Abstract

We present a rapid and flexible framework for the accurate calculation of the detection efficiency of fluorescence emission in isotropic media as well as in the vicinity of dielectric or metallic interfaces. The framework accounts for the dipole characteristics of the emitted fluorescence and yields the absolute detection efficiency by taking into account the total power radiated by the fluorophore. This analysis proved to be useful for quantitative measurements, i.e. the fluorescence detection at a glass–water interface for total internal reflection fluorescence microscopy in an epi- and a trans-illumination configuration.

© 2008 Optical Society of America

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  1. D. Magde, W.W. Webb, E. Elson, "Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29,705 (1972).
    [CrossRef]
  2. R. Rigler, E. S. Elson, Fluorescence Correlation Spectroscopy: Theory and Applications, Springer Ser. Chem. Phys. 65, ISBN 3-540-67433-0 (2001).
  3. P. Kask, K. Palo, D. Ullmann, K. Gall, "Fluorescence-intensity distribution analysis and its application in biomolecular detection technology," PNAS 96, 13756-13761 (1999).
    [CrossRef] [PubMed]
  4. Y. Chen, J. D. Muller, P. T. C. So, and E. Gratton, "The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy," Biophys. J. 77, 553-567 (1999).
    [CrossRef] [PubMed]
  5. C. Eggeling, P. Kask, D. Winkler, S. Jager, "Rapid Analysis of Forster Resonance Energy Transfer by Two-Color Global Fluorescence Correlation Spectroscopy: Trypsin Proteinase Reaction," Biophys. J. 89, 605-618 (2005).
    [CrossRef] [PubMed]
  6. J. Enderlein, I. Gregor, D. Patra, T. Dertinger, U. B. Kaupp, "Performance of Fluorescence Correlation Spectroscopy for Measuring Diffusion and Concentration," ChemPhysChem 6, 2324-2336 (2005).
    [CrossRef] [PubMed]
  7. P. Torok, P. D. Higdon, and T. Wilson, "Theory for confocal and conventional microscopes imaging small dielectric scatterers," J. Mod. Opt. 45, 1681-1698 (1998).
    [CrossRef]
  8. P. D. Higdon, P. Torok, and T. Wilson, "Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes," J. Microsc. 193, 127-141 (1999).
    [CrossRef]
  9. P. Torok, "Propagation of electromagnetic dipole waves through dielectric interfaces," Opt. Lett. 25, 1463-1465 (2000).
    [CrossRef]
  10. J. Enderlein, M. Bohmer, "Influence of interfacedipole interactions on the efficiency of fluorescence light collection near surfaces," Opt. Lett. 28, 941-943 (2003).
    [CrossRef] [PubMed]
  11. P. Debye, "Das Verhalten von Lichtwellen in der N¨ahe eines Brennpunktes oder einer Brennlinie," Ann. Phys. 30, 755-776 (1909).
    [CrossRef]
  12. E. Wolf, "Electromagnetic diffraction in optical systems, I. An integral representation of the image field," Proc. R. Soc. London Ser. A 253, 349-357 (1959).
    [CrossRef]
  13. B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems, II. Structure of the image field in an aplanatic system," Proc. R. Soc. London Ser. A 253, 358-379 (1959).
    [CrossRef]
  14. M. Leutenegger, R. Rao, R. A. Leitgeb, and T. Lasser, "Fast focus field calculations," Opt. Express 14, 11277-11291 (2006).
    [CrossRef] [PubMed]
  15. The subscript refers to the wavelength used for calculating the corresponding quantity.
  16. B. Valeur, Molecular fluorescence: principles and applications, (Wiley-VCH, 2002) ISBN 3-527-29919-X .
  17. J. Widengren and P. Schwille, "Characterization of Photoinduced Isomerization and Back-Isomerization of the Cyanine Dye Cy5 by Fluorescence Correlation Spectroscopy," J. Phys. Chem. A 104, 6416-6428 (2000).
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  18. C. Eggeling, A. Volkmer, C. A. M. Seidel, "Molecular Photobleaching Kinetics of Rhodamine 6G by One- and Two-Photon Induced Confocal Fluorescence Microscopy," Chem. Phys. Chem. 6, 791-804 (2005).
    [CrossRef] [PubMed]
  19. W. Lukosz and R. E. Kunz, "Light-emission by magnetic and electric dipoles close to a plane interface: 1. Total radiated power," J. Opt. Soc. Am. 67, 1607-1615 (1977).
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  20. W. Lukosz, "Light-emission by magnetic and electric dipoles close to a plane dielectric interface: 3. Radiationpatterns of dipoles with arbitrary orientation," J. Opt. Soc. Am. 69, 1495-1503 (1979).
    [CrossRef]
  21. T. P. Burghardt and N. L. Thompson, "Effect of planar dielectric interfaces on fluorescence emission and detection. Evanescent excitation with high-aperture collection," Biophys. J. 46, 729-737 (1984).
    [CrossRef] [PubMed]
  22. E. H. Hellen and D. Axelrod, "Fluorescence emission at dielectric and metal-film interfaces," J. Opt. Soc. Am. B 4, 337-350 (1987).
    [CrossRef]
  23. L. Novotny, "Allowed and forbidden light in near-field optics. II. Interacting dipolar particles," J. Opt. Soc. Am. A 14, 105-113 (1997).
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  24. J. Mertz, "Radiative absorption, fluorescence, and scattering of a classical dipole near a lossless interface: a unified description," J. Opt. Soc. Am. B 17, 1906-1913 (2000).
    [CrossRef]
  25. G. W. Ford and W. H. Weber, "Electromagnetic interactions of molecules with metal surfaces," Phys. Rep. 113, 195-287 (1984).
    [CrossRef]
  26. J. Enderlein and T. Ruckstuhl, "The efficiency of surface-plasmon coupled emission for sensitive fluorescence detection," Opt. Express 13, 8855-8865 (2005).
    [CrossRef] [PubMed]
  27. F. D. Stefani, K. Vasilev, N. Bocchio, N. Stoyanova, and M. Kreiter, "Surface-plasmon-mediated single-molecule fluorescence through a thin metallic film," Phys. Rev. Lett. 94, 023005 (2005).
    [CrossRef] [PubMed]
  28. E. H. Hellen and D. Axelrod, "Fluorescence emission at dielectric and metal-film interfaces," J. Opt. Soc. Am. B 4, 337-350 (1987).
    [CrossRef]
  29. J. R. Lakowicz, J. Malicka, I. Gryczynski, Z. Gryczynski, C. D. Geddes, "Radiative decay engineering: the role of photonic mode density in biotechnology," J. Phys. D: Appl. Phys. 36, R240-R249 (2003).
    [CrossRef]
  30. For single photon detectors SPCM-AQR-xx by PerkinElmer, qd ? 55% at ?fl = 525nm, respectively qd? 45% for the PDM 50CT by Micro Photon Devices.
  31. Typical values: ? 85% transmission through the dichroic mirror and ? 90% transmission through the emission bandpass filter with a bandwidth covering ? 60% of the fluorescence spectrum, that is T fl? 45% in total.
  32. R. J. Potton, "Reciprocity in optics," Rep. Prog. Phys. 67, 717-754 (2004).
    [CrossRef]
  33. J.-L. Kaiser, E. Quertemont, and R. Chevallier, "Light propagation in the pseudo-paraxial Fresnel approximation," Opt. Commun. 233, 261-269 (2004).
    [CrossRef]
  34. A. E. Siegman, Lasers, (Oxford Univ. Press,1986) ISBN 0-19-855713-2.
  35. D. Mas, J. Garcia, C. Ferreira, L. M. Bernardo, and F. Marinho, "Fast algorithms for free-space diffraction patterns calculation," Opt. Commun. 164, 233-245 (1999).
    [CrossRef]
  36. The Royal microscope standard limits the clear objective aperture to less than 16mm. The shortest commercial tube length is 164mm (Carl Zeiss), which yields an image NA < 8mm/164mm = 0.049.
  37. According to previous notes qdT fl? 23% typically.
  38. 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, 3294-3302 (2003).
    [CrossRef] [PubMed]
  39. M. Leutenegger, H. Blom, J. Widengren, C. Eggeling, M. Gosch, R. A. Leitgeb, T. Lasser, "Dual-color total internal reflection fluorescence cross-correlation spectroscopy," J. Biomed. Opt. 11, 040502 (2006).
    [CrossRef] [PubMed]
  40. K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. Gosch, T. Lasser, "Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule," Opt. Express 13, 7415-7423 (2005).
    [CrossRef] [PubMed]

2006 (2)

M. Leutenegger, H. Blom, J. Widengren, C. Eggeling, M. Gosch, R. A. Leitgeb, T. Lasser, "Dual-color total internal reflection fluorescence cross-correlation spectroscopy," J. Biomed. Opt. 11, 040502 (2006).
[CrossRef] [PubMed]

M. Leutenegger, R. Rao, R. A. Leitgeb, and T. Lasser, "Fast focus field calculations," Opt. Express 14, 11277-11291 (2006).
[CrossRef] [PubMed]

2005 (6)

K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. Gosch, T. Lasser, "Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule," Opt. Express 13, 7415-7423 (2005).
[CrossRef] [PubMed]

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

C. Eggeling, P. Kask, D. Winkler, S. Jager, "Rapid Analysis of Forster Resonance Energy Transfer by Two-Color Global Fluorescence Correlation Spectroscopy: Trypsin Proteinase Reaction," Biophys. J. 89, 605-618 (2005).
[CrossRef] [PubMed]

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, U. B. Kaupp, "Performance of Fluorescence Correlation Spectroscopy for Measuring Diffusion and Concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

C. Eggeling, A. Volkmer, C. A. M. Seidel, "Molecular Photobleaching Kinetics of Rhodamine 6G by One- and Two-Photon Induced Confocal Fluorescence Microscopy," Chem. Phys. Chem. 6, 791-804 (2005).
[CrossRef] [PubMed]

F. D. Stefani, K. Vasilev, N. Bocchio, N. Stoyanova, and M. Kreiter, "Surface-plasmon-mediated single-molecule fluorescence through a thin metallic film," Phys. Rev. Lett. 94, 023005 (2005).
[CrossRef] [PubMed]

2004 (2)

R. J. Potton, "Reciprocity in optics," Rep. Prog. Phys. 67, 717-754 (2004).
[CrossRef]

J.-L. Kaiser, E. Quertemont, and R. Chevallier, "Light propagation in the pseudo-paraxial Fresnel approximation," Opt. Commun. 233, 261-269 (2004).
[CrossRef]

2003 (3)

J. R. Lakowicz, J. Malicka, I. Gryczynski, Z. Gryczynski, C. D. Geddes, "Radiative decay engineering: the role of photonic mode density in biotechnology," J. Phys. D: Appl. Phys. 36, R240-R249 (2003).
[CrossRef]

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, 3294-3302 (2003).
[CrossRef] [PubMed]

J. Enderlein, M. Bohmer, "Influence of interfacedipole interactions on the efficiency of fluorescence light collection near surfaces," Opt. Lett. 28, 941-943 (2003).
[CrossRef] [PubMed]

2000 (3)

1999 (4)

D. Mas, J. Garcia, C. Ferreira, L. M. Bernardo, and F. Marinho, "Fast algorithms for free-space diffraction patterns calculation," Opt. Commun. 164, 233-245 (1999).
[CrossRef]

P. Kask, K. Palo, D. Ullmann, K. Gall, "Fluorescence-intensity distribution analysis and its application in biomolecular detection technology," PNAS 96, 13756-13761 (1999).
[CrossRef] [PubMed]

Y. Chen, J. D. Muller, P. T. C. So, and E. Gratton, "The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy," Biophys. J. 77, 553-567 (1999).
[CrossRef] [PubMed]

P. D. Higdon, P. Torok, and T. Wilson, "Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes," J. Microsc. 193, 127-141 (1999).
[CrossRef]

1998 (1)

P. Torok, P. D. Higdon, and T. Wilson, "Theory for confocal and conventional microscopes imaging small dielectric scatterers," J. Mod. Opt. 45, 1681-1698 (1998).
[CrossRef]

1997 (1)

1987 (2)

1984 (2)

T. P. Burghardt and N. L. Thompson, "Effect of planar dielectric interfaces on fluorescence emission and detection. Evanescent excitation with high-aperture collection," Biophys. J. 46, 729-737 (1984).
[CrossRef] [PubMed]

G. W. Ford and W. H. Weber, "Electromagnetic interactions of molecules with metal surfaces," Phys. Rep. 113, 195-287 (1984).
[CrossRef]

1979 (1)

1977 (1)

1972 (1)

D. Magde, W.W. Webb, E. Elson, "Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29,705 (1972).
[CrossRef]

1959 (2)

E. Wolf, "Electromagnetic diffraction in optical systems, I. An integral representation of the image field," Proc. R. Soc. London Ser. A 253, 349-357 (1959).
[CrossRef]

B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems, II. Structure of the image field in an aplanatic system," Proc. R. Soc. London Ser. A 253, 358-379 (1959).
[CrossRef]

1909 (1)

P. Debye, "Das Verhalten von Lichtwellen in der N¨ahe eines Brennpunktes oder einer Brennlinie," Ann. Phys. 30, 755-776 (1909).
[CrossRef]

Axelrod, D.

Bernardo, L. M.

D. Mas, J. Garcia, C. Ferreira, L. M. Bernardo, and F. Marinho, "Fast algorithms for free-space diffraction patterns calculation," Opt. Commun. 164, 233-245 (1999).
[CrossRef]

Blom, H.

M. Leutenegger, H. Blom, J. Widengren, C. Eggeling, M. Gosch, R. A. Leitgeb, T. Lasser, "Dual-color total internal reflection fluorescence cross-correlation spectroscopy," J. Biomed. Opt. 11, 040502 (2006).
[CrossRef] [PubMed]

Bocchio, N.

F. D. Stefani, K. Vasilev, N. Bocchio, N. Stoyanova, and M. Kreiter, "Surface-plasmon-mediated single-molecule fluorescence through a thin metallic film," Phys. Rev. Lett. 94, 023005 (2005).
[CrossRef] [PubMed]

Bohmer, M.

Burghardt, T. P.

T. P. Burghardt and N. L. Thompson, "Effect of planar dielectric interfaces on fluorescence emission and detection. Evanescent excitation with high-aperture collection," Biophys. J. 46, 729-737 (1984).
[CrossRef] [PubMed]

Chen, Y.

Y. Chen, J. D. Muller, P. T. C. So, and E. Gratton, "The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy," Biophys. J. 77, 553-567 (1999).
[CrossRef] [PubMed]

Chevallier, R.

J.-L. Kaiser, E. Quertemont, and R. Chevallier, "Light propagation in the pseudo-paraxial Fresnel approximation," Opt. Commun. 233, 261-269 (2004).
[CrossRef]

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, 3294-3302 (2003).
[CrossRef] [PubMed]

Debye, P.

P. Debye, "Das Verhalten von Lichtwellen in der N¨ahe eines Brennpunktes oder einer Brennlinie," Ann. Phys. 30, 755-776 (1909).
[CrossRef]

Dertinger, T.

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, U. B. Kaupp, "Performance of Fluorescence Correlation Spectroscopy for Measuring Diffusion and Concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

Eggeling, C.

M. Leutenegger, H. Blom, J. Widengren, C. Eggeling, M. Gosch, R. A. Leitgeb, T. Lasser, "Dual-color total internal reflection fluorescence cross-correlation spectroscopy," J. Biomed. Opt. 11, 040502 (2006).
[CrossRef] [PubMed]

C. Eggeling, A. Volkmer, C. A. M. Seidel, "Molecular Photobleaching Kinetics of Rhodamine 6G by One- and Two-Photon Induced Confocal Fluorescence Microscopy," Chem. Phys. Chem. 6, 791-804 (2005).
[CrossRef] [PubMed]

C. Eggeling, P. Kask, D. Winkler, S. Jager, "Rapid Analysis of Forster Resonance Energy Transfer by Two-Color Global Fluorescence Correlation Spectroscopy: Trypsin Proteinase Reaction," Biophys. J. 89, 605-618 (2005).
[CrossRef] [PubMed]

Elson, E.

D. Magde, W.W. Webb, E. Elson, "Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29,705 (1972).
[CrossRef]

Enderlein, J.

Ferreira, C.

D. Mas, J. Garcia, C. Ferreira, L. M. Bernardo, and F. Marinho, "Fast algorithms for free-space diffraction patterns calculation," Opt. Commun. 164, 233-245 (1999).
[CrossRef]

Ford, G. W.

G. W. Ford and W. H. Weber, "Electromagnetic interactions of molecules with metal surfaces," Phys. Rep. 113, 195-287 (1984).
[CrossRef]

Gall, K.

P. Kask, K. Palo, D. Ullmann, K. Gall, "Fluorescence-intensity distribution analysis and its application in biomolecular detection technology," PNAS 96, 13756-13761 (1999).
[CrossRef] [PubMed]

Garcia, J.

D. Mas, J. Garcia, C. Ferreira, L. M. Bernardo, and F. Marinho, "Fast algorithms for free-space diffraction patterns calculation," Opt. Commun. 164, 233-245 (1999).
[CrossRef]

Geddes, C. D.

J. R. Lakowicz, J. Malicka, I. Gryczynski, Z. Gryczynski, C. D. Geddes, "Radiative decay engineering: the role of photonic mode density in biotechnology," J. Phys. D: Appl. Phys. 36, R240-R249 (2003).
[CrossRef]

Gosch, M.

M. Leutenegger, H. Blom, J. Widengren, C. Eggeling, M. Gosch, R. A. Leitgeb, T. Lasser, "Dual-color total internal reflection fluorescence cross-correlation spectroscopy," J. Biomed. Opt. 11, 040502 (2006).
[CrossRef] [PubMed]

K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. Gosch, T. Lasser, "Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule," Opt. Express 13, 7415-7423 (2005).
[CrossRef] [PubMed]

Gratton, E.

Y. Chen, J. D. Muller, P. T. C. So, and E. Gratton, "The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy," Biophys. J. 77, 553-567 (1999).
[CrossRef] [PubMed]

Gregor, I.

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, U. B. Kaupp, "Performance of Fluorescence Correlation Spectroscopy for Measuring Diffusion and Concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

Gryczynski, I.

J. R. Lakowicz, J. Malicka, I. Gryczynski, Z. Gryczynski, C. D. Geddes, "Radiative decay engineering: the role of photonic mode density in biotechnology," J. Phys. D: Appl. Phys. 36, R240-R249 (2003).
[CrossRef]

Gryczynski, Z.

J. R. Lakowicz, J. Malicka, I. Gryczynski, Z. Gryczynski, C. D. Geddes, "Radiative decay engineering: the role of photonic mode density in biotechnology," J. Phys. D: Appl. Phys. 36, R240-R249 (2003).
[CrossRef]

Hassler, K.

Hellen, E. H.

Higdon, P. D.

P. D. Higdon, P. Torok, and T. Wilson, "Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes," J. Microsc. 193, 127-141 (1999).
[CrossRef]

P. Torok, P. D. Higdon, and T. Wilson, "Theory for confocal and conventional microscopes imaging small dielectric scatterers," J. Mod. Opt. 45, 1681-1698 (1998).
[CrossRef]

Jager, S.

C. Eggeling, P. Kask, D. Winkler, S. Jager, "Rapid Analysis of Forster Resonance Energy Transfer by Two-Color Global Fluorescence Correlation Spectroscopy: Trypsin Proteinase Reaction," Biophys. J. 89, 605-618 (2005).
[CrossRef] [PubMed]

Kaiser, J.-L.

J.-L. Kaiser, E. Quertemont, and R. Chevallier, "Light propagation in the pseudo-paraxial Fresnel approximation," Opt. Commun. 233, 261-269 (2004).
[CrossRef]

Kask, P.

C. Eggeling, P. Kask, D. Winkler, S. Jager, "Rapid Analysis of Forster Resonance Energy Transfer by Two-Color Global Fluorescence Correlation Spectroscopy: Trypsin Proteinase Reaction," Biophys. J. 89, 605-618 (2005).
[CrossRef] [PubMed]

P. Kask, K. Palo, D. Ullmann, K. Gall, "Fluorescence-intensity distribution analysis and its application in biomolecular detection technology," PNAS 96, 13756-13761 (1999).
[CrossRef] [PubMed]

Kaupp, U. B.

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, U. B. Kaupp, "Performance of Fluorescence Correlation Spectroscopy for Measuring Diffusion and Concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

Kreiter, M.

F. D. Stefani, K. Vasilev, N. Bocchio, N. Stoyanova, and M. Kreiter, "Surface-plasmon-mediated single-molecule fluorescence through a thin metallic film," Phys. Rev. Lett. 94, 023005 (2005).
[CrossRef] [PubMed]

Kunz, R. E.

Lakowicz, J. R.

J. R. Lakowicz, J. Malicka, I. Gryczynski, Z. Gryczynski, C. D. Geddes, "Radiative decay engineering: the role of photonic mode density in biotechnology," J. Phys. D: Appl. Phys. 36, R240-R249 (2003).
[CrossRef]

Lasser, T.

Leitgeb, R. A.

M. Leutenegger, H. Blom, J. Widengren, C. Eggeling, M. Gosch, R. A. Leitgeb, T. Lasser, "Dual-color total internal reflection fluorescence cross-correlation spectroscopy," J. Biomed. Opt. 11, 040502 (2006).
[CrossRef] [PubMed]

M. Leutenegger, R. Rao, R. A. Leitgeb, and T. Lasser, "Fast focus field calculations," Opt. Express 14, 11277-11291 (2006).
[CrossRef] [PubMed]

Leutenegger, M.

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, 3294-3302 (2003).
[CrossRef] [PubMed]

Lukosz, W.

Magde, D.

D. Magde, W.W. Webb, E. Elson, "Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29,705 (1972).
[CrossRef]

Malicka, J.

J. R. Lakowicz, J. Malicka, I. Gryczynski, Z. Gryczynski, C. D. Geddes, "Radiative decay engineering: the role of photonic mode density in biotechnology," J. Phys. D: Appl. Phys. 36, R240-R249 (2003).
[CrossRef]

Marinho, F.

D. Mas, J. Garcia, C. Ferreira, L. M. Bernardo, and F. Marinho, "Fast algorithms for free-space diffraction patterns calculation," Opt. Commun. 164, 233-245 (1999).
[CrossRef]

Mas, D.

D. Mas, J. Garcia, C. Ferreira, L. M. Bernardo, and F. Marinho, "Fast algorithms for free-space diffraction patterns calculation," Opt. Commun. 164, 233-245 (1999).
[CrossRef]

Mertz, J.

Muller, J. D.

Y. Chen, J. D. Muller, P. T. C. So, and E. Gratton, "The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy," Biophys. J. 77, 553-567 (1999).
[CrossRef] [PubMed]

Novotny, L.

Palo, K.

P. Kask, K. Palo, D. Ullmann, K. Gall, "Fluorescence-intensity distribution analysis and its application in biomolecular detection technology," PNAS 96, 13756-13761 (1999).
[CrossRef] [PubMed]

Patra, D.

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, U. B. Kaupp, "Performance of Fluorescence Correlation Spectroscopy for Measuring Diffusion and Concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

Potton, R. J.

R. J. Potton, "Reciprocity in optics," Rep. Prog. Phys. 67, 717-754 (2004).
[CrossRef]

Quertemont, E.

J.-L. Kaiser, E. Quertemont, and R. Chevallier, "Light propagation in the pseudo-paraxial Fresnel approximation," Opt. Commun. 233, 261-269 (2004).
[CrossRef]

Rao, R.

Richards, B.

B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems, II. Structure of the image field in an aplanatic system," Proc. R. Soc. London Ser. A 253, 358-379 (1959).
[CrossRef]

Rigler, P.

Rigler, R.

Ruckstuhl, T.

Schwille, P.

J. Widengren and P. Schwille, "Characterization of Photoinduced Isomerization and Back-Isomerization of the Cyanine Dye Cy5 by Fluorescence Correlation Spectroscopy," J. Phys. Chem. A 104, 6416-6428 (2000).
[CrossRef]

Seidel, C. A. M.

C. Eggeling, A. Volkmer, C. A. M. Seidel, "Molecular Photobleaching Kinetics of Rhodamine 6G by One- and Two-Photon Induced Confocal Fluorescence Microscopy," Chem. Phys. Chem. 6, 791-804 (2005).
[CrossRef] [PubMed]

So, P. T. C.

Y. Chen, J. D. Muller, P. T. C. So, and E. Gratton, "The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy," Biophys. J. 77, 553-567 (1999).
[CrossRef] [PubMed]

Stefani, F. D.

F. D. Stefani, K. Vasilev, N. Bocchio, N. Stoyanova, and M. Kreiter, "Surface-plasmon-mediated single-molecule fluorescence through a thin metallic film," Phys. Rev. Lett. 94, 023005 (2005).
[CrossRef] [PubMed]

Stoyanova, N.

F. D. Stefani, K. Vasilev, N. Bocchio, N. Stoyanova, and M. Kreiter, "Surface-plasmon-mediated single-molecule fluorescence through a thin metallic film," Phys. Rev. Lett. 94, 023005 (2005).
[CrossRef] [PubMed]

T¨or¨ok, P.

P. D. Higdon, P. Torok, and T. Wilson, "Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes," J. Microsc. 193, 127-141 (1999).
[CrossRef]

P. Torok, P. D. Higdon, and T. Wilson, "Theory for confocal and conventional microscopes imaging small dielectric scatterers," J. Mod. Opt. 45, 1681-1698 (1998).
[CrossRef]

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, 3294-3302 (2003).
[CrossRef] [PubMed]

T. P. Burghardt and N. L. Thompson, "Effect of planar dielectric interfaces on fluorescence emission and detection. Evanescent excitation with high-aperture collection," Biophys. J. 46, 729-737 (1984).
[CrossRef] [PubMed]

Torok, P.

Ullmann, D.

P. Kask, K. Palo, D. Ullmann, K. Gall, "Fluorescence-intensity distribution analysis and its application in biomolecular detection technology," PNAS 96, 13756-13761 (1999).
[CrossRef] [PubMed]

Vasilev, K.

F. D. Stefani, K. Vasilev, N. Bocchio, N. Stoyanova, and M. Kreiter, "Surface-plasmon-mediated single-molecule fluorescence through a thin metallic film," Phys. Rev. Lett. 94, 023005 (2005).
[CrossRef] [PubMed]

Volkmer, A.

C. Eggeling, A. Volkmer, C. A. M. Seidel, "Molecular Photobleaching Kinetics of Rhodamine 6G by One- and Two-Photon Induced Confocal Fluorescence Microscopy," Chem. Phys. Chem. 6, 791-804 (2005).
[CrossRef] [PubMed]

Webb, W.W.

D. Magde, W.W. Webb, E. Elson, "Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29,705 (1972).
[CrossRef]

Weber, W. H.

G. W. Ford and W. H. Weber, "Electromagnetic interactions of molecules with metal surfaces," Phys. Rep. 113, 195-287 (1984).
[CrossRef]

Widengren, J.

M. Leutenegger, H. Blom, J. Widengren, C. Eggeling, M. Gosch, R. A. Leitgeb, T. Lasser, "Dual-color total internal reflection fluorescence cross-correlation spectroscopy," J. Biomed. Opt. 11, 040502 (2006).
[CrossRef] [PubMed]

J. Widengren and P. Schwille, "Characterization of Photoinduced Isomerization and Back-Isomerization of the Cyanine Dye Cy5 by Fluorescence Correlation Spectroscopy," J. Phys. Chem. A 104, 6416-6428 (2000).
[CrossRef]

Wilson, T.

P. D. Higdon, P. Torok, and T. Wilson, "Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes," J. Microsc. 193, 127-141 (1999).
[CrossRef]

P. Torok, P. D. Higdon, and T. Wilson, "Theory for confocal and conventional microscopes imaging small dielectric scatterers," J. Mod. Opt. 45, 1681-1698 (1998).
[CrossRef]

Winkler, D.

C. Eggeling, P. Kask, D. Winkler, S. Jager, "Rapid Analysis of Forster Resonance Energy Transfer by Two-Color Global Fluorescence Correlation Spectroscopy: Trypsin Proteinase Reaction," Biophys. J. 89, 605-618 (2005).
[CrossRef] [PubMed]

Wolf, E.

E. Wolf, "Electromagnetic diffraction in optical systems, I. An integral representation of the image field," Proc. R. Soc. London Ser. A 253, 349-357 (1959).
[CrossRef]

B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems, II. Structure of the image field in an aplanatic system," Proc. R. Soc. London Ser. A 253, 358-379 (1959).
[CrossRef]

Ann. Phys. (1)

P. Debye, "Das Verhalten von Lichtwellen in der N¨ahe eines Brennpunktes oder einer Brennlinie," Ann. Phys. 30, 755-776 (1909).
[CrossRef]

Biophys. J. (4)

T. P. Burghardt and N. L. Thompson, "Effect of planar dielectric interfaces on fluorescence emission and detection. Evanescent excitation with high-aperture collection," Biophys. J. 46, 729-737 (1984).
[CrossRef] [PubMed]

Y. Chen, J. D. Muller, P. T. C. So, and E. Gratton, "The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy," Biophys. J. 77, 553-567 (1999).
[CrossRef] [PubMed]

C. Eggeling, P. Kask, D. Winkler, S. Jager, "Rapid Analysis of Forster Resonance Energy Transfer by Two-Color Global Fluorescence Correlation Spectroscopy: Trypsin Proteinase Reaction," Biophys. J. 89, 605-618 (2005).
[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, 3294-3302 (2003).
[CrossRef] [PubMed]

Chem. Phys. Chem. (1)

C. Eggeling, A. Volkmer, C. A. M. Seidel, "Molecular Photobleaching Kinetics of Rhodamine 6G by One- and Two-Photon Induced Confocal Fluorescence Microscopy," Chem. Phys. Chem. 6, 791-804 (2005).
[CrossRef] [PubMed]

ChemPhysChem (1)

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, U. B. Kaupp, "Performance of Fluorescence Correlation Spectroscopy for Measuring Diffusion and Concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

M. Leutenegger, H. Blom, J. Widengren, C. Eggeling, M. Gosch, R. A. Leitgeb, T. Lasser, "Dual-color total internal reflection fluorescence cross-correlation spectroscopy," J. Biomed. Opt. 11, 040502 (2006).
[CrossRef] [PubMed]

J. Microsc. (1)

P. D. Higdon, P. Torok, and T. Wilson, "Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes," J. Microsc. 193, 127-141 (1999).
[CrossRef]

J. Mod. Opt. (1)

P. Torok, P. D. Higdon, and T. Wilson, "Theory for confocal and conventional microscopes imaging small dielectric scatterers," J. Mod. Opt. 45, 1681-1698 (1998).
[CrossRef]

J. Opt. Soc. Am. (2)

J. Opt. Soc. Am. A (1)

J. Opt. Soc. Am. B (3)

J. Phys. Chem. A (1)

J. Widengren and P. Schwille, "Characterization of Photoinduced Isomerization and Back-Isomerization of the Cyanine Dye Cy5 by Fluorescence Correlation Spectroscopy," J. Phys. Chem. A 104, 6416-6428 (2000).
[CrossRef]

J. Phys. D: Appl. Phys. (1)

J. R. Lakowicz, J. Malicka, I. Gryczynski, Z. Gryczynski, C. D. Geddes, "Radiative decay engineering: the role of photonic mode density in biotechnology," J. Phys. D: Appl. Phys. 36, R240-R249 (2003).
[CrossRef]

Opt. Commun. (2)

J.-L. Kaiser, E. Quertemont, and R. Chevallier, "Light propagation in the pseudo-paraxial Fresnel approximation," Opt. Commun. 233, 261-269 (2004).
[CrossRef]

D. Mas, J. Garcia, C. Ferreira, L. M. Bernardo, and F. Marinho, "Fast algorithms for free-space diffraction patterns calculation," Opt. Commun. 164, 233-245 (1999).
[CrossRef]

Opt. Express (3)

Opt. Lett. (2)

Phys. Rep. (1)

G. W. Ford and W. H. Weber, "Electromagnetic interactions of molecules with metal surfaces," Phys. Rep. 113, 195-287 (1984).
[CrossRef]

Phys. Rev. Lett. (2)

F. D. Stefani, K. Vasilev, N. Bocchio, N. Stoyanova, and M. Kreiter, "Surface-plasmon-mediated single-molecule fluorescence through a thin metallic film," Phys. Rev. Lett. 94, 023005 (2005).
[CrossRef] [PubMed]

D. Magde, W.W. Webb, E. Elson, "Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29,705 (1972).
[CrossRef]

PNAS (1)

P. Kask, K. Palo, D. Ullmann, K. Gall, "Fluorescence-intensity distribution analysis and its application in biomolecular detection technology," PNAS 96, 13756-13761 (1999).
[CrossRef] [PubMed]

Proc. R. Soc. London Ser. A (2)

E. Wolf, "Electromagnetic diffraction in optical systems, I. An integral representation of the image field," Proc. R. Soc. London Ser. A 253, 349-357 (1959).
[CrossRef]

B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems, II. Structure of the image field in an aplanatic system," Proc. R. Soc. London Ser. A 253, 358-379 (1959).
[CrossRef]

Rep. Prog. Phys. (1)

R. J. Potton, "Reciprocity in optics," Rep. Prog. Phys. 67, 717-754 (2004).
[CrossRef]

Other (8)

The subscript refers to the wavelength used for calculating the corresponding quantity.

B. Valeur, Molecular fluorescence: principles and applications, (Wiley-VCH, 2002) ISBN 3-527-29919-X .

R. Rigler, E. S. Elson, Fluorescence Correlation Spectroscopy: Theory and Applications, Springer Ser. Chem. Phys. 65, ISBN 3-540-67433-0 (2001).

The Royal microscope standard limits the clear objective aperture to less than 16mm. The shortest commercial tube length is 164mm (Carl Zeiss), which yields an image NA < 8mm/164mm = 0.049.

According to previous notes qdT fl? 23% typically.

A. E. Siegman, Lasers, (Oxford Univ. Press,1986) ISBN 0-19-855713-2.

For single photon detectors SPCM-AQR-xx by PerkinElmer, qd ? 55% at ?fl = 525nm, respectively qd? 45% for the PDM 50CT by Micro Photon Devices.

Typical values: ? 85% transmission through the dichroic mirror and ? 90% transmission through the emission bandpass filter with a bandwidth covering ? 60% of the fluorescence spectrum, that is T fl? 45% in total.

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Figures (8)

Fig. 1.
Fig. 1.

Dipole µ⃗ located at r⃗ 0 above the first interface. n 1 is the refraction index of the upper half-space (z>0) around the dipole. nm is the refraction index of the lower half-space (z<-d) and ni the refraction indices of the intermediate layers (-d<z<0).

Fig. 2.
Fig. 2.

Coupling of the dipole moment µ⃗ with the electric fields. Ed represents the direct dipole field, Er the reflected field and Et the transmitted field. s⃗ is perpendicular to the incidence plane, whereas p⃗, q⃗ and o⃗ are parallel to the incidence plane.

Fig. 3.
Fig. 3.

Enhanced power dissipated by a vertical and a horizontal dipole near an interface.

Fig. 4.
Fig. 4.

Radiated angular power density ∝|E⃗k |2 for a horizontal dipole along the x-axis (arrow). The dipole is located at the glass–water interface.

Fig. 5.
Fig. 5.

Calculation of the electromagnetic field in the pinhole plane P. The objective and the tube lens are represented by their principal planes (thin lines; refraction loci ≡ principal planes), the object focus F o , the aperture A in the back-focal plane of the objective, and the image focus F i .

Fig. 6.
Fig. 6.

Electric fields in the aperture of a 100×1.45 NA oil immersion objective (a) and at the pinhole (b) for a dipole at the cover glass–sample (water) interface emitting at a wavelength of λfl =525nm. The circle in (b) indicates a pinhole of 50µm in diameter. The left half-pictures show the field of the horizontal dipole. The field of the vertical dipole is shown in the right half-pictures.

Fig. 7.
Fig. 7.

Detection efficiency (through the cover glass) of isotropically oriented fluorophores achieved with two immersion objectives focused on the cover glass–water interface. The iso-surfaces show the efficiencies Qfl (r⃗)=e -1…-4 Qfl (0) in the sample. The oil immersion objective (a) has a peak detection efficiency of ≈24% and the water immersion objective (b) of ≈14%.

Fig. 8.
Fig. 8.

Detection efficiency of isotropically oriented fluorophores for immersion objectives focused on the cover glass–sample interface. White dotted lines outline an efficiency of 20% and 15%, white solid lines of 10%, black solid lines of 5%, dotted lines of 2% and 1% and ticked lines of 0.5%, respectively.

Equations (30)

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Γ ( r , Ω ) = Γ em ( r , Ω ) + Γ nr
P = ω 2 Im ( μ * · E ( r 0 ) )
× E ( r ) = μ 0 t H ( r )   × H ( r ) + 0 1 t E ( r ) = j ( r )
E ( r ) = E k exp ( i k · r ) d k
k × ( k × E k ) + k 1 2 E k = ω 2 μ 0 μ δ ( r r 0 ) .
E k = μ 0 exp ( i k · r 0 ) ω 2 k 1 2 ( μ + k × ( k × μ ) k 2 k 1 2 ) .
E ( r ) = μ 0 ω 2 k 1 2 d k xy exp ( i k xy · ( r r 0 ) ) + d k z ( μ + k × ( k × μ ) k z 2 k 1 z 2 ) exp ( i k z ( z z 0 ) )
E ( r ) = 2 π μ 0 ω 2 k 1 2 d k xy exp ( i k xy · ( r r 0 ) )
× { δ ( z z 0 ) μ z + i 2 k 1 z k 1 × ( k 1 × μ ) exp ( i k 1 z z z 0 ) } .
E ( r 0 ) = i π μ 0 ω 2 k 1 2 k xy < k 1 d k xy k 1 z k 1 × ( k 1 × μ )
P = π 2 μ 0 ω 3 2 k 1 2 0 k 1 d k xy k xy k 1 z ( 2 k xy 2 μ z 2 + ( 2 k 1 2 k xy 2 ) μ xy 2 ) ,
P = 2 3 π 2 μ 0 ω 3 k 1 μ 2 .
k 1 × ( k 1 × μ ) = ( p · μ ) p + ( s · μ ) s
E ( r ) = i π μ 0 ω 2 k 1 2 d k xy k 1 z exp ( i k xy · ( r r 0 ) i k 1 z ( z z 0 ) ) { ( p · μ ) p + ( s · μ ) s } .
E ( r ) = i π μ 0 ω 2 k 1 2 d k xy k 1 z exp ( i k xy · ( r r 0 ) + i k 1 z z 0 ) ×
{ ( p · μ ) ( exp ( i k 1 z z ) p + r 1 m p exp ( i k 1 z z ) q ) + ( s · μ ) ( exp ( i k 1 z z ) + r 1 m s exp ( i k 1 z z ) ) s }
P ( r , Ω ) = π 2 μ 0 ω 3 2 k 1 2 Re 0 d k xy k xy k 1 z { 2 k xy 2 ( 1 + r 1 m p exp ( 2 i k 1 z z ) ) μ z 2
+ [ k 1 2 ( 1 + r 1 m s exp ( 2 i k 1 z z ) ) + μ xy 2 k 1 z 2 ( 1 r 1 m p exp ( 2 i k 1 z z ) ) ] μ xy 2 }
Δ P z ( r , Ω ) = π 2 μ 0 ω 3 k 1 2 Re 0 d k xy k xy k 1 z exp ( 2 i k 1 z z ) k xy 2 r 1 m p μ z 2 and
Δ P xy ( r , Ω ) = π 2 μ 0 ω 3 2 k 1 2 Re 0 d k xy k xy k 1 z exp ( 2 i k 1 z z ) ( k 1 2 r 1 m s k 1 z 2 r 1 m p ) μ xy 2 .
γ ( r , Ω ) = 1 + 3 4 k 1 3 Re 0 d k xy k xy k 1 z exp ( 2 i k 1 z z ) { 2 k xy 2 r 1 m p cos 2 Θ + ( k 1 2 r 1 m s k 1 z 2 r 1 m p ) sin 2 Θ }
q f l ( r , Ω ) = γ fl ( r , Ω ) q fl γ fl q fl + 1 q fl
R fl ( r , Ω ) = q fl τ S 1 P S 1 = γ fl ( r , Ω ) q fl τ S 1 P S 1 ,
E k = i π μ 0 ω 2 k 1 2 exp ( i k 1 · r 0 ) k 1 z
× { ( q · μ + ( p · μ ) r 1 m p exp ( 2 i k 1 z z 0 ) ) q + ( s · μ ) ( 1 + r 1 m s exp ( 2 i k 1 z z 0 ) ) s }
E k = i π μ 0 ω 2 k 1 2 exp ( i k 1 · r 0 ) k m z ( ( p · μ ) t m 1 p O + ( s · μ ) t m 1 s s ) exp ( i k mz d )
x , y = R k 0 N A k x , y and d k xy = ( k 0 N A R ) 2 d x d y .
E a p , s ( x , y ) = k 0 N A R t ta p , s E o p , s ( k x , k y ) .
( z p z a ) 3 π 4 λ max ( ( x a x p ) 2 + ( y a y p ) 2 ) 2 .
Q fl ( r ) = Ω Q fl ( r , Ω ) P ( Ω ) 1 3 ( Q fl ( r , Ω x ) + Q fl ( r , Ω y ) + Q fl ( r , Ω z ) ) .

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