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Fluorescence Correlation Spectroscopy (FCS)


Fluorescence Correlation Spectroscopy uses the information contained in intensity fluctuations over time. Fluorescently labeled molecules emit bursts of photons while traveling through the excitation volume due to Brownian motion. Correlations of fluctuations in the intensities of detected fluorescence over time directly offer information on the concentration or average number of molecules and the diffusion time of the molecules (size of the PSF). Furthermore fluctuations in intensity can be caused by triplet states, conformational motions, or chemical reactions and can be used to determine reaction rates, hydrodynamic radii, diffusion coefficients, or singlet-triplet dynamics. Changes in diffusion time can reveal information on binding kinetics.


  • Magde D., Elson E.L., Webb W.W. "Thermodynamic Fluctuations in a Reacting System: Measurement by Fluorescence Correlation Spectroscopy." Physical Review Letters, 1972. 29(11): 705.
  • Elson E.L., Magde D. "Fluorescence correlation spectroscopy. I. Conceptual basis and theory." Biopolymers, 1974. 13(1): 1-27.

Pulsed Interleaved Excitation-Fluorescence Cross-Correlation Spectroscopy (PIE-FCCS)

Fluorescence Cross-Correlation Measurements determine the correlation between different colors rather than the autocorrelation of only one (FCS) and can therefore show, whether differently labeled particles diffuse together. Cross-Correlations can be used to observe molecular interactions independent of the diffusion time in vitro or in cellulo.
Pulsed interleaved excitation ensures, that the excitation source of each photon can be recognized and thus spectral cross-talk and direct excitation can be distinguished from cross-correlation.


  • Eigen M., Rigler R. "Sorting single molecules: Application to diagnostics and evolutionary biotechnology." Proc. Natl. Acad. Sci. U.S.A., 1994. 91: 5740-5747.
  • Schwille P., Meyer-Almes F.J., Rigler R. "Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution." Biophysical Journal, 1997. 72(4): 1878-1886.
  • Foo Y.H., Naredi-Rainer N., Lamb D.C., Ahmed S., Wohland T. "Factors Affecting the Quantification of Biomolecular Interactions by Fluorescence Cross-Correlation Spectroscopy." Biophysical Journal, 2012. 102: 1174-1183.

Fluorescence Lifetime Correlation Spectroscopy (FLCS)

The short time of the excitation pulse in pulsed interleaved excitation and picosecond time-resolved detection is used for fluorescence lifetime correlation spectroscopy to determine the fluorescence lifetime of the fluorophore. It is thus possible to perform cross-correlation analysis on spectrally overlapping fluorophores, which differ in their lifetimes, by application of lifetime-specific filters.


  • Kapusta P., Wahl M., Benda A., Hof M., Enderlein, J. "Fluorescence lifetime correlation spectroscopy." J. Fluoresc., 2007, 17: 43-48.
  • Kapusta P., Machan R., Benda A., Hof M. "Fluorescence Lifetime Correlation Spectroscopy (FLCS): Concepts, Applications and Outlook." Int. J. Mol. Sci., 2012, 13: 12890-12910.

Species-FCS

Similar to FLCS, Species-FCS uses the information from pulsed excitation. Filters can be generated taking into account the fluorescence anisotropy and the spectrally resolved fluorescence decays. Species-FCS can thus distinguish different populations with similar fluorescence lifetimes and calculate background-free correlation curves. Since Species-FCS relies on changes in anisotropy, it is well suited for molecular interaction studies.


  • Felekyan S., Kalinin S., Sanabria H., Valeri A., Seidel C.A.M. "Filtered FCS: Species Auto- and Cross-Correlation Functions Highlight Binding and Dynamics in Biomolecules." ChemPhysChem, 2012. 13: 1036-1053.

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