Abstract

Fluorescence fluctuation spectroscopy is applied to study molecules passing through a small observation volume, usually subjected to diffusive or convective motion in a liquid phase. We suggest that such a technique could be used to measure the areal absolute concentration of fluorophores deposited on a substrate or embedded in a thin film, with a resolution of a few micrometers. The principle is to translate the solid substrate in front of a confocal fluorescence microscope objective and to record the subsequent fluctuations of the fluorescence intensity. The validity of this concept is investigated on model substrates (fluorescent microspheres) and DNA biochips.

© 2006 Optical Society of America

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References

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2005

Y. Xiao, V. Buschmann, and K. D. Weston, Anal. Chem. 77, 36 (2005).
[CrossRef]

J. P. Skinner, Y. Chen, and J. D. Müller, Biophys. J. 89, 1288 (2005).
[CrossRef] [PubMed]

2004

Q. Ruan, M. A. Cheng, M. Levi, E. Gratton, and W. W. Mantulin, Biophys. J. 87, 1260 (2004).
[CrossRef] [PubMed]

J. D. Müller, Biophys. J. 86, 3981 (2004).
[CrossRef] [PubMed]

2001

1999

Y. Chen, J. D. Müller, P. T. C. So, and E. Gratton, Biophys. J. 77, 553 (1999).
[CrossRef] [PubMed]

P. Kask, K. Palo, D. Ullmann, and K. Gall, Proc. Natl. Acad. Sci. U.S.A. 96, 13756 (1999).
[CrossRef] [PubMed]

1998

X. S. Xie and J. K. Trautman, Annu. Rev. Phys. Chem. 49, 441 (1998).
[CrossRef]

1993

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, Biophys. J. 65, 1135 (1993).
[CrossRef] [PubMed]

1986

N. O. Petersen, Biophys. J. 49, 809 (1986).
[CrossRef] [PubMed]

1978

D. Magde, W. W. Webb, and E. L. Elson, Biopolymers 17, 361 (1978).
[CrossRef]

Buschmann, V.

Y. Xiao, V. Buschmann, and K. D. Weston, Anal. Chem. 77, 36 (2005).
[CrossRef]

Chen, Y.

J. P. Skinner, Y. Chen, and J. D. Müller, Biophys. J. 89, 1288 (2005).
[CrossRef] [PubMed]

Y. Chen, J. D. Müller, P. T. C. So, and E. Gratton, Biophys. J. 77, 553 (1999).
[CrossRef] [PubMed]

Cheng, M. A.

Q. Ruan, M. A. Cheng, M. Levi, E. Gratton, and W. W. Mantulin, Biophys. J. 87, 1260 (2004).
[CrossRef] [PubMed]

Elson, E. L.

D. Magde, W. W. Webb, and E. L. Elson, Biopolymers 17, 361 (1978).
[CrossRef]

Gall, K.

P. Kask, K. Palo, D. Ullmann, and K. Gall, Proc. Natl. Acad. Sci. U.S.A. 96, 13756 (1999).
[CrossRef] [PubMed]

Gratton, E.

Q. Ruan, M. A. Cheng, M. Levi, E. Gratton, and W. W. Mantulin, Biophys. J. 87, 1260 (2004).
[CrossRef] [PubMed]

Y. Chen, J. D. Müller, P. T. C. So, and E. Gratton, Biophys. J. 77, 553 (1999).
[CrossRef] [PubMed]

Höddelius, P. L.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, Biophys. J. 65, 1135 (1993).
[CrossRef] [PubMed]

Kask, P.

P. Kask, K. Palo, D. Ullmann, and K. Gall, Proc. Natl. Acad. Sci. U.S.A. 96, 13756 (1999).
[CrossRef] [PubMed]

Levi, M.

Q. Ruan, M. A. Cheng, M. Levi, E. Gratton, and W. W. Mantulin, Biophys. J. 87, 1260 (2004).
[CrossRef] [PubMed]

Magde, D.

D. Magde, W. W. Webb, and E. L. Elson, Biopolymers 17, 361 (1978).
[CrossRef]

Magnusson, K. E.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, Biophys. J. 65, 1135 (1993).
[CrossRef] [PubMed]

Mantulin, W. W.

Q. Ruan, M. A. Cheng, M. Levi, E. Gratton, and W. W. Mantulin, Biophys. J. 87, 1260 (2004).
[CrossRef] [PubMed]

Müller, J. D.

J. P. Skinner, Y. Chen, and J. D. Müller, Biophys. J. 89, 1288 (2005).
[CrossRef] [PubMed]

J. D. Müller, Biophys. J. 86, 3981 (2004).
[CrossRef] [PubMed]

Y. Chen, J. D. Müller, P. T. C. So, and E. Gratton, Biophys. J. 77, 553 (1999).
[CrossRef] [PubMed]

Palo, K.

P. Kask, K. Palo, D. Ullmann, and K. Gall, Proc. Natl. Acad. Sci. U.S.A. 96, 13756 (1999).
[CrossRef] [PubMed]

Petersen, N. O.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, Biophys. J. 65, 1135 (1993).
[CrossRef] [PubMed]

N. O. Petersen, Biophys. J. 49, 809 (1986).
[CrossRef] [PubMed]

Ruan, Q.

Q. Ruan, M. A. Cheng, M. Levi, E. Gratton, and W. W. Mantulin, Biophys. J. 87, 1260 (2004).
[CrossRef] [PubMed]

Seger, O.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, Biophys. J. 65, 1135 (1993).
[CrossRef] [PubMed]

Skinner, J. P.

J. P. Skinner, Y. Chen, and J. D. Müller, Biophys. J. 89, 1288 (2005).
[CrossRef] [PubMed]

So, P. T. C.

Y. Chen, J. D. Müller, P. T. C. So, and E. Gratton, Biophys. J. 77, 553 (1999).
[CrossRef] [PubMed]

Trautman, J. K.

X. S. Xie and J. K. Trautman, Annu. Rev. Phys. Chem. 49, 441 (1998).
[CrossRef]

Ullmann, D.

P. Kask, K. Palo, D. Ullmann, and K. Gall, Proc. Natl. Acad. Sci. U.S.A. 96, 13756 (1999).
[CrossRef] [PubMed]

Webb, W. W.

W. W. Webb, Appl. Opt. 40, 3969 (2001).
[CrossRef]

D. Magde, W. W. Webb, and E. L. Elson, Biopolymers 17, 361 (1978).
[CrossRef]

Weston, K. D.

Y. Xiao, V. Buschmann, and K. D. Weston, Anal. Chem. 77, 36 (2005).
[CrossRef]

Wiseman, P. W.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, Biophys. J. 65, 1135 (1993).
[CrossRef] [PubMed]

Xiao, Y.

Y. Xiao, V. Buschmann, and K. D. Weston, Anal. Chem. 77, 36 (2005).
[CrossRef]

Xie, X. S.

X. S. Xie and J. K. Trautman, Annu. Rev. Phys. Chem. 49, 441 (1998).
[CrossRef]

Anal. Chem.

Y. Xiao, V. Buschmann, and K. D. Weston, Anal. Chem. 77, 36 (2005).
[CrossRef]

Annu. Rev. Phys. Chem.

X. S. Xie and J. K. Trautman, Annu. Rev. Phys. Chem. 49, 441 (1998).
[CrossRef]

Appl. Opt.

Biophys. J.

J. D. Müller, Biophys. J. 86, 3981 (2004).
[CrossRef] [PubMed]

Y. Chen, J. D. Müller, P. T. C. So, and E. Gratton, Biophys. J. 77, 553 (1999).
[CrossRef] [PubMed]

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, Biophys. J. 65, 1135 (1993).
[CrossRef] [PubMed]

N. O. Petersen, Biophys. J. 49, 809 (1986).
[CrossRef] [PubMed]

Q. Ruan, M. A. Cheng, M. Levi, E. Gratton, and W. W. Mantulin, Biophys. J. 87, 1260 (2004).
[CrossRef] [PubMed]

J. P. Skinner, Y. Chen, and J. D. Müller, Biophys. J. 89, 1288 (2005).
[CrossRef] [PubMed]

Biopolymers

D. Magde, W. W. Webb, and E. L. Elson, Biopolymers 17, 361 (1978).
[CrossRef]

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

P. Kask, K. Palo, D. Ullmann, and K. Gall, Proc. Natl. Acad. Sci. U.S.A. 96, 13756 (1999).
[CrossRef] [PubMed]

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Figures (2)

Fig. 1
Fig. 1

Fluorescence data obtained by translating a sample containing highly diluted nanospheres: (a) fluorescence count rate versus position; (b) autocorrelation functions obtained by translating the sample at various speeds; the solid curves are the experimental data (speeds decrease from left to right) and the dashed curves are the theoretical curves [Eq. (1)] with various characteristic times, τ c , given in the inset.

Fig. 2
Fig. 2

Scan of fluorescent DNA molecules deposited on a glass coverslip. The solid curve is the local count rate, averaged over distances of 5 μ m ( Δ t = 0.1 s ) . The solid curve with error bars is the local number of fluorescent molecules at the focal spot estimated from the fluctuations of the fluorescence using Eq. (4). Error bars are estimated according to Müller (Ref. [3]). The horizontal dashed line is the number of molecules given by Eq. (2) with the autocorrelation function calculated over the whole scan.

Equations (4)

Equations on this page are rendered with MathJax. Learn more.

G ( τ ) = 1 + [ G ( 0 ) 1 ] exp [ ( τ τ c ) 2 ] .
N = γ 2 G ( 0 ) 1 ,
N = γ 2 k 2 Δ k 2 k .
ϵ eff = ϵ ( 1 + Δ ϵ 2 ϵ 2 ) .

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