Abstract

Fluorescence correlation spectroscopy (FCS) is a powerful spectroscopic technique for studying samples at dilute fluorophore concentrations down to single molecules. The standard way of data acquisition, at such low concentrations, is an asynchronous photon counting mode that generates data only when a photon is detected. A significant problem is how to efficiently convert such asynchronously recorded photon count data into a FCS curve. This problem becomes even more challenging for more complex correlation analysis such as the recently introduced combination of FCS and time-correlated single-photon counting (TCSPC). Here, we present, analyze, and apply an algorithm that is highly efficient and can easily be adapted to arbitrarily complex correlation analysis.

© 2003 Optical Society of America

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References

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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]

Appl. Phys. B.

K. Schätzel �??Correlation techniques in dynamic light scattering,�?? Appl. Phys. B. 42, 193-213 (1987).
[CrossRef]

Biophys. J.

P. Schwille, F.J. Meyer-Almes, R. Rigler �??Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,�?? Biophys. J. 72, 1878-86 (1997).
[CrossRef] [PubMed]

D.C. Lamb, A. Schenk, C. Röcker, C. Scalfi-Happ, G.U. Nienhaus �??Sensitivity Enhancement in Fluorescence Correlation Spectroscopy of Multiple Species Using Time-Gated Detection,�?? Biophys. J. 79, 1129-38 (2000).
[CrossRef] [PubMed]

Chem. Phys. Lett.

M. Böhmer, M. Wahl, H.J. Rahn, R. Erdmann, J. Enderlein �??Time-resolved fluorescence correlation spectroscopy,�?? Chem. Phys. Lett. 353, 439-45 (2002).
[CrossRef]

J. Biotechnol.

C. Eggeling, S. Berger, L. Brand, J.R. Fries, J. Schaffer, A. Volkmer, C.A.M. Seidel �??Data registration and selective single-molecule anaylsis using mulit-parameter fluorescence detection,�?? J. Biotechnol. 86, 163-80 (2001).
[CrossRef] [PubMed]

J. Mod. Opt.

K. Schätzel, M. Drewel, S. Stimac �??Photon correlation measurements at large lag times: Improving statistical accuracy,�?? J. Mod. Opt. 35, 711-8 (1988).
[CrossRef]

Rev. Sci. Instrum.

M. Höbel, J. Ricka �??Dead-time and afterpulsing correction in multiphoton timing with nonideal detectors,�?? Rev. Sci. Instrum. 65, 2326-36 (1994).
[CrossRef]

W. Becker, H. Hickl, C. Zander, K.H. Drexhage, M. Sauer, S. Siebert, J. Wolfrum �??Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC),�?? Rev. Sci. Instrum. 70, 1835-41 (1999).
[CrossRef]

M. Böhmer, F. Pampaloni, M. Wahl, H.J. Rahn, R. Erdmann, J. Enderlein �??Advanced Time-Resolved Confocal Scanning Device For Ultrasensitive Fluorescence Detection,�?? Rev. Sci. Instrum. 72, 4145-52 (2001).
[CrossRef]

J.S. Eid, J.D. Müller, E. Gratton �??Data acquisition card for fluctuation correlation spectroscopy allowing full access to the detected photon sequence,�?? Rev. Sci. Instrum. 71, 361-8 (2000).
[CrossRef]

D. Magatti, F. Ferri �??25 ns software correlator for photon and fluorescence correlation spectroscopy,�?? Rev. Sci. Instrum. 74, 1135-44 (2003).
[CrossRef]

Other

M. Böhmer, J. Enderlein �??Single molecule detection on surfaces with the confocal laser scanning microscope,�?? in ref.[1], pp.145-83.

R. Rigler, E. Elson (Eds.) Fluorescence Correlation Spectroscopy (Springer, New York/Berlin, 2001).
[CrossRef]

C. Zander, J. Enderlein, R.A. Keller (Eds.) Single-Molecule Detection in Solution - Methods and Applications (VCH-Wiley, Berlin/New York, 2002).
[CrossRef]

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Figures (3)

Fig. 1.
Fig. 1.

Count-rate dependence of the estimated ratio Lbin /Ltttr of the computation times, Eqs.(3,4), for the conventional bin-and-correlate algorithm and the algorithm presented in this paper. The assumed minimum time resolution of the experiment is 100 ns, and the lag time vector is given by Eq.(2) with B=10 and ncasc =17.

Fig. 2.
Fig. 2.

Comparison of autocorrelation functions calculated with different algorithms. The blue line shows the result of the bin-and-correlate algorithm, red dots indicate the result of the time-tag-tocorrelation algorithm without time-scale coarsening, and the light-green line is the result of the time-tag-to-correlation algorithm with time-scale coarsening.

Fig. 3.
Fig. 3.

Dependence of computation time on count rate. The blue dots correspond to six measurements at the different excitation powers as indicated in the text. The line is a unity slope fit to the last five data points (assuming a linear dependence of computation time on count rate).

Equations (4)

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g ( τ ) = I ( t ) I ( t + τ )
τ j = { 1 if j = 1 τ j 1 + 2 ( j 1 ) B if j > 1
L bin ~ k T τ k
L tttr ~ B k N 2 k [ 1 exp ( 2 k N T ) ]

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