Statistical Analysis of Diffusion Coefficient Determination by Fluorescence Correlation Spectroscopy Article Received: 17 June 2004 Accepted: 29 June 2004 DOI:
10.1007/s10895-005-2633-0 Cite this article as: Enderlein, J., Gregor, I., Patra, D. et al. J Fluoresc (2005) 15: 415. doi:10.1007/s10895-005-2633-0 Abstract
Fluorescence correlation spectroscopy (FCS) has become an important and widely used technique for many applications in physics, chemistry, and biology. The parameter most frequently addressed by FCS is the diffusion of molecules in solution. Due to the highly non-linear connection between the diffusion coefficient and a measured autocorrelation function, it is extremely difficult to analyse the accuracy of the diffusion-coefficient determination in a FCS experiment. Here, we present a simplified analysis based on some general maximum-likelihood considerations, and numerical result are given for the dependence of the accuracy of the diffusion-coefficient determination on sample concentration, brightness, and measurement time. Optimal concentration values for performing FCS are found.
Key Words Fluorescence correlation spectroscopy diffusion coefficient statistical accuracy References
D. Magde, E. Elson, and W. W. Webb (1972). Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy.
Phys. Rev. Lett
E. L. Elson and D. Magde (1974). Fluorescence Correlation Spectroscopy. I. Conceptual basis and theory.
D. Magde, E. Elson, and W. W. Webb (1974). Fluorescence Correlation Spectroscopy. II. An experimental realization.
N. L. Thompson (1991). Fluorescence correlation spectroscopy. in J. R. Lakowicz (Ed.),
Topics in Fluorescence Spectroscopy 1,
Plenum Press, New York, pp. 337–378.
J. Widengren and ¨. Mets (2002). Conceptual basis of Fluorescence Correlation Spectroscopy and related techniques as tools in bioscience. in C. Zander, J. Enderlein, and R. A. Keller (Eds.),
Single-Molecule Detection in Solution—Methods and Applications, Wiley-VCH, Berlin, pp. 69–95.
J. Korlach, P. Schwille, W. W. Webb, and G. W. Feigenson (1999). Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy.
Proc. Nat. Acad. Sci. USA 69
C. Gell, D. J. Brockwell, G. S. Beddard, S. E. Radford, A. P. Kalverda, and D. A. Smith (2001). Accurate use of single molecule fluorescence correlation spectroscopy to determine molecular diffusion times.
N. Yoshida, M. Tamura, and M. Kinjo (2000). Fluorescence Correlation Spectroscopy: A new tool for probing the microenvironment of the internal space of organelles.
J. Widengren and R. Rigler (1998). Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solution and on cell surfaces.
Cell. Mol. Biol
S. Bj¨rling, M. Kinjo, Z. F¨ldes-Papp, E. Hagman, P. Thyberg, and R. Rigler (1998). Fluorescence Correlation Spectroscopy of enzymatic DNA polymerization.
K. H¨sler, O. P¨nke, and W. Junge (1999). On the stator of rotary ATP synthase: The binding strength of subunit δ to αβ
as determined by Fluorescence Correlation Spectroscopy.
P. Schwille, J. Bieschke, and F. Oehlenschlager (1997). Kinetic investigations by Fluorescence Correlation Spectroscopy: The analytical and diagnostic potential of diffusion studies.
T. Wohland, K. Friedrich, R. Hovius, and H. Vogel (1999). Study of ligand-receptor interactions by Fluorescence Correlation Spectroscopy with different fluorophores: Evidence that the homopentameric 5-hydroxytryptamine type 3 as receptor binds only one ligand.
K. G. Heinze, M. Rarbach, M. Jahnz, and P. Schwille (2002). Two-photon fluorescence coincidence analysis: Rapid measurements of enzyme kinetics.
U. Kettling, A. Koltermann, P. Schwille, and M. Eigen (1998). Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy.
Proc. Natl. Acad. Sci. USA 95
J. Widengren, ¨. Mets, and R. Rigler (1999). Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy.
U. Haupts, S. Maiti, P. Schwille, and W. W. Webb (1998). Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy.
Proc. Nat. Acad. Sci. USA 95
A. A. Heikal, S. T. Hess, G. S. Baird, R. Y. Tsien, and W. W. Webb (2000). Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: Coral red (dsRed) and yellow (Citrine).
Proc. Natl. Acad. Sci. USA 97
R. Brock, G. Vàmosi, G. Vereb, and T. M. Jovin (1999). Rapid characterization of green fluorescent protein fusion proteins on the molecular and cellular level by fluorescence correlation microscopy.
Proc. Natl. Acad. Sci. USA 96
A. A. Heikal, S. T. Hess, and W. W. Webb (2001). Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): Acid–base specificity.
P. Schwille, S. Kummer, A. A. Heikal, W. E. Moerner, and W. W. Webb (2000). Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins.
Proc. Natl. Acad. Sci. USA 97
J. Widengren and C. A. M Seidel (2000). Manipulation and characterization of photo-induced transient states of Merocyanine 540 by fluorescence correlation spectroscopy.
Phys. Chem. Chem. Phys
S. Huang, A. A. Heikal, and W. W. Webb (2002). Two-Photon Fluorescence Spectroscopy and Microscopy of NAD(P)H and Flavoprotein.
F. Malvezzi-Campeggi, M. Jahnz, K. G. Heinze, P. Dittrich, and P. Schwille (2001). Light-induced flickering of dsRed provides evidence for distinct and interconvertible fluorescent states.
J. Widengren and P. Schwille (2000). Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy.
J. Phys. Chem. A 104
P. Schwille (2001). Fluorescence correlation spectroscopy and its potential for intracellular applications.
Cell. Biochem. Biophys
S. T. Hess, S. Huang, A. A. Heikal, and W. W. Webb (2002). Biological and chemical applications of Fluorescence Correlation Spectroscopy: A review.
R. Rigler and E. Elson (Eds.) (2001).
Fluorescence Correlation Spectroscopy
, Springer, Berlin.
D. E. Koppel (1974). Statistical accuracy in fluorescence correlation spectroscopy.
Phys. Rev. A 10
H. Qian (1990). On the statistics of fluorescence correlation spectroscopy.
P. Kask, R. G¨nther, and P. Axhausen (1997). Statistical accuracy in fluorescence fluctuation experiments
Eur. Biophys. J
U. Meseth, T. Wohland, R. Rigler, and H. Vogel (1999). Resolution of fluorescence correlation measurements.
, 1619– 1631.
T. Wohland, R. Rigler, and H. Vogel (2001). The standard deviation in fluorescence correlation spectroscopy.
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