation (ALEX) Förster resonance energy transfer (FRET) Biomolecular interactions 



We thank N.K. Lee for providing DNA samples and L. LeReste for assistance. This work was funded by NIH grant GM069709-01 to S.W. and A.N.K., DOE grants 02ER63339 and 04ER63938 to S.W., and EPSRC grant EP/D058775, EU Marie Curie Fellowship MIRG-CT-2005-031079, and a UK Bionanotechnology IRC grant to A.N.K. M.H. was supported by a DAAD fellowship.


  1. Antonik M, Felekyan S, Gaiduk A, Seidel CA (2006) Separating structural heterogeneities from stochastic variations in fluorescence resonance energy transfer distributions via photon distribution analysis. J Phys Chem B 110:6970–6978CrossRefGoogle Scholar
  2. Bates M, Blosser TR, Zhuang X (2005) Short-range spectroscopic ruler based on a single-molecule optical switch. Phys Rev Lett 94:108101CrossRefADSGoogle Scholar
  3. Deniz AA, Dahan M, Grunwell JR, Ha T, Faulhaber AE, Chemla DS, Weiss S, Schultz PG (1999) Single-pair fluorescence resonance energy transfer on freely diffusing molecules: observation of Förster distance dependence and subpopulations. Proc Natl Acad Sci USA 96:3670–3675CrossRefADSGoogle Scholar
  4. Doose S, Tsay JM, Pinaud F, Weiss S (2005) Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy. Anal Chem 77:2235–2242CrossRefGoogle Scholar
  5. Ha T (2001) Single-molecule fluorescence resonance energy transfer. Methods 25:78–86CrossRefGoogle Scholar
  6. Heilemann M, Margeat E, Kasper R, Sauer M, Tinnefeld P (2005) Carbocyanine dyes as efficient reversible single-molecule optical switch. J Am Chem Soc 127:3801–3806CrossRefGoogle Scholar
  7. Kapanidis AN, Lee NK, Laurence TA, Doose S, Margeat E, Weiss S (2004) Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc Natl Acad Sci USA 101:8936–8941CrossRefADSGoogle Scholar
  8. Kapanidis AN, Laurence TA, Lee NK, Margeat E, Kong X, Weiss S (2005a) Alternating-laser excitation of single molecules. Acc Chem Res 38:523–533CrossRefGoogle Scholar
  9. Kapanidis AN, Margeat E, Laurence TA, Doose S, Ho SO, Mukhopadhyay J, Kortkhonjia E, Mekler V, Ebright RH, Weiss S (2005b) Retention of transcription initiation factor [sigma]70 in transcription elongation: single-molecule analysis. Mol Cell 20:347–356CrossRefGoogle Scholar
  10. Kukolka F, Muller BK, Paternoster S, Arndt A, Niemeyer CM, Bräuchle C, Lamb DC (2006) A single-molecule Förster resonance energy transfer analysis of fluorescent DNA-protein conjugates for nanobiotechnology. Small 2:1083–1089CrossRefGoogle Scholar
  11. Laurence TA, Kong X, Jäger M, Weiss S (2005) Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins. Proc Natl Acad Sci USA 102:17348–17353CrossRefADSGoogle Scholar
  12. Lee NK, Kapanidis AN, Wang Y, Michalet X, Mukhopadhyay J, Ebright RH, Weiss S (2005) Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. Biophys J 88:2939–2953CrossRefGoogle Scholar
  13. Lee NK, Kapanidis AN, Koh HR, Korlann Y, Ho SO, Kim Y, Gassman N, Kim SK, Weiss S (2007) Three-color alternating-laser excitation of single molecules: simultaneous monitoring of multiple interactions and distances. Biophys J 92:303–312CrossRefGoogle Scholar
  14. Li H, Ying L, Green JJ, Balasubramanian S, Klenerman D (2003) Ultrasensitive coincidence fluorescence detection of single DNA molecules. Anal Chem 75:1664–1670CrossRefGoogle Scholar
  15. Muller BK, Zaychikov E, Bräuchle C, Lamb DC (2005) Pulsed interleaved excitation. Biophys J 89:3508–3522CrossRefGoogle Scholar
  16. Nir E, Michalet X, Hamadani K, Laurence TA, Neuhauser D, Kovchegov Y, Weiss S (2006) Shot-noise limited single-molecule FRET histogram: comparison between theory and experiments. J Phys Chem B 110:22103–22224CrossRefGoogle Scholar
  17. Ruttinger S, Macdonald R, Kramer B, Koberling F, Roos M, Hildt E (2006) Accurate single-pair Forster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy. J Biomed Optics 11: 024012CrossRefADSGoogle Scholar
  18. Schwille P, Meyer-Almes FJ, Rigler R (1997) Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys J 72:1878–1886CrossRefGoogle Scholar

Copyright information

© EBSA 2007

Authors and Affiliations

  1. 1.Clarendon Laboratory, Department of Physics and IRC in BionanotechnologyOxford UniversityOxfordUK
  2. 2.Department of Chemistry and BiochemistryUniversity of California Los AngelesLos AngelesUSA
  3. 3.Department of PhysiologyUniversity of California Los AngelesLos AngelesUSA
  4. 4.Applied Laser Physics and Laser SpectroscopyUniversity of BielefeldBielefeldGermany

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