Abstract

Fluorescence Correlation Spectroscopy (FCS) demands a high rate of photon detection per molecule, low background, and large fluctuations of fluorescence associated with translational motion. The new approach presented here, Surface Plasmon Assisted Microscope (SPAM), meets these requirements by drastically limiting the observation volume. In this method, the observational layer is made so thin that fluctuations are mostly due to the axial motion of molecules. This is conveniently realized by placing a sample on a thin metal film and illuminating it with a laser beam through an aqueous medium. The excited fluorophores close to the surface couple (via near-field interactions) to surface plasmons in the metal. Propagated surface plasmons decouple on opposite side of the metal film as a far-field radiation and emit in directional manner. Fluorescence is collected with a high Numerical Aperture objective. A confocal aperture inserted in its conjugate image plane reduces lateral dimensions of the detection volume to a diffraction limit. The thickness of the detection layer is reduced further by metal quenching of excited fluorophores at a close proximity (about 30 nm) to the surface. We used a suspension of fluorescent microspheres to show that FCS-SPAM is an efficient method to measure molecular diffusion.

© 2008 Optical Society of America

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  1. R. Rigler and E. L. Elson, Fluorescence Correlation Spectroscopy: Theory and Applications, (Berlin: Springer, 2001).
    [CrossRef]
  2. M. Auer, K., J. Moore, F. J. Meyer-Almes, R. Guenther, A. J. Pope, and K. A. Stoeckli, "Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS," Drug Discov. Today 3, 457-465 (1998).
    [CrossRef]
  3. N. L. Thompson, T. P. Burghardt, and D. Axelrod, "Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy," Biophys J. 33, 435-454 (1981).
    [CrossRef] [PubMed]
  4. A. M. Lieto, R. C. Cush, and N. L. Thompson, "Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy," Biophys J. 85, 3294-302 (2003).
    [CrossRef] [PubMed]
  5. R. L. Hansen and J. M. Harris, "Measuring reversible adsorption kinetics of small molecules at solid/liquid interfaces by total internal reflection fluorescence correlation spectroscopy," Anal. Chem. 70, 4247-4256 (1998).
    [CrossRef]
  6. T. Ruckstuhl and S. Seeger, "Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy," Opt. Lett. 29, 569-571 (2004).
    [CrossRef] [PubMed]
  7. K. Hassler, T. Anhut, R. Rigler, M. Gosch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys J. 88, L01-3 (2005).
    [CrossRef]
  8. P. Schwille, "TIR-FCS: staying on the surface can sometimes be better," Biophys J. 85, 2783-2784 (2003).
    [CrossRef] [PubMed]
  9. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, (Springer, 2006).
    [CrossRef]
  10. J. Borejdo, N. Calander, Z. Gryczynski, and I. Gryczynski, "Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope," Opt. Express 14, 7878-7888 (2006).
    [CrossRef] [PubMed]
  11. I. Gryczynski, J. Malicka, Z. Gryczynski, and J. R. Lakowicz, "Surface Plasmon-Coupled Emission with Gold Films," J. Phys. Chem. 108, 12568-12574 (2004).
    [CrossRef]
  12. I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowcz, N. Calander, and Z. Gryczynski, "Two-photon induced surface plasmon-coupled emission," Thin Solid Films 491, 173-176 (2005).
    [CrossRef]
  13. Z. Gryczynski, J. Borejdo, E. Matveeva, N. Calander, R. Grygorczyk, J. Harper, and I. Gryczynski, "Minimization of Detection Volume by Surface Plasmon-Coupled Emission," Proc. SPIE S1-S10 (2006).
  14. Z. Gryczynski, J. Borejdo, N. Calander, E. G. Matveeva, and I. Gryczynski, "Minimization of Detection Volume by Surface Plasmon-Coupled Emission," Anal. Biochem 356, 125-131 (2006).
    [CrossRef] [PubMed]
  15. J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, Application of Surface Plasmon Coupled Emission to Study of Muscle, Biophys. J. 91, 2626-2635 (2006).
    [CrossRef] [PubMed]
  16. M. J. Natan, and A. L. Lyon, "Surface Plasmon Resonance Biosensing with Colloidal Au Amplification," Metal Nanoparticles, D. l. Feldheim and C. A. Foss, eds., 183-205 (2002).
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    [CrossRef] [PubMed]
  18. V. Kiessling, B. Muller, and P. Fromherz, "Extracellular resistance in cells adhesion measured with a transistor probe," Langmuir 16, 3517-3521 (2000).
    [CrossRef]
  19. TFC-Calc, Optical Coating Design Software, Software Spectra, Inc.: Portland, OR 97229.
  20. C. Tanford, Physical Chemistry of Macromolecules, (John Wiley & Sons, 1963).
  21. D. Braun and P. Fromherz, "Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye," Biophys J. 87, 1351-1359 (2004).
    [CrossRef] [PubMed]
  22. Y. Iwanaga, D. Braun, and P. Fromherz, "No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of Dil," Eur Biophys J. 30, 17-26 (2001).
    [CrossRef] [PubMed]

2006

Z. Gryczynski, J. Borejdo, N. Calander, E. G. Matveeva, and I. Gryczynski, "Minimization of Detection Volume by Surface Plasmon-Coupled Emission," Anal. Biochem 356, 125-131 (2006).
[CrossRef] [PubMed]

J. Borejdo, N. Calander, Z. Gryczynski, and I. Gryczynski, "Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope," Opt. Express 14, 7878-7888 (2006).
[CrossRef] [PubMed]

2005

J. Enderlein and T. Ruckstuhl, "The efficiency of surface-plasmon coupled emission for sensitive fluorescence detection," Opt. Express 13, 8855-8865 (2005).
[CrossRef] [PubMed]

I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowcz, N. Calander, and Z. Gryczynski, "Two-photon induced surface plasmon-coupled emission," Thin Solid Films 491, 173-176 (2005).
[CrossRef]

2004

I. Gryczynski, J. Malicka, Z. Gryczynski, and J. R. Lakowicz, "Surface Plasmon-Coupled Emission with Gold Films," J. Phys. Chem. 108, 12568-12574 (2004).
[CrossRef]

T. Ruckstuhl and S. Seeger, "Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy," Opt. Lett. 29, 569-571 (2004).
[CrossRef] [PubMed]

D. Braun and P. Fromherz, "Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye," Biophys J. 87, 1351-1359 (2004).
[CrossRef] [PubMed]

2003

P. Schwille, "TIR-FCS: staying on the surface can sometimes be better," Biophys J. 85, 2783-2784 (2003).
[CrossRef] [PubMed]

A. M. Lieto, R. C. Cush, and N. L. Thompson, "Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy," Biophys J. 85, 3294-302 (2003).
[CrossRef] [PubMed]

2001

Y. Iwanaga, D. Braun, and P. Fromherz, "No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of Dil," Eur Biophys J. 30, 17-26 (2001).
[CrossRef] [PubMed]

2000

V. Kiessling, B. Muller, and P. Fromherz, "Extracellular resistance in cells adhesion measured with a transistor probe," Langmuir 16, 3517-3521 (2000).
[CrossRef]

1998

R. L. Hansen and J. M. Harris, "Measuring reversible adsorption kinetics of small molecules at solid/liquid interfaces by total internal reflection fluorescence correlation spectroscopy," Anal. Chem. 70, 4247-4256 (1998).
[CrossRef]

M. Auer, K., J. Moore, F. J. Meyer-Almes, R. Guenther, A. J. Pope, and K. A. Stoeckli, "Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS," Drug Discov. Today 3, 457-465 (1998).
[CrossRef]

1981

N. L. Thompson, T. P. Burghardt, and D. Axelrod, "Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy," Biophys J. 33, 435-454 (1981).
[CrossRef] [PubMed]

Anhut, T.

K. Hassler, T. Anhut, R. Rigler, M. Gosch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys J. 88, L01-3 (2005).
[CrossRef]

Auer, M.

M. Auer, K., J. Moore, F. J. Meyer-Almes, R. Guenther, A. J. Pope, and K. A. Stoeckli, "Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS," Drug Discov. Today 3, 457-465 (1998).
[CrossRef]

Axelrod, D.

N. L. Thompson, T. P. Burghardt, and D. Axelrod, "Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy," Biophys J. 33, 435-454 (1981).
[CrossRef] [PubMed]

Borejdo, J.

Z. Gryczynski, J. Borejdo, N. Calander, E. G. Matveeva, and I. Gryczynski, "Minimization of Detection Volume by Surface Plasmon-Coupled Emission," Anal. Biochem 356, 125-131 (2006).
[CrossRef] [PubMed]

J. Borejdo, N. Calander, Z. Gryczynski, and I. Gryczynski, "Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope," Opt. Express 14, 7878-7888 (2006).
[CrossRef] [PubMed]

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, Application of Surface Plasmon Coupled Emission to Study of Muscle, Biophys. J. 91, 2626-2635 (2006).
[CrossRef] [PubMed]

Braun, D.

D. Braun and P. Fromherz, "Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye," Biophys J. 87, 1351-1359 (2004).
[CrossRef] [PubMed]

Y. Iwanaga, D. Braun, and P. Fromherz, "No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of Dil," Eur Biophys J. 30, 17-26 (2001).
[CrossRef] [PubMed]

Burghardt, T. P.

N. L. Thompson, T. P. Burghardt, and D. Axelrod, "Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy," Biophys J. 33, 435-454 (1981).
[CrossRef] [PubMed]

Calander, N.

Z. Gryczynski, J. Borejdo, N. Calander, E. G. Matveeva, and I. Gryczynski, "Minimization of Detection Volume by Surface Plasmon-Coupled Emission," Anal. Biochem 356, 125-131 (2006).
[CrossRef] [PubMed]

J. Borejdo, N. Calander, Z. Gryczynski, and I. Gryczynski, "Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope," Opt. Express 14, 7878-7888 (2006).
[CrossRef] [PubMed]

I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowcz, N. Calander, and Z. Gryczynski, "Two-photon induced surface plasmon-coupled emission," Thin Solid Films 491, 173-176 (2005).
[CrossRef]

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, Application of Surface Plasmon Coupled Emission to Study of Muscle, Biophys. J. 91, 2626-2635 (2006).
[CrossRef] [PubMed]

Cush, R. C.

A. M. Lieto, R. C. Cush, and N. L. Thompson, "Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy," Biophys J. 85, 3294-302 (2003).
[CrossRef] [PubMed]

Enderlein, J.

Fromherz, P.

D. Braun and P. Fromherz, "Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye," Biophys J. 87, 1351-1359 (2004).
[CrossRef] [PubMed]

Y. Iwanaga, D. Braun, and P. Fromherz, "No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of Dil," Eur Biophys J. 30, 17-26 (2001).
[CrossRef] [PubMed]

V. Kiessling, B. Muller, and P. Fromherz, "Extracellular resistance in cells adhesion measured with a transistor probe," Langmuir 16, 3517-3521 (2000).
[CrossRef]

Goldys, E. M.

I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowcz, N. Calander, and Z. Gryczynski, "Two-photon induced surface plasmon-coupled emission," Thin Solid Films 491, 173-176 (2005).
[CrossRef]

Gosch, M.

K. Hassler, T. Anhut, R. Rigler, M. Gosch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys J. 88, L01-3 (2005).
[CrossRef]

Gryczynski, I.

Z. Gryczynski, J. Borejdo, N. Calander, E. G. Matveeva, and I. Gryczynski, "Minimization of Detection Volume by Surface Plasmon-Coupled Emission," Anal. Biochem 356, 125-131 (2006).
[CrossRef] [PubMed]

J. Borejdo, N. Calander, Z. Gryczynski, and I. Gryczynski, "Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope," Opt. Express 14, 7878-7888 (2006).
[CrossRef] [PubMed]

I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowcz, N. Calander, and Z. Gryczynski, "Two-photon induced surface plasmon-coupled emission," Thin Solid Films 491, 173-176 (2005).
[CrossRef]

I. Gryczynski, J. Malicka, Z. Gryczynski, and J. R. Lakowicz, "Surface Plasmon-Coupled Emission with Gold Films," J. Phys. Chem. 108, 12568-12574 (2004).
[CrossRef]

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, Application of Surface Plasmon Coupled Emission to Study of Muscle, Biophys. J. 91, 2626-2635 (2006).
[CrossRef] [PubMed]

Gryczynski, Z.

Z. Gryczynski, J. Borejdo, N. Calander, E. G. Matveeva, and I. Gryczynski, "Minimization of Detection Volume by Surface Plasmon-Coupled Emission," Anal. Biochem 356, 125-131 (2006).
[CrossRef] [PubMed]

J. Borejdo, N. Calander, Z. Gryczynski, and I. Gryczynski, "Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope," Opt. Express 14, 7878-7888 (2006).
[CrossRef] [PubMed]

I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowcz, N. Calander, and Z. Gryczynski, "Two-photon induced surface plasmon-coupled emission," Thin Solid Films 491, 173-176 (2005).
[CrossRef]

I. Gryczynski, J. Malicka, Z. Gryczynski, and J. R. Lakowicz, "Surface Plasmon-Coupled Emission with Gold Films," J. Phys. Chem. 108, 12568-12574 (2004).
[CrossRef]

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, Application of Surface Plasmon Coupled Emission to Study of Muscle, Biophys. J. 91, 2626-2635 (2006).
[CrossRef] [PubMed]

Hansen, R. L.

R. L. Hansen and J. M. Harris, "Measuring reversible adsorption kinetics of small molecules at solid/liquid interfaces by total internal reflection fluorescence correlation spectroscopy," Anal. Chem. 70, 4247-4256 (1998).
[CrossRef]

Harris, J. M.

R. L. Hansen and J. M. Harris, "Measuring reversible adsorption kinetics of small molecules at solid/liquid interfaces by total internal reflection fluorescence correlation spectroscopy," Anal. Chem. 70, 4247-4256 (1998).
[CrossRef]

Hassler, K.

K. Hassler, T. Anhut, R. Rigler, M. Gosch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys J. 88, L01-3 (2005).
[CrossRef]

Iwanaga, Y.

Y. Iwanaga, D. Braun, and P. Fromherz, "No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of Dil," Eur Biophys J. 30, 17-26 (2001).
[CrossRef] [PubMed]

Kiessling, V.

V. Kiessling, B. Muller, and P. Fromherz, "Extracellular resistance in cells adhesion measured with a transistor probe," Langmuir 16, 3517-3521 (2000).
[CrossRef]

Lakowcz, J. R.

I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowcz, N. Calander, and Z. Gryczynski, "Two-photon induced surface plasmon-coupled emission," Thin Solid Films 491, 173-176 (2005).
[CrossRef]

Lakowicz, J. R.

I. Gryczynski, J. Malicka, Z. Gryczynski, and J. R. Lakowicz, "Surface Plasmon-Coupled Emission with Gold Films," J. Phys. Chem. 108, 12568-12574 (2004).
[CrossRef]

Lasser, T.

K. Hassler, T. Anhut, R. Rigler, M. Gosch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys J. 88, L01-3 (2005).
[CrossRef]

Lieto, A. M.

A. M. Lieto, R. C. Cush, and N. L. Thompson, "Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy," Biophys J. 85, 3294-302 (2003).
[CrossRef] [PubMed]

Malicka, J.

I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowcz, N. Calander, and Z. Gryczynski, "Two-photon induced surface plasmon-coupled emission," Thin Solid Films 491, 173-176 (2005).
[CrossRef]

I. Gryczynski, J. Malicka, Z. Gryczynski, and J. R. Lakowicz, "Surface Plasmon-Coupled Emission with Gold Films," J. Phys. Chem. 108, 12568-12574 (2004).
[CrossRef]

Matveeva, E. G.

Z. Gryczynski, J. Borejdo, N. Calander, E. G. Matveeva, and I. Gryczynski, "Minimization of Detection Volume by Surface Plasmon-Coupled Emission," Anal. Biochem 356, 125-131 (2006).
[CrossRef] [PubMed]

Muller, B.

V. Kiessling, B. Muller, and P. Fromherz, "Extracellular resistance in cells adhesion measured with a transistor probe," Langmuir 16, 3517-3521 (2000).
[CrossRef]

Muthu, P.

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, Application of Surface Plasmon Coupled Emission to Study of Muscle, Biophys. J. 91, 2626-2635 (2006).
[CrossRef] [PubMed]

Rigler, R.

K. Hassler, T. Anhut, R. Rigler, M. Gosch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys J. 88, L01-3 (2005).
[CrossRef]

Ruckstuhl, T.

Schwille, P.

P. Schwille, "TIR-FCS: staying on the surface can sometimes be better," Biophys J. 85, 2783-2784 (2003).
[CrossRef] [PubMed]

Seeger, S.

Thompson, N. L.

A. M. Lieto, R. C. Cush, and N. L. Thompson, "Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy," Biophys J. 85, 3294-302 (2003).
[CrossRef] [PubMed]

N. L. Thompson, T. P. Burghardt, and D. Axelrod, "Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy," Biophys J. 33, 435-454 (1981).
[CrossRef] [PubMed]

Anal. Biochem

Z. Gryczynski, J. Borejdo, N. Calander, E. G. Matveeva, and I. Gryczynski, "Minimization of Detection Volume by Surface Plasmon-Coupled Emission," Anal. Biochem 356, 125-131 (2006).
[CrossRef] [PubMed]

Anal. Chem.

R. L. Hansen and J. M. Harris, "Measuring reversible adsorption kinetics of small molecules at solid/liquid interfaces by total internal reflection fluorescence correlation spectroscopy," Anal. Chem. 70, 4247-4256 (1998).
[CrossRef]

Biophys J.

P. Schwille, "TIR-FCS: staying on the surface can sometimes be better," Biophys J. 85, 2783-2784 (2003).
[CrossRef] [PubMed]

N. L. Thompson, T. P. Burghardt, and D. Axelrod, "Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy," Biophys J. 33, 435-454 (1981).
[CrossRef] [PubMed]

A. M. Lieto, R. C. Cush, and N. L. Thompson, "Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy," Biophys J. 85, 3294-302 (2003).
[CrossRef] [PubMed]

D. Braun and P. Fromherz, "Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye," Biophys J. 87, 1351-1359 (2004).
[CrossRef] [PubMed]

Drug Discov. Today

M. Auer, K., J. Moore, F. J. Meyer-Almes, R. Guenther, A. J. Pope, and K. A. Stoeckli, "Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS," Drug Discov. Today 3, 457-465 (1998).
[CrossRef]

Eur Biophys J.

Y. Iwanaga, D. Braun, and P. Fromherz, "No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of Dil," Eur Biophys J. 30, 17-26 (2001).
[CrossRef] [PubMed]

J. Phys. Chem.

I. Gryczynski, J. Malicka, Z. Gryczynski, and J. R. Lakowicz, "Surface Plasmon-Coupled Emission with Gold Films," J. Phys. Chem. 108, 12568-12574 (2004).
[CrossRef]

Langmuir

V. Kiessling, B. Muller, and P. Fromherz, "Extracellular resistance in cells adhesion measured with a transistor probe," Langmuir 16, 3517-3521 (2000).
[CrossRef]

Opt. Express

Opt. Lett.

Thin Solid Films

I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowcz, N. Calander, and Z. Gryczynski, "Two-photon induced surface plasmon-coupled emission," Thin Solid Films 491, 173-176 (2005).
[CrossRef]

Other

Z. Gryczynski, J. Borejdo, E. Matveeva, N. Calander, R. Grygorczyk, J. Harper, and I. Gryczynski, "Minimization of Detection Volume by Surface Plasmon-Coupled Emission," Proc. SPIE S1-S10 (2006).

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, Application of Surface Plasmon Coupled Emission to Study of Muscle, Biophys. J. 91, 2626-2635 (2006).
[CrossRef] [PubMed]

M. J. Natan, and A. L. Lyon, "Surface Plasmon Resonance Biosensing with Colloidal Au Amplification," Metal Nanoparticles, D. l. Feldheim and C. A. Foss, eds., 183-205 (2002).

TFC-Calc, Optical Coating Design Software, Software Spectra, Inc.: Portland, OR 97229.

C. Tanford, Physical Chemistry of Macromolecules, (John Wiley & Sons, 1963).

R. Rigler and E. L. Elson, Fluorescence Correlation Spectroscopy: Theory and Applications, (Berlin: Springer, 2001).
[CrossRef]

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, (Springer, 2006).
[CrossRef]

K. Hassler, T. Anhut, R. Rigler, M. Gosch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys J. 88, L01-3 (2005).
[CrossRef]

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Figures (7)

Fig. 1.
Fig. 1.

RK-SPAM-FCS microscope. The insert at right presents the concept for surface plasmon coupled emission. The excited fluorophores close to the surface couple (via near-field interactions) to surface plasmons in the metal. Propagated surface plasmons decouple on opposite side of the metal film as a far-field radiation and emit in directional manner.

Fig. 2.
Fig. 2.

Background rejection by SPAM. 0.5 mM Rhodamine 800 added as background obscures the image in ordinary TIRF (A). SPAM in RK configuration eliminates much of the background contribution (B). Myofibrils (0.1 mg/mL) were labeled with 100 nM Alexa647-phalloidin +10 µM unlabeled phalloidin for 5 min at room temperature, then extensively washed with buffer containing 50 mM KCl, 2 mM MgCl2, 1 mM DTT, 10 mM TRIS pH 7.0. 633 nm excitation, 1.65 NA×100 Olympus objective, sapphire substrate, 1.78 Refractive Index immersion oil. The bars are 5 µm and 10 µm in A & B, respectively.

Fig. 3.
Fig. 3.

The intensity fluctuations caused by diffusion of 100 nm spheres through detection volume in RK SPAM experiment on gold substrate. Inset: 100 nm microspheres in RK SPAM, 100x, NA=1.65 objective.

Fig. 4.
Fig. 4.

Autocorrelation function of data shown in Fig. 3.

Fig. 5.
Fig. 5.

TIRF autocorrelation function.

Fig. 6.
Fig. 6.

Comparison of RK-SPAM on Au and TIRF. The modeling parameters for RK Au are d0=170 nm, d1=30 nm, and for TIRF d0=135 nm. Red: The RK-SPAM detection signal=intensity at emission into the sapphire prism. Normal incidence (θ=0°). Blue: TIRF excitation. Angle of incidence (θ=49.5°) at maximum intensity of the evanescent wave. Metal is 50 nm layer of gold. The red curve is magnified by a factor of 10.

Fig. 7.
Fig. 7.

Fit of theoretical FCS curves to experimental data. Bi-exponential for SPAM a)) and single-exponential for TIRF (b). Note that the y-axis is linear.

Equations (2)

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R nn ( t ) = ( 1 + Dt σ 2 ) 1 ( ( 1 2 Dt d n 2 ) erfc ( Dt d n 2 ) exp ( Dt d n 2 ) + 4 Dt π d n 2 )
R nm ( t ) = ( 1 + Dt σ 2 ) 1 ( d m d m d n erfc ( Dt d m 2 ) exp ( Dt d m 2 ) + d n d n d m erfc ( Dt d n 2 ) exp ( Dt d n 2 ) )

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