Abstract

Fluorescence correlation spectroscopy (FCS) is carried out with an electron multiplying CCD (EMCCD). This new strategy is compared to standard detection by an avalanche photo diode showing good agreement with respect to the resulting autocorrelation curves. Applying different readout modes, a time resolution of 20 µs can be achieved, which is sufficient to resolve the diffusion of free dye in solution. The advantages of implementing EMCCD cameras in wide-field ultra low light imaging, as well as in multi-spot confocal laser scanning microscopy, can consequently also be exploited for spatially resolved FCS. First proof-of-principle FCS measurements with two excitation volumes demonstrate the advantage of the flexible CCD area detection.

© 2006 Optical Society of America

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References

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  1. D. Magde, W. W. Webb, and E. Elson, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
    [Crossref]
  2. R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1993).
    [Crossref]
  3. J. Widengren, R. Rigler, and Ü. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4(3), 255–258 (1994).
    [Crossref]
  4. U. Haupts, S. Maiti, P. Schwille, and W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(23), 13573–13578 (1998).
    [Crossref]
  5. P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72, 1878–1886 (1997).
    [Crossref] [PubMed]
  6. M. Brinkmeier, K. Dörre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
    [Crossref]
  7. H. Blom, M. Johansson, A.-S. Hedman, L. Lundberg, A. Hanning, S. H°ard, and R. Rigler, “Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2×2 fan-out element,” Appl. Opt. 41(16), 3336–3342 (2002).
    [Crossref]
  8. M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2×2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
    [Crossref]

2004 (1)

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2×2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[Crossref]

2002 (1)

H. Blom, M. Johansson, A.-S. Hedman, L. Lundberg, A. Hanning, S. H°ard, and R. Rigler, “Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2×2 fan-out element,” Appl. Opt. 41(16), 3336–3342 (2002).
[Crossref]

1999 (1)

M. Brinkmeier, K. Dörre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
[Crossref]

1998 (1)

U. Haupts, S. Maiti, P. Schwille, and W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(23), 13573–13578 (1998).
[Crossref]

1997 (1)

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72, 1878–1886 (1997).
[Crossref] [PubMed]

1994 (1)

J. Widengren, R. Rigler, and Ü. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4(3), 255–258 (1994).
[Crossref]

1993 (1)

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1993).
[Crossref]

1972 (1)

D. Magde, W. W. Webb, and E. Elson, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Anhut, T.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2×2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[Crossref]

Besse, P.-A.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2×2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[Crossref]

Blom, H.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2×2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[Crossref]

H. Blom, M. Johansson, A.-S. Hedman, L. Lundberg, A. Hanning, S. H°ard, and R. Rigler, “Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2×2 fan-out element,” Appl. Opt. 41(16), 3336–3342 (2002).
[Crossref]

Brinkmeier, M.

M. Brinkmeier, K. Dörre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
[Crossref]

Dörre, K.

M. Brinkmeier, K. Dörre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
[Crossref]

Eigen, M.

M. Brinkmeier, K. Dörre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
[Crossref]

Elson, E.

D. Magde, W. W. Webb, and E. Elson, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Gösch, M.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2×2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[Crossref]

H°ard, S.

H. Blom, M. Johansson, A.-S. Hedman, L. Lundberg, A. Hanning, S. H°ard, and R. Rigler, “Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2×2 fan-out element,” Appl. Opt. 41(16), 3336–3342 (2002).
[Crossref]

Hanning, A.

H. Blom, M. Johansson, A.-S. Hedman, L. Lundberg, A. Hanning, S. H°ard, and R. Rigler, “Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2×2 fan-out element,” Appl. Opt. 41(16), 3336–3342 (2002).
[Crossref]

Haupts, U.

U. Haupts, S. Maiti, P. Schwille, and W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(23), 13573–13578 (1998).
[Crossref]

Hedman, A.-S.

H. Blom, M. Johansson, A.-S. Hedman, L. Lundberg, A. Hanning, S. H°ard, and R. Rigler, “Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2×2 fan-out element,” Appl. Opt. 41(16), 3336–3342 (2002).
[Crossref]

Johansson, M.

H. Blom, M. Johansson, A.-S. Hedman, L. Lundberg, A. Hanning, S. H°ard, and R. Rigler, “Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2×2 fan-out element,” Appl. Opt. 41(16), 3336–3342 (2002).
[Crossref]

Kask, P.

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1993).
[Crossref]

Lasser, T.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2×2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[Crossref]

Lundberg, L.

H. Blom, M. Johansson, A.-S. Hedman, L. Lundberg, A. Hanning, S. H°ard, and R. Rigler, “Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2×2 fan-out element,” Appl. Opt. 41(16), 3336–3342 (2002).
[Crossref]

Magde, D.

D. Magde, W. W. Webb, and E. Elson, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Maiti, S.

U. Haupts, S. Maiti, P. Schwille, and W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(23), 13573–13578 (1998).
[Crossref]

Mets, Ü.

J. Widengren, R. Rigler, and Ü. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4(3), 255–258 (1994).
[Crossref]

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1993).
[Crossref]

Meyer-Almes, F. J.

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72, 1878–1886 (1997).
[Crossref] [PubMed]

Popovic, R. S.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2×2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[Crossref]

Rigler, R.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2×2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[Crossref]

H. Blom, M. Johansson, A.-S. Hedman, L. Lundberg, A. Hanning, S. H°ard, and R. Rigler, “Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2×2 fan-out element,” Appl. Opt. 41(16), 3336–3342 (2002).
[Crossref]

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72, 1878–1886 (1997).
[Crossref] [PubMed]

J. Widengren, R. Rigler, and Ü. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4(3), 255–258 (1994).
[Crossref]

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1993).
[Crossref]

Rochas, A.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2×2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[Crossref]

Schwille, P.

U. Haupts, S. Maiti, P. Schwille, and W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(23), 13573–13578 (1998).
[Crossref]

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72, 1878–1886 (1997).
[Crossref] [PubMed]

Serov, A.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2×2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[Crossref]

Stephan, J.

M. Brinkmeier, K. Dörre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
[Crossref]

Webb, W. W.

U. Haupts, S. Maiti, P. Schwille, and W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(23), 13573–13578 (1998).
[Crossref]

D. Magde, W. W. Webb, and E. Elson, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Widengren, J.

J. Widengren, R. Rigler, and Ü. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4(3), 255–258 (1994).
[Crossref]

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1993).
[Crossref]

Anal. Chem. (1)

M. Brinkmeier, K. Dörre, J. Stephan, and M. Eigen, “Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures,” Anal. Chem. 71(3), 609–616 (1999).
[Crossref]

Appl. Opt. (1)

H. Blom, M. Johansson, A.-S. Hedman, L. Lundberg, A. Hanning, S. H°ard, and R. Rigler, “Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2×2 fan-out element,” Appl. Opt. 41(16), 3336–3342 (2002).
[Crossref]

Biophys. J. (1)

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72, 1878–1886 (1997).
[Crossref] [PubMed]

Eur. Biophys. J. (1)

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1993).
[Crossref]

J. Biomed. Opt. (1)

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2×2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[Crossref]

J. Fluoresc. (1)

J. Widengren, R. Rigler, and Ü. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4(3), 255–258 (1994).
[Crossref]

Phys. Rev. Lett. (1)

D. Magde, W. W. Webb, and E. Elson, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Proc. Natl. Acad. Sci. U.S.A. (1)

U. Haupts, S. Maiti, P. Schwille, and W. W. Webb, “Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 95(23), 13573–13578 (1998).
[Crossref]

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Figures (5)

Fig. 1.
Fig. 1.

Optical setup to perform FCS with two different detectors (BS: beam splitter, BF: bandpass filter, TL: tube lens).

Fig. 2.
Fig. 2.

Integrated CCD images over time (left) and extracted signal traces (right) from various number of CCD-pixels (pixel size 24 µm×24 µm) for a measurement of fluorescent quantum dots. The CCD is operated in kinetic mode (usual frame transfer). The bottom trace shows the count rate from a standard APD measurement with a 50 µm diameter fiber.

Fig. 3.
Fig. 3.

Autocorrelation curves for the CCD signal (see Fig. 2) and fit results in comparison to standard APD detection.

Fig. 4.
Fig. 4.

The fast kinetic mode of the CCD with 20 µs time resolution allows to perform FCS for commonly used fluorescent probes like eGFP (left) and Alexa Fluor 488 (right). The fit results for CCD detection (2) show a good agreement with APD detection (1).

Fig. 5.
Fig. 5.

Two-spot FCS for Alexa Fluor 488, simultaneously detected by the CCD (2,4) and successively by an APD (1,3) for comparison.

Equations (1)

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G ( τ ) = 1 N ( 1 + τ τ D ) 1 ( 1 + 1 S P 2 τ τ D ) 1 2

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