Abstract

We report the design and application of a prism-based detection system for fluorescence (cross) correlation spectroscopy. The system utilizes a single laser wavelength for the simultaneous excitation of several dyes of different emission spectra. Fluorescence light is spectrally separated with a prismatic setup, and wavelengths are selected by scanning a fiber-coupled avalanche photodiode across the image spots. Multicolor autocorrelations are demonstrated with standard and tandem dyes, and fluorescence cross-correlation measurements of biotinylated nanocontainers and streptavidin are presented. This spectrometer offers high optical stability and no focal volume mismatch for the multicolor detection of molecular dynamics and interactions, with single-molecule sensitivity.

© 2006 Optical Society of America

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  1. D. Magde, E. L. Elson, and W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
    [CrossRef]
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    [CrossRef] [PubMed]
  3. T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, Proc. Natl. Acad. Sci. U.S.A. 97, 9461 (2000).
    [CrossRef] [PubMed]
  4. P. Schwille, F. Meyer-Almes, and R. Rigler, Biophys. J. 72, 1878 (1997).
    [CrossRef] [PubMed]
  5. L. C. Hwang and T. Wohland, ChemPhysChem 5, 549 (2004).
    [CrossRef] [PubMed]
  6. L. C. Hwang and T. Wohland, J. Chem. Phys. 122, 114708 (2005).
    [CrossRef] [PubMed]
  7. L. C. Hwang, M. Gösch, T. Lasser, and T. Wohland, have submitted a paper called ''Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect, higher order interactions using single wavelength laser excitation,'' to Biophys. J.
  8. SCHOTT, http://www.schott.com/opticslowbardevices/english/products/flash/abbediagrammlowbarflash.htm.
  9. P. Rigler and M. Meier, J. Am. Chem. Soc. 128, 367 (2006).
    [CrossRef] [PubMed]
  10. C. Nardin, T. Hirt, J. Leukel, and W. Meier, Langmuir 16, 1035 (2000).
    [CrossRef]
  11. M. Burkhardt, K. G. Heinze, and P. Schwille, Opt. Lett. 30, 2266 (2005).
    [CrossRef] [PubMed]

2006

P. Rigler and M. Meier, J. Am. Chem. Soc. 128, 367 (2006).
[CrossRef] [PubMed]

2005

2004

L. C. Hwang and T. Wohland, ChemPhysChem 5, 549 (2004).
[CrossRef] [PubMed]

2002

N. L. Thompson, A. M. Lieto, and N. W. Allen, Curr. Opin. Struct. Biol. 12, 634 (2002).
[CrossRef] [PubMed]

2000

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, Proc. Natl. Acad. Sci. U.S.A. 97, 9461 (2000).
[CrossRef] [PubMed]

C. Nardin, T. Hirt, J. Leukel, and W. Meier, Langmuir 16, 1035 (2000).
[CrossRef]

1997

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

1972

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

Alivisatos, A. P.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, Proc. Natl. Acad. Sci. U.S.A. 97, 9461 (2000).
[CrossRef] [PubMed]

Allen, N. W.

N. L. Thompson, A. M. Lieto, and N. W. Allen, Curr. Opin. Struct. Biol. 12, 634 (2002).
[CrossRef] [PubMed]

Burkhardt, M.

Chemla, D. S.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, Proc. Natl. Acad. Sci. U.S.A. 97, 9461 (2000).
[CrossRef] [PubMed]

Elson, E. L.

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

Gösch, M.

L. C. Hwang, M. Gösch, T. Lasser, and T. Wohland, have submitted a paper called ''Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect, higher order interactions using single wavelength laser excitation,'' to Biophys. J.

Heinze, K. G.

Hirt, T.

C. Nardin, T. Hirt, J. Leukel, and W. Meier, Langmuir 16, 1035 (2000).
[CrossRef]

Hwang, L. C.

L. C. Hwang and T. Wohland, J. Chem. Phys. 122, 114708 (2005).
[CrossRef] [PubMed]

L. C. Hwang and T. Wohland, ChemPhysChem 5, 549 (2004).
[CrossRef] [PubMed]

L. C. Hwang, M. Gösch, T. Lasser, and T. Wohland, have submitted a paper called ''Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect, higher order interactions using single wavelength laser excitation,'' to Biophys. J.

Lacoste, T. D.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, Proc. Natl. Acad. Sci. U.S.A. 97, 9461 (2000).
[CrossRef] [PubMed]

Lasser, T.

L. C. Hwang, M. Gösch, T. Lasser, and T. Wohland, have submitted a paper called ''Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect, higher order interactions using single wavelength laser excitation,'' to Biophys. J.

Leukel, J.

C. Nardin, T. Hirt, J. Leukel, and W. Meier, Langmuir 16, 1035 (2000).
[CrossRef]

Lieto, A. M.

N. L. Thompson, A. M. Lieto, and N. W. Allen, Curr. Opin. Struct. Biol. 12, 634 (2002).
[CrossRef] [PubMed]

Magde, D.

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

Meier, M.

P. Rigler and M. Meier, J. Am. Chem. Soc. 128, 367 (2006).
[CrossRef] [PubMed]

Meier, W.

C. Nardin, T. Hirt, J. Leukel, and W. Meier, Langmuir 16, 1035 (2000).
[CrossRef]

Meyer-Almes, F.

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

Michalet, X.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, Proc. Natl. Acad. Sci. U.S.A. 97, 9461 (2000).
[CrossRef] [PubMed]

Nardin, C.

C. Nardin, T. Hirt, J. Leukel, and W. Meier, Langmuir 16, 1035 (2000).
[CrossRef]

Pinaud, F.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, Proc. Natl. Acad. Sci. U.S.A. 97, 9461 (2000).
[CrossRef] [PubMed]

Rigler, P.

P. Rigler and M. Meier, J. Am. Chem. Soc. 128, 367 (2006).
[CrossRef] [PubMed]

Rigler, R.

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

Schwille, P.

M. Burkhardt, K. G. Heinze, and P. Schwille, Opt. Lett. 30, 2266 (2005).
[CrossRef] [PubMed]

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

Thompson, N. L.

N. L. Thompson, A. M. Lieto, and N. W. Allen, Curr. Opin. Struct. Biol. 12, 634 (2002).
[CrossRef] [PubMed]

Webb, W. W.

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

Weiss, S.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, Proc. Natl. Acad. Sci. U.S.A. 97, 9461 (2000).
[CrossRef] [PubMed]

Wohland, T.

L. C. Hwang and T. Wohland, J. Chem. Phys. 122, 114708 (2005).
[CrossRef] [PubMed]

L. C. Hwang and T. Wohland, ChemPhysChem 5, 549 (2004).
[CrossRef] [PubMed]

L. C. Hwang, M. Gösch, T. Lasser, and T. Wohland, have submitted a paper called ''Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect, higher order interactions using single wavelength laser excitation,'' to Biophys. J.

Biophys. J.

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

ChemPhysChem

L. C. Hwang and T. Wohland, ChemPhysChem 5, 549 (2004).
[CrossRef] [PubMed]

Curr. Opin. Struct. Biol.

N. L. Thompson, A. M. Lieto, and N. W. Allen, Curr. Opin. Struct. Biol. 12, 634 (2002).
[CrossRef] [PubMed]

J. Am. Chem. Soc.

P. Rigler and M. Meier, J. Am. Chem. Soc. 128, 367 (2006).
[CrossRef] [PubMed]

J. Chem. Phys.

L. C. Hwang and T. Wohland, J. Chem. Phys. 122, 114708 (2005).
[CrossRef] [PubMed]

Langmuir

C. Nardin, T. Hirt, J. Leukel, and W. Meier, Langmuir 16, 1035 (2000).
[CrossRef]

Opt. Lett.

Phys. Rev. Lett.

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

Proc. Natl. Acad. Sci. U.S.A.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, Proc. Natl. Acad. Sci. U.S.A. 97, 9461 (2000).
[CrossRef] [PubMed]

Other

L. C. Hwang, M. Gösch, T. Lasser, and T. Wohland, have submitted a paper called ''Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect, higher order interactions using single wavelength laser excitation,'' to Biophys. J.

SCHOTT, http://www.schott.com/opticslowbardevices/english/products/flash/abbediagrammlowbarflash.htm.

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Figures (3)

Fig. 1
Fig. 1

Optical setup of prism-based fluorescence correlation spectrometer. A single laser wavelength excites differently emitting fluorophores. The emitted light is collimated and chromatically dispersed by an isosceles prism and focused onto an array of optical fibers that is coupled to avalanche photodiodes. The inset shows (a) the normalized emission spectra of Rhodamine green (RhG), R-phycoerythin (RPE) and Alexa 647-R-phycoerythrin-streptavidin (AXSA); (b)–(d) the colored autocorrelation data curves recorded by scanning a 100 μ m optical fiber along the imaged spots from green, yellow, to red wavelengths, respectively. The black curves show the experimental fits. F, excitation filter; Obj, microscope objective, L1–L5, lenses; DM, dichroic mirror; PH, pinhole; P, dispersive prism; OF, optical fibers; APD1–3, avalanche photodiodes.

Fig. 2
Fig. 2

(a) Plot of lateral displacement y ( λ ) against wavelength caused by prism dispersion. The nonlinear dispersion depends on the glass properties of the prism. (b) Plot of transmission T ( λ ) versus wavelength shows the laser line 488 nm and the spectral bandwidths calculated for optic fibers with core diameters of 105 μ m with cladding and buffer diameters of 250 μ m .

Fig. 3
Fig. 3

Cross-correlation data (gray curves) and their fitting curves (black curves) between green and red channels and their intensity traces. (a) Cross-correlation curves show binding of biotinylated RhG nanocontainers and AXSA. Inset, schematic drawing of the dual-color complex. Red intensity spikes show oligomerization of AXSA to the nanocontainers. (b) Negative control shows neither cross correlation nor intensity spikes from binding. Correlations were measured at 60 s each for 20 × with a laser line of 488 nm at power 50 μ W .

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