Abstract

We have developed a filterless multicolor detection unit for fluorescence correlation spectroscopy (FCS). This grating-based setup is continuously tunable for multicolor separation and is thus a powerful alternative to the classical cascade of dichroic mirrors and filters. Our tailored platform allows for accommodation of up to 15 detection channels covering the entire visible spectral range. As a proof of principle, we successfully demonstrate simultaneous FCS of four distinct fluorescent quantum dot species being mixed in aqueous solution. Grating-based detection allows for spectral high-resolution FCS in a stable and compact setup and is a feasible tool for quantitative investigation of complex biomolecular dynamics on a single molecule level.

© 2005 Optical Society of America

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    [CrossRef] [PubMed]
  2. D. Magde, E. Elson, and W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
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    [CrossRef] [PubMed]
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    [CrossRef]
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  6. T. Kohl, K. G. Heinze, R. Kuhlemann, A. Koltermann, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 99, 12,161 (2002).
    [CrossRef]
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    [CrossRef]
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    [CrossRef]
  9. D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, Science 300, 1434 (2003).
    [CrossRef] [PubMed]

2004 (2)

K. G. Heinze, M. Jahnz, and P. Schwille, Biophys. J. 86, 506 (2004).
[CrossRef]

S. A. Kim, K. G. Heinze, M. N. Waxham, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 101, 105 (2004).
[CrossRef]

2003 (1)

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, Science 300, 1434 (2003).
[CrossRef] [PubMed]

2002 (1)

T. Kohl, K. G. Heinze, R. Kuhlemann, A. Koltermann, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 99, 12,161 (2002).
[CrossRef]

2000 (1)

S. Weiss, Nat. Struct. Biol. 7, 724 (2000).
[CrossRef] [PubMed]

1998 (1)

W. Schrof, J. F. Klingler, S. Rozouvan, and D. Horn, Phys. Rev. E 57, R2523 (1998).
[CrossRef]

1997 (1)

P. Schwille, F. J. Meyer-Almes, and R. Rigler, Biophys. J. 72, 1878 (1997).
[CrossRef] [PubMed]

1993 (1)

R. Rigler, U. Mets, J. Widengren, and P. Kask, Eur. Biophys. J. 22, 169 (1993).

1972 (1)

D. Magde, E. Elson, and W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
[CrossRef]

Bruchez, M. P.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, Science 300, 1434 (2003).
[CrossRef] [PubMed]

Clark, S. W.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, Science 300, 1434 (2003).
[CrossRef] [PubMed]

Elson, E.

D. Magde, E. Elson, and W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
[CrossRef]

Heinze, K. G.

K. G. Heinze, M. Jahnz, and P. Schwille, Biophys. J. 86, 506 (2004).
[CrossRef]

S. A. Kim, K. G. Heinze, M. N. Waxham, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 101, 105 (2004).
[CrossRef]

T. Kohl, K. G. Heinze, R. Kuhlemann, A. Koltermann, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 99, 12,161 (2002).
[CrossRef]

Horn, D.

W. Schrof, J. F. Klingler, S. Rozouvan, and D. Horn, Phys. Rev. E 57, R2523 (1998).
[CrossRef]

Jahnz, M.

K. G. Heinze, M. Jahnz, and P. Schwille, Biophys. J. 86, 506 (2004).
[CrossRef]

Kask, P.

R. Rigler, U. Mets, J. Widengren, and P. Kask, Eur. Biophys. J. 22, 169 (1993).

Kim, S. A.

S. A. Kim, K. G. Heinze, M. N. Waxham, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 101, 105 (2004).
[CrossRef]

Klingler, J. F.

W. Schrof, J. F. Klingler, S. Rozouvan, and D. Horn, Phys. Rev. E 57, R2523 (1998).
[CrossRef]

Kohl, T.

T. Kohl, K. G. Heinze, R. Kuhlemann, A. Koltermann, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 99, 12,161 (2002).
[CrossRef]

Koltermann, A.

T. Kohl, K. G. Heinze, R. Kuhlemann, A. Koltermann, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 99, 12,161 (2002).
[CrossRef]

Kuhlemann, R.

T. Kohl, K. G. Heinze, R. Kuhlemann, A. Koltermann, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 99, 12,161 (2002).
[CrossRef]

Larson, D. R.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, Science 300, 1434 (2003).
[CrossRef] [PubMed]

Magde, D.

D. Magde, E. Elson, and W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
[CrossRef]

Mets, U.

R. Rigler, U. Mets, J. Widengren, and P. Kask, Eur. Biophys. J. 22, 169 (1993).

Meyer-Almes, F. J.

P. Schwille, F. J. Meyer-Almes, and R. Rigler, Biophys. J. 72, 1878 (1997).
[CrossRef] [PubMed]

Rigler, R.

P. Schwille, F. J. Meyer-Almes, and R. Rigler, Biophys. J. 72, 1878 (1997).
[CrossRef] [PubMed]

R. Rigler, U. Mets, J. Widengren, and P. Kask, Eur. Biophys. J. 22, 169 (1993).

Rozouvan, S.

W. Schrof, J. F. Klingler, S. Rozouvan, and D. Horn, Phys. Rev. E 57, R2523 (1998).
[CrossRef]

Schrof, W.

W. Schrof, J. F. Klingler, S. Rozouvan, and D. Horn, Phys. Rev. E 57, R2523 (1998).
[CrossRef]

Schwille, P.

K. G. Heinze, M. Jahnz, and P. Schwille, Biophys. J. 86, 506 (2004).
[CrossRef]

S. A. Kim, K. G. Heinze, M. N. Waxham, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 101, 105 (2004).
[CrossRef]

T. Kohl, K. G. Heinze, R. Kuhlemann, A. Koltermann, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 99, 12,161 (2002).
[CrossRef]

P. Schwille, F. J. Meyer-Almes, and R. Rigler, Biophys. J. 72, 1878 (1997).
[CrossRef] [PubMed]

Waxham, M. N.

S. A. Kim, K. G. Heinze, M. N. Waxham, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 101, 105 (2004).
[CrossRef]

Webb, W. W.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, Science 300, 1434 (2003).
[CrossRef] [PubMed]

D. Magde, E. Elson, and W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
[CrossRef]

Weiss, S.

S. Weiss, Nat. Struct. Biol. 7, 724 (2000).
[CrossRef] [PubMed]

Widengren, J.

R. Rigler, U. Mets, J. Widengren, and P. Kask, Eur. Biophys. J. 22, 169 (1993).

Williams, R. M.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, Science 300, 1434 (2003).
[CrossRef] [PubMed]

Wise, F. W.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, Science 300, 1434 (2003).
[CrossRef] [PubMed]

Zipfel, W. R.

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, Science 300, 1434 (2003).
[CrossRef] [PubMed]

Biophys. J. (2)

P. Schwille, F. J. Meyer-Almes, and R. Rigler, Biophys. J. 72, 1878 (1997).
[CrossRef] [PubMed]

K. G. Heinze, M. Jahnz, and P. Schwille, Biophys. J. 86, 506 (2004).
[CrossRef]

Eur. Biophys. J. (1)

R. Rigler, U. Mets, J. Widengren, and P. Kask, Eur. Biophys. J. 22, 169 (1993).

Nat. Struct. Biol. (1)

S. Weiss, Nat. Struct. Biol. 7, 724 (2000).
[CrossRef] [PubMed]

Phys. Rev. E (1)

W. Schrof, J. F. Klingler, S. Rozouvan, and D. Horn, Phys. Rev. E 57, R2523 (1998).
[CrossRef]

Phys. Rev. Lett. (1)

D. Magde, E. Elson, and W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
[CrossRef]

Proc. Natl. Acad. Sci. U.S.A. (2)

T. Kohl, K. G. Heinze, R. Kuhlemann, A. Koltermann, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 99, 12,161 (2002).
[CrossRef]

S. A. Kim, K. G. Heinze, M. N. Waxham, and P. Schwille, Proc. Natl. Acad. Sci. U.S.A. 101, 105 (2004).
[CrossRef]

Science (1)

D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise, and W. W. Webb, Science 300, 1434 (2003).
[CrossRef] [PubMed]

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Figures (3)

Fig. 1
Fig. 1

Optical setup for filter-free multicolor FCS. Four differently emitting Qdots, mixed in a sample, are efficiently excited with a single argon laser line. Emission is spectrally split by a diffraction grating and projected onto several fibers arranged in a linear array and detected by multiple APDs independently. Inset, normalized emission spectra (bottom) and the spectral collection range for each fiber in the array (top), shown by a scaled photograph.

Fig. 2
Fig. 2

Calibration of the detection by successively recording several subspectra of the TMR emission for different positions of a single 100 μ m fiber in the dispersed spectrum (first order) of the grating. The spectra were acquired with a fiber-coupled spectrometer. The full spectrum was detected by positioning the fiber in the zeroth diffraction order of the grating.

Fig. 3
Fig. 3

Autocorrelation curves of the four Qdots, quasi-simultaneously recorded with the respective fibers of the array (scattered plots) and fitted using a 3D diffusion model (solid curves). Efficient excitation was achieved with a standard 488 nm argon laser line and a focal power density of 1.4 kW cm 2 . The measurement time was 200 s.

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