Journal of Fluorescence

, Volume 20, Issue 1, pp 105–114 | Cite as

On the Resolution Capabilities and Limits of Fluorescence Lifetime Correlation Spectroscopy (FLCS) Measurements

  • Steffen Rüttinger
  • Peter Kapusta
  • Matthias Patting
  • Michael Wahl
  • Rainer Macdonald
Original Paper


Quantitative tests were performed in order to explore the practical limits of FLCS. We demonstrate that: a) FLCS yields precise and correct concentration values from as low as picomolar to micromolar concentrations; b) it is possible to separate four signal components in a single detector setup; c) diffusion times differing only 25% from each other can be resolved by separating a two component mixture based on the different fluorescence lifetimes of both components; d) most of the inherent technical limitations of conventional FCS are easily overcome by FLCS employing a single detector channel confocal detection scheme.


Fluorescence lifetime correlation spectroscopy (FLCS) Fluorescence correlation spectroscopy (FCS) Single molecule detection (SMD) Confocal laser scanning microscopy (CLSM) 



We gratefully acknowledge financial support from the German ministry of Education and Research (BMBF, Biophotonik Project 13N8850) and the German ministry of Economics (BMWi, grant MNPQ 12/06).

Supplementary material

10895_2009_528_MOESM1_ESM.doc (34 kb)
Supplemental Material 1 (DOC 33.5 kb)


  1. 1.
    Magde D, Elson EL, Webb WW (1972) Thermodynamic fluctuations in a reacting system & measurement by fluorescence correlation spectroscopy. Phys Rev Lett 29(11):705–708. doi: 10.1103/PhysRevLett.29.705 CrossRefGoogle Scholar
  2. 2.
    Elson EL, Magde D (1974) Fluorescence correlation spectroscopy: I. conceptual basis and theory. Biopolymers 13:1–27. doi: 10.1002/bip.1974.360130102 CrossRefGoogle Scholar
  3. 3.
    Magde D, Elson EL, Webb WW (1974) Fluorescence correlation spectroscopy: II. An experimental realization. Biopolymers 13:29–61. doi: 10.1002/bip.1974.360130103 CrossRefPubMedGoogle Scholar
  4. 4.
    Rigler R, Widengren J (1990) Ultrasensitive detection of single molecules by fluorescence correlation spectroscopy. Bioscience 3:180–183Google Scholar
  5. 5.
    Thompson NL (1991) Topics in fluorescence spectroscopy, vol 1. Plenum, New York, p 337CrossRefGoogle Scholar
  6. 6.
    Widengren J, Mets Ü (2002) In: Zander C, Enderlein J, Keller RA (eds) Wiley-VCH, ISBN3-527-40310-8, pp 69–120Google Scholar
  7. 7.
    Meseth U, Wohland T, Rigler R, Vogel H (1999) Resolution of fluorescence correlation measurements. Biophys J 76(3):1619–1631. doi: 10.1016/S0006-3495(99)77321-2 CrossRefPubMedGoogle Scholar
  8. 8.
    Schwille P, Meyer-Almes FJ, Rigler R (1997) Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys J 72(4):1878–1886. doi: 10.1016/S0006-3495(97)78833-7 CrossRefPubMedGoogle Scholar
  9. 9.
    Schwille P, Bieschke J, Oehlenschlager F (1997) Kinetic investigations by fluorescence correlation spectroscopy: the analytical and diagnostic potential of diffusion studies. Biophys Chem 66(2-3):211–228. doi: 10.1016/S0301-4622(97)00061-6 CrossRefPubMedGoogle Scholar
  10. 10.
    Rigler R, Földes-Papp Z, Meyer-Almes FJ, Sammet C, Volcker M, Schnetz A (1998) Fluorescence cross-correlation: a new concept for polymerase chain reaction. J Biotechnol 63(2):97–109. doi: 10.1016/S0168-1656(98)00079-0 CrossRefPubMedGoogle Scholar
  11. 11.
    Kettling U, Koltermann A, Schwille P, Eigen M (1998) Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc Natl Acad Sci USA 95(4):1416–1420. doi: 10.1073/pnas.95.4.1416 CrossRefPubMedGoogle Scholar
  12. 12.
    Koltermann A, Kettling U, Bieschke J, Winkler T, Eigen M (1998) Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: high throughput screening for enzyme activity. Proc Natl Acad Sci USA 95(4):1421–1426. doi: 10.1073/pnas.95.4.1421 CrossRefPubMedGoogle Scholar
  13. 13.
    Winkler T, Kettling U, Koltermann A, Eigen M (1999) Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening. Proc Natl Acad Sci USA 96(4):1375–1378. doi: 10.1073/pnas.96.4.1375 CrossRefPubMedGoogle Scholar
  14. 14.
    Heinze KG, Koltermann A, Schwille P (2000) Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis. Proc Natl Acad Sci USA 97(19):10377–10382. doi: 10.1073/pnas.180317197 CrossRefPubMedGoogle Scholar
  15. 15.
    Bieschke J, Giese A, Schulz-Schaeffer W, Zerr I, Poser S, Eigen M, Kretzschmar H (2000) Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets. Proc Natl Acad Sci USA 97(10):5468–5473. doi: 10.1073/pnas.97.10.5468 CrossRefPubMedGoogle Scholar
  16. 16.
    Medina MA, Schwille P (2002) Fluorescence correlation spectroscopy for the detection and study of single molecules in biology. Bioessays 24(8):758–764. doi: 10.1002/bies.10118 CrossRefPubMedGoogle Scholar
  17. 17.
    Kohl T, Heinze KG, Kuhlemann R, Koltermann A, Schwille P (2002) A Protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins. Proc Natl Acad Sci USA 99(19):12161–12166. doi: 10.1073/pnas.192433499 CrossRefPubMedGoogle Scholar
  18. 18.
    Koppel D (1974) Statistical accuracy in fluorescence correlation spectroscopy. Phys Rev A 10:1938–1945CrossRefGoogle Scholar
  19. 19.
    Rüttinger S (2006) Confocal microscopy and quantitative single molecule techniques for metrology in molecular medicine. PhD thesis, TU-BerlinGoogle Scholar
  20. 20.
    Lamb DC, Schenk A, Röcker C, Scalfi-Happ C, Nienhaus GU (2000) Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection. Biophys J 79:1129–1138. doi: 10.1016/S0006-3495(00)76366-1 CrossRefPubMedGoogle Scholar
  21. 21.
    Lamb DC, Müller BK, Bräuchle CH (2005) Enhancing the sensitivity of fluorescence correlation spectroscopy by using time-correlated single photon counting. Curr Pharm Biotechnol 6:405–414. doi: 10.2174/138920105774370625 CrossRefPubMedGoogle Scholar
  22. 22.
    Höbel M, Ricka J (1994) Dead-time and afterpulsing correction in multiphoton timing with nonideal detectors. Rev Sci Instrum 65(7):2326–2336. doi: 10.1063/1.1144684 CrossRefGoogle Scholar
  23. 23.
    Enderlein J, Gregor I (2005) Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence correlation spectroscopy. Rev Sci Instrum 76:033102. doi: 10.1063/1.1863399 CrossRefGoogle Scholar
  24. 24.
    Zhao M, Jin L, Chen B, Ding Y, Ma H, Chen D (2003) Afterpulsing and its correction in fluorescence correlation spectroscopy experiments. Appl Opt 42(19):4031–4036. doi: 10.1364/AO.42.004031 CrossRefPubMedGoogle Scholar
  25. 25.
    Böhmer M, Wahl M, Rahn H-J, Erdmann R, Enderlein J (2002) Time-resolved fluorescence correlation spectroscopy. Chem Phys Lett 353:439–445. doi: 10.1016/S0009-2614(02)00044-1 CrossRefGoogle Scholar
  26. 26.
    Benda A, Hof M, Wahl M, Patting M, Erdmann R, Kapusta P (2005) TCSPC upgrade of a confocal FCS microscope. Rev Sci Instrum 76:33106. doi: 10.1063/1.1866814 CrossRefGoogle Scholar
  27. 27.
    Benda A, Fagulová V, Deyneka A, Enderlein J, Hof M (2006) Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: New perspectives in supported phospholipid bilayer research. Langmuir 22:9580–9585. doi: 10.1021/la061573d CrossRefPubMedGoogle Scholar
  28. 28.
    Humpolíčková J, Benda A, Sykora J, Macháň R, Kral T, Gasinska B, Enderlein J, Hof M (2008) Equilibrium dynamics of spermine-induced plasmid DNA condensation revealed by fluorescence lifetime correlation spectroscopy. Biophys J 94:L17–L19. doi: 10.1529/biophysj.107.122408 CrossRefPubMedGoogle Scholar
  29. 29.
    Humpolícková J, Beranová L, Stepánek M, Benda A, Procházka K, Hof M (2008) Fluorescence lifetime correlation spectroscopy reveals compaction mechanism of 10 and 49 kbp DNA and differences between polycation and cationic surfactant. J Phys Chem B 112:16823–16829CrossRefGoogle Scholar
  30. 30.
    Kapusta P, Wahl M, Benda A, Hof M, Enderlein J (2007) Fluorescence lifetime correlation spectroscopy. J Fluoresc 17:43–48. doi: 10.1007/s10895-006-0145-1 CrossRefPubMedGoogle Scholar
  31. 31.
    Gregor I, Enderlein J (2007) Time-resolved methods in biophysics. Fluorescence lifetime correlation spectroscopy. Photochem Photobiol Sci 6:0013–0018CrossRefGoogle Scholar
  32. 32.
    Wahl M, Gregor I, Patting M, Enderlein J (2003) Fast calculation of fluorescence correlation data with asynchronous time-correlated single-photon counting. Opt Express 11:3583–3591PubMedCrossRefGoogle Scholar
  33. 33.
    Koberling F, Krämer B, Tannert S, Rüttinger S, Ortmann U, Patting M, Wahl M, Ewers B, Kapusta P, Erdmann R (2008) Recent advances in time-correlated single-photon counting. Proceedings of SPIE, 6862:686209Google Scholar
  34. 34.
    Wahl M, Koberling F, Patting M, Rahn H, Erdmann R (2004) Time-resolved confocal fluorescence imaging and spectroscopy system with single molecule sensitivity and sub-micrometer resolution. Curr Pharm Biotechnol 5:299–308. doi: 10.2174/1389201043376841 CrossRefPubMedGoogle Scholar
  35. 35.
    Wahl M, Rahn H, Gregor I, Erdmann R, Enderlein J (2007) Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels. Rev Sci Instrum 78:033106. doi: 10.1063/1.2715948 CrossRefPubMedGoogle Scholar
  36. 36.
    Dertinger T, Pacheco V, von der Hocht I, Hartmann R, Gregor I, Enderlein J (2007) Two focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. ChemPhysChem 8:433–443. doi: 10.1002/cphc.200600638 CrossRefPubMedGoogle Scholar
  37. 37.
    Rüttinger S, Buschmann V, Krämer B, Erdmann R, Macdonald R, Koberling F (2007) Determination of the confocal volume for quantitative fluorescence correlation spectroscopy. Proceedings of SPIE 6630:66300DGoogle Scholar
  38. 38.
    Rüttinger S, Buschmann V, Krämer B, Erdmann R, Macdonald R, Koberling F (2008) Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy. J Microsc 232(2):334–352Google Scholar
  39. 39.

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Steffen Rüttinger
    • 1
  • Peter Kapusta
    • 2
  • Matthias Patting
    • 2
  • Michael Wahl
    • 2
  • Rainer Macdonald
    • 1
  1. 1.Physikalisch-Technische BundesanstaltBerlinGermany
  2. 2.PicoQuant GmbHBerlinGermany

Personalised recommendations