Abstract

For the observation of single molecule dynamics with fluorescence fluctuation spectroscopy (FFS) very low fluorophore concentrations are necessary. For in vitro measurements, this requirement is easy to fulfill. In biology however, micromolar concentrations are often encountered and may pose a real challenge to conventional FFS methods based on confocal instrumentation. We show a higher confinement of the sampling volume in the near-field of sub-wavelength sized apertures in a thin gold film. The gold apertures have been measured and characterized with fluorescence correlation spectroscopy (FCS), indicating light confinement beyond the far-field diffraction limit. We measured a reduction of the effective sampling volume by an order of magnitude compared to confocal instrumentation.

© 2006 Optical Society of America

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  1. R. Rigler et al., “Fluorescence Correlation Spectroscopy with high Count Rate and low Background - Analysis of Translational Diffusion,” Eur. Biophys. J. Biophys. Lett. 22, 169–175 (1993).
    [Crossref]
  2. S. Weiss, “Fluorescence Spectroscopy of Single Biomolecules,” Science 283, 1676–1683 (1999).
    [Crossref] [PubMed]
  3. P. Kask, K. Palo, D. Ullmann, and 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. Müller, P.T.C. So, and E. Gratton, “The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy,” Biophys. J. 77, 553–567 (1999).
    [Crossref] [PubMed]
  5. L.N. Hillesheim and J.D. Müller, “The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy with Non-Ideal Photodetectors,” Biophys. J. 85, 1948–1958 (2003).
    [Crossref] [PubMed]
  6. T.A. Laurence, A.N. Kapanidis, X. Kong, D.S. Chemla, and S. Weiss, “Photon Arrival-Time Interval Distribution (PAID): A Novel Tool for Analyzing Molecular Interactions,” J. Phys. Chem. B 108, 3051–3067 (2004).
    [Crossref]
  7. K. Starchev, J. Ricka, and J. Buffle, “Noise on Fluorescence Correlation Spectroscopy,” J. Coll. Interf. Science 233, 50–55 (2001).
    [Crossref]
  8. D.E. Koppel, “Statistical accuracy in fluorescence correlation spectroscopy,” Phys. Rev. A 10, 1938–1945 (1974).
    [Crossref]
  9. M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, and W.W. Webb, “Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations,” Science 299, 682–686 (2003).
    [Crossref] [PubMed]
  10. M. Foquet, J. Korlach, W.R. Zipfel, W.W. Webb, and H.G. Craighead, “Focal Volume Confinement by Submicrometer-Sized Fluidic Channels,” Anal. Chem. 76, 1618–1626 (2004).
    [Crossref] [PubMed]
  11. T. Ruckstuhl and S. Seeger, “Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy,” Opt. Lett. 29, 569–571 (2004).
    [Crossref] [PubMed]
  12. K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High Count Rates with Total Internal Reflection Fluorescence Correlation Spectroscopy,” Biophys. J. 88, L1–L3 (2005).
    [Crossref]
  13. K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. Gösch, and 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]
  14. M. Paulus and O.J.F. Martin, “Light propagation and scattering in stratified media: a Green’s tensor approach,” J. Opt. Soc. Am. A 18, 854–861 (2001).
    [Crossref]
  15. H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T.W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005).
    [Crossref] [PubMed]
  16. J. Wenger, P.-F. Lenne, E. Popov, H. Rigneault, J. Dintinger, and T.W. Ebbesen, “Single molecule fluorescence in rectangular nano-apertures,” Opt. Express 13, 7035–7044 (2005).
    [Crossref] [PubMed]
  17. D. Amarie, N.D. Rawlinson, W.L. Schaich, B. Dragnea, and S.C. Jacobson, “Three-Dimensional Mapping of the Light Intensity Transmitted through Nanoapertures,” Nanolett. 5, 1227–1230 (2005).
    [Crossref]
  18. A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
    [Crossref]
  19. O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65, 251–297 (2002).
    [Crossref]
  20. K. Bacia, I.V. Majoul, and P. Schwille, “Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis,” Biophys. J. 83, 1184–1193 (2002).
    [Crossref] [PubMed]
  21. J. Widgengren, Ü. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: A theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
    [Crossref]
  22. T. Wohland, R. Rigler, and H. Vogel, “The Standard Deviation in Fluorescence Correlation Spectroscopy,” Biophys. J. 80, 2987–2999 (2001).
    [Crossref] [PubMed]
  23. E. Bismuto, E. Gratton, and D.C. Lamb, “Dynamics of ANS Binding to Tuna Apomyoglobin Measured with Fluorescence Correlation Spectroscopy,” Biophys. J. 81, 3510–3521 (2001).
    [Crossref] [PubMed]
  24. J. Enderlein, I. Gregor, D. Patra, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharmaceut. Biotech. 5, 155–161 (2004).
    [Crossref]
  25. G.T. Boyd, Z.H. Yu, and Y.R. Shen, “Photoinduced luminescence from the noble metals and its enhancement on roughned surfaces,” Phys. Rev. B 33, 7923–7936 (1986).
    [Crossref]
  26. K. Kneipp, H. Kneipp, I. Itzkan, R.R. Dasari, and M.S. Feld, “Surface-enhanced Raman scattering and biophysics,” J. Phys. Condens. Matter 14, R597–R624 (2002).
    [Crossref]

2005 (6)

H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T.W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005).
[Crossref] [PubMed]

J. Wenger, P.-F. Lenne, E. Popov, H. Rigneault, J. Dintinger, and T.W. Ebbesen, “Single molecule fluorescence in rectangular nano-apertures,” Opt. Express 13, 7035–7044 (2005).
[Crossref] [PubMed]

D. Amarie, N.D. Rawlinson, W.L. Schaich, B. Dragnea, and S.C. Jacobson, “Three-Dimensional Mapping of the Light Intensity Transmitted through Nanoapertures,” Nanolett. 5, 1227–1230 (2005).
[Crossref]

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High Count Rates with Total Internal Reflection Fluorescence Correlation Spectroscopy,” Biophys. J. 88, L1–L3 (2005).
[Crossref]

K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. Gösch, and 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]

2004 (4)

T.A. Laurence, A.N. Kapanidis, X. Kong, D.S. Chemla, and S. Weiss, “Photon Arrival-Time Interval Distribution (PAID): A Novel Tool for Analyzing Molecular Interactions,” J. Phys. Chem. B 108, 3051–3067 (2004).
[Crossref]

M. Foquet, J. Korlach, W.R. Zipfel, W.W. Webb, and H.G. Craighead, “Focal Volume Confinement by Submicrometer-Sized Fluidic Channels,” Anal. Chem. 76, 1618–1626 (2004).
[Crossref] [PubMed]

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

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharmaceut. Biotech. 5, 155–161 (2004).
[Crossref]

2003 (2)

M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, and W.W. Webb, “Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations,” Science 299, 682–686 (2003).
[Crossref] [PubMed]

L.N. Hillesheim and J.D. Müller, “The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy with Non-Ideal Photodetectors,” Biophys. J. 85, 1948–1958 (2003).
[Crossref] [PubMed]

2002 (3)

O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65, 251–297 (2002).
[Crossref]

K. Bacia, I.V. Majoul, and P. Schwille, “Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis,” Biophys. J. 83, 1184–1193 (2002).
[Crossref] [PubMed]

K. Kneipp, H. Kneipp, I. Itzkan, R.R. Dasari, and M.S. Feld, “Surface-enhanced Raman scattering and biophysics,” J. Phys. Condens. Matter 14, R597–R624 (2002).
[Crossref]

2001 (4)

M. Paulus and O.J.F. Martin, “Light propagation and scattering in stratified media: a Green’s tensor approach,” J. Opt. Soc. Am. A 18, 854–861 (2001).
[Crossref]

T. Wohland, R. Rigler, and H. Vogel, “The Standard Deviation in Fluorescence Correlation Spectroscopy,” Biophys. J. 80, 2987–2999 (2001).
[Crossref] [PubMed]

E. Bismuto, E. Gratton, and D.C. Lamb, “Dynamics of ANS Binding to Tuna Apomyoglobin Measured with Fluorescence Correlation Spectroscopy,” Biophys. J. 81, 3510–3521 (2001).
[Crossref] [PubMed]

K. Starchev, J. Ricka, and J. Buffle, “Noise on Fluorescence Correlation Spectroscopy,” J. Coll. Interf. Science 233, 50–55 (2001).
[Crossref]

1999 (3)

S. Weiss, “Fluorescence Spectroscopy of Single Biomolecules,” Science 283, 1676–1683 (1999).
[Crossref] [PubMed]

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

Y. Chen, J.D. Müller, P.T.C. So, and E. Gratton, “The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy,” Biophys. J. 77, 553–567 (1999).
[Crossref] [PubMed]

1995 (1)

J. Widgengren, Ü. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: A theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
[Crossref]

1993 (1)

R. Rigler et al., “Fluorescence Correlation Spectroscopy with high Count Rate and low Background - Analysis of Translational Diffusion,” Eur. Biophys. J. Biophys. Lett. 22, 169–175 (1993).
[Crossref]

1986 (1)

G.T. Boyd, Z.H. Yu, and Y.R. Shen, “Photoinduced luminescence from the noble metals and its enhancement on roughned surfaces,” Phys. Rev. B 33, 7923–7936 (1986).
[Crossref]

1974 (1)

D.E. Koppel, “Statistical accuracy in fluorescence correlation spectroscopy,” Phys. Rev. A 10, 1938–1945 (1974).
[Crossref]

Amarie, D.

D. Amarie, N.D. Rawlinson, W.L. Schaich, B. Dragnea, and S.C. Jacobson, “Three-Dimensional Mapping of the Light Intensity Transmitted through Nanoapertures,” Nanolett. 5, 1227–1230 (2005).
[Crossref]

Anhut, T.

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High Count Rates with Total Internal Reflection Fluorescence Correlation Spectroscopy,” Biophys. J. 88, L1–L3 (2005).
[Crossref]

Bacia, K.

K. Bacia, I.V. Majoul, and P. Schwille, “Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis,” Biophys. J. 83, 1184–1193 (2002).
[Crossref] [PubMed]

Baida, F.

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

Bernal, M.P.

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

Bismuto, E.

E. Bismuto, E. Gratton, and D.C. Lamb, “Dynamics of ANS Binding to Tuna Apomyoglobin Measured with Fluorescence Correlation Spectroscopy,” Biophys. J. 81, 3510–3521 (2001).
[Crossref] [PubMed]

Bonnet, G.

O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65, 251–297 (2002).
[Crossref]

Bonod, N.

H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T.W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005).
[Crossref] [PubMed]

Boyd, G.T.

G.T. Boyd, Z.H. Yu, and Y.R. Shen, “Photoinduced luminescence from the noble metals and its enhancement on roughned surfaces,” Phys. Rev. B 33, 7923–7936 (1986).
[Crossref]

Buffle, J.

K. Starchev, J. Ricka, and J. Buffle, “Noise on Fluorescence Correlation Spectroscopy,” J. Coll. Interf. Science 233, 50–55 (2001).
[Crossref]

Capoulade, J.

H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T.W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005).
[Crossref] [PubMed]

Chemla, D.S.

T.A. Laurence, A.N. Kapanidis, X. Kong, D.S. Chemla, and S. Weiss, “Photon Arrival-Time Interval Distribution (PAID): A Novel Tool for Analyzing Molecular Interactions,” J. Phys. Chem. B 108, 3051–3067 (2004).
[Crossref]

Chen, Y.

Y. Chen, J.D. Müller, P.T.C. So, and E. Gratton, “The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy,” Biophys. J. 77, 553–567 (1999).
[Crossref] [PubMed]

Craighead, H.G.

M. Foquet, J. Korlach, W.R. Zipfel, W.W. Webb, and H.G. Craighead, “Focal Volume Confinement by Submicrometer-Sized Fluidic Channels,” Anal. Chem. 76, 1618–1626 (2004).
[Crossref] [PubMed]

M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, and W.W. Webb, “Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations,” Science 299, 682–686 (2003).
[Crossref] [PubMed]

Dasari, R.R.

K. Kneipp, H. Kneipp, I. Itzkan, R.R. Dasari, and M.S. Feld, “Surface-enhanced Raman scattering and biophysics,” J. Phys. Condens. Matter 14, R597–R624 (2002).
[Crossref]

Dintinger, J.

H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T.W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005).
[Crossref] [PubMed]

J. Wenger, P.-F. Lenne, E. Popov, H. Rigneault, J. Dintinger, and T.W. Ebbesen, “Single molecule fluorescence in rectangular nano-apertures,” Opt. Express 13, 7035–7044 (2005).
[Crossref] [PubMed]

Dragnea, B.

D. Amarie, N.D. Rawlinson, W.L. Schaich, B. Dragnea, and S.C. Jacobson, “Three-Dimensional Mapping of the Light Intensity Transmitted through Nanoapertures,” Nanolett. 5, 1227–1230 (2005).
[Crossref]

Dwir, B.

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

Ebbesen, T.W.

J. Wenger, P.-F. Lenne, E. Popov, H. Rigneault, J. Dintinger, and T.W. Ebbesen, “Single molecule fluorescence in rectangular nano-apertures,” Opt. Express 13, 7035–7044 (2005).
[Crossref] [PubMed]

H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T.W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005).
[Crossref] [PubMed]

Enderlein, J.

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharmaceut. Biotech. 5, 155–161 (2004).
[Crossref]

Feld, M.S.

K. Kneipp, H. Kneipp, I. Itzkan, R.R. Dasari, and M.S. Feld, “Surface-enhanced Raman scattering and biophysics,” J. Phys. Condens. Matter 14, R597–R624 (2002).
[Crossref]

Fitter, J.

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharmaceut. Biotech. 5, 155–161 (2004).
[Crossref]

Foquet, M.

M. Foquet, J. Korlach, W.R. Zipfel, W.W. Webb, and H.G. Craighead, “Focal Volume Confinement by Submicrometer-Sized Fluidic Channels,” Anal. Chem. 76, 1618–1626 (2004).
[Crossref] [PubMed]

M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, and W.W. Webb, “Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations,” Science 299, 682–686 (2003).
[Crossref] [PubMed]

Gall, K.

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

Gösch, M.

Gratton, E.

E. Bismuto, E. Gratton, and D.C. Lamb, “Dynamics of ANS Binding to Tuna Apomyoglobin Measured with Fluorescence Correlation Spectroscopy,” Biophys. J. 81, 3510–3521 (2001).
[Crossref] [PubMed]

Y. Chen, J.D. Müller, 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, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharmaceut. Biotech. 5, 155–161 (2004).
[Crossref]

Hassler, K.

Hillesheim, L.N.

L.N. Hillesheim and J.D. Müller, “The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy with Non-Ideal Photodetectors,” Biophys. J. 85, 1948–1958 (2003).
[Crossref] [PubMed]

Hoffmann, P.

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

Itzkan, I.

K. Kneipp, H. Kneipp, I. Itzkan, R.R. Dasari, and M.S. Feld, “Surface-enhanced Raman scattering and biophysics,” J. Phys. Condens. Matter 14, R597–R624 (2002).
[Crossref]

Jacobson, S.C.

D. Amarie, N.D. Rawlinson, W.L. Schaich, B. Dragnea, and S.C. Jacobson, “Three-Dimensional Mapping of the Light Intensity Transmitted through Nanoapertures,” Nanolett. 5, 1227–1230 (2005).
[Crossref]

Kapanidis, A.N.

T.A. Laurence, A.N. Kapanidis, X. Kong, D.S. Chemla, and S. Weiss, “Photon Arrival-Time Interval Distribution (PAID): A Novel Tool for Analyzing Molecular Interactions,” J. Phys. Chem. B 108, 3051–3067 (2004).
[Crossref]

Kask, P.

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

Kneipp, H.

K. Kneipp, H. Kneipp, I. Itzkan, R.R. Dasari, and M.S. Feld, “Surface-enhanced Raman scattering and biophysics,” J. Phys. Condens. Matter 14, R597–R624 (2002).
[Crossref]

Kneipp, K.

K. Kneipp, H. Kneipp, I. Itzkan, R.R. Dasari, and M.S. Feld, “Surface-enhanced Raman scattering and biophysics,” J. Phys. Condens. Matter 14, R597–R624 (2002).
[Crossref]

Kong, X.

T.A. Laurence, A.N. Kapanidis, X. Kong, D.S. Chemla, and S. Weiss, “Photon Arrival-Time Interval Distribution (PAID): A Novel Tool for Analyzing Molecular Interactions,” J. Phys. Chem. B 108, 3051–3067 (2004).
[Crossref]

Koppel, D.E.

D.E. Koppel, “Statistical accuracy in fluorescence correlation spectroscopy,” Phys. Rev. A 10, 1938–1945 (1974).
[Crossref]

Korlach, J.

M. Foquet, J. Korlach, W.R. Zipfel, W.W. Webb, and H.G. Craighead, “Focal Volume Confinement by Submicrometer-Sized Fluidic Channels,” Anal. Chem. 76, 1618–1626 (2004).
[Crossref] [PubMed]

M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, and W.W. Webb, “Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations,” Science 299, 682–686 (2003).
[Crossref] [PubMed]

Krichevsky, O.

O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65, 251–297 (2002).
[Crossref]

Labeke, D. Van

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

Lamb, D.C.

E. Bismuto, E. Gratton, and D.C. Lamb, “Dynamics of ANS Binding to Tuna Apomyoglobin Measured with Fluorescence Correlation Spectroscopy,” Biophys. J. 81, 3510–3521 (2001).
[Crossref] [PubMed]

Lasser, T.

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High Count Rates with Total Internal Reflection Fluorescence Correlation Spectroscopy,” Biophys. J. 88, L1–L3 (2005).
[Crossref]

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. Gösch, and 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]

Laurence, T.A.

T.A. Laurence, A.N. Kapanidis, X. Kong, D.S. Chemla, and S. Weiss, “Photon Arrival-Time Interval Distribution (PAID): A Novel Tool for Analyzing Molecular Interactions,” J. Phys. Chem. B 108, 3051–3067 (2004).
[Crossref]

Lenne, P.-F.

H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T.W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005).
[Crossref] [PubMed]

J. Wenger, P.-F. Lenne, E. Popov, H. Rigneault, J. Dintinger, and T.W. Ebbesen, “Single molecule fluorescence in rectangular nano-apertures,” Opt. Express 13, 7035–7044 (2005).
[Crossref] [PubMed]

Leutenegger, M.

K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. Gösch, and 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]

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

Levene, M.J.

M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, and W.W. Webb, “Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations,” Science 299, 682–686 (2003).
[Crossref] [PubMed]

Majoul, I.V.

K. Bacia, I.V. Majoul, and P. Schwille, “Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis,” Biophys. J. 83, 1184–1193 (2002).
[Crossref] [PubMed]

Martin, O.J.F.

Mets, Ü.

J. Widgengren, Ü. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: A theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
[Crossref]

Müller, J.D.

L.N. Hillesheim and J.D. Müller, “The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy with Non-Ideal Photodetectors,” Biophys. J. 85, 1948–1958 (2003).
[Crossref] [PubMed]

Y. Chen, J.D. Müller, P.T.C. So, and E. Gratton, “The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy,” Biophys. J. 77, 553–567 (1999).
[Crossref] [PubMed]

Palo, K.

P. Kask, K. Palo, D. Ullmann, and 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, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharmaceut. Biotech. 5, 155–161 (2004).
[Crossref]

Paulus, M.

Perentes, A.

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

Popov, E.

J. Wenger, P.-F. Lenne, E. Popov, H. Rigneault, J. Dintinger, and T.W. Ebbesen, “Single molecule fluorescence in rectangular nano-apertures,” Opt. Express 13, 7035–7044 (2005).
[Crossref] [PubMed]

H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T.W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005).
[Crossref] [PubMed]

Rao, R.

Rawlinson, N.D.

D. Amarie, N.D. Rawlinson, W.L. Schaich, B. Dragnea, and S.C. Jacobson, “Three-Dimensional Mapping of the Light Intensity Transmitted through Nanoapertures,” Nanolett. 5, 1227–1230 (2005).
[Crossref]

Ricka, J.

K. Starchev, J. Ricka, and J. Buffle, “Noise on Fluorescence Correlation Spectroscopy,” J. Coll. Interf. Science 233, 50–55 (2001).
[Crossref]

Rigler, P.

Rigler, R.

K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. Gösch, and 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]

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High Count Rates with Total Internal Reflection Fluorescence Correlation Spectroscopy,” Biophys. J. 88, L1–L3 (2005).
[Crossref]

T. Wohland, R. Rigler, and H. Vogel, “The Standard Deviation in Fluorescence Correlation Spectroscopy,” Biophys. J. 80, 2987–2999 (2001).
[Crossref] [PubMed]

J. Widgengren, Ü. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: A theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
[Crossref]

R. Rigler et al., “Fluorescence Correlation Spectroscopy with high Count Rate and low Background - Analysis of Translational Diffusion,” Eur. Biophys. J. Biophys. Lett. 22, 169–175 (1993).
[Crossref]

Rigneault, H.

H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T.W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005).
[Crossref] [PubMed]

J. Wenger, P.-F. Lenne, E. Popov, H. Rigneault, J. Dintinger, and T.W. Ebbesen, “Single molecule fluorescence in rectangular nano-apertures,” Opt. Express 13, 7035–7044 (2005).
[Crossref] [PubMed]

Ruckstuhl, T.

Russey, M.

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

Salvi, J.

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

Schaich, W.L.

D. Amarie, N.D. Rawlinson, W.L. Schaich, B. Dragnea, and S.C. Jacobson, “Three-Dimensional Mapping of the Light Intensity Transmitted through Nanoapertures,” Nanolett. 5, 1227–1230 (2005).
[Crossref]

Schwille, P.

K. Bacia, I.V. Majoul, and P. Schwille, “Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis,” Biophys. J. 83, 1184–1193 (2002).
[Crossref] [PubMed]

Seeger, S.

Shen, Y.R.

G.T. Boyd, Z.H. Yu, and Y.R. Shen, “Photoinduced luminescence from the noble metals and its enhancement on roughned surfaces,” Phys. Rev. B 33, 7923–7936 (1986).
[Crossref]

So, P.T.C.

Y. Chen, J.D. Müller, P.T.C. So, and E. Gratton, “The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy,” Biophys. J. 77, 553–567 (1999).
[Crossref] [PubMed]

Starchev, K.

K. Starchev, J. Ricka, and J. Buffle, “Noise on Fluorescence Correlation Spectroscopy,” J. Coll. Interf. Science 233, 50–55 (2001).
[Crossref]

Turner, S.W.

M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, and W.W. Webb, “Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations,” Science 299, 682–686 (2003).
[Crossref] [PubMed]

Ullmann, D.

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

Utke, I.

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

Vogel, H.

T. Wohland, R. Rigler, and H. Vogel, “The Standard Deviation in Fluorescence Correlation Spectroscopy,” Biophys. J. 80, 2987–2999 (2001).
[Crossref] [PubMed]

Webb, W.W.

M. Foquet, J. Korlach, W.R. Zipfel, W.W. Webb, and H.G. Craighead, “Focal Volume Confinement by Submicrometer-Sized Fluidic Channels,” Anal. Chem. 76, 1618–1626 (2004).
[Crossref] [PubMed]

M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, and W.W. Webb, “Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations,” Science 299, 682–686 (2003).
[Crossref] [PubMed]

Weiss, S.

T.A. Laurence, A.N. Kapanidis, X. Kong, D.S. Chemla, and S. Weiss, “Photon Arrival-Time Interval Distribution (PAID): A Novel Tool for Analyzing Molecular Interactions,” J. Phys. Chem. B 108, 3051–3067 (2004).
[Crossref]

S. Weiss, “Fluorescence Spectroscopy of Single Biomolecules,” Science 283, 1676–1683 (1999).
[Crossref] [PubMed]

Wenger, J.

J. Wenger, P.-F. Lenne, E. Popov, H. Rigneault, J. Dintinger, and T.W. Ebbesen, “Single molecule fluorescence in rectangular nano-apertures,” Opt. Express 13, 7035–7044 (2005).
[Crossref] [PubMed]

H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T.W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005).
[Crossref] [PubMed]

Widgengren, J.

J. Widgengren, Ü. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: A theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
[Crossref]

Wohland, T.

T. Wohland, R. Rigler, and H. Vogel, “The Standard Deviation in Fluorescence Correlation Spectroscopy,” Biophys. J. 80, 2987–2999 (2001).
[Crossref] [PubMed]

Yu, Z.H.

G.T. Boyd, Z.H. Yu, and Y.R. Shen, “Photoinduced luminescence from the noble metals and its enhancement on roughned surfaces,” Phys. Rev. B 33, 7923–7936 (1986).
[Crossref]

Zipfel, W.R.

M. Foquet, J. Korlach, W.R. Zipfel, W.W. Webb, and H.G. Craighead, “Focal Volume Confinement by Submicrometer-Sized Fluidic Channels,” Anal. Chem. 76, 1618–1626 (2004).
[Crossref] [PubMed]

Anal. Chem. (1)

M. Foquet, J. Korlach, W.R. Zipfel, W.W. Webb, and H.G. Craighead, “Focal Volume Confinement by Submicrometer-Sized Fluidic Channels,” Anal. Chem. 76, 1618–1626 (2004).
[Crossref] [PubMed]

Biophys. J. (6)

Y. Chen, J.D. Müller, P.T.C. So, and E. Gratton, “The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy,” Biophys. J. 77, 553–567 (1999).
[Crossref] [PubMed]

L.N. Hillesheim and J.D. Müller, “The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy with Non-Ideal Photodetectors,” Biophys. J. 85, 1948–1958 (2003).
[Crossref] [PubMed]

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High Count Rates with Total Internal Reflection Fluorescence Correlation Spectroscopy,” Biophys. J. 88, L1–L3 (2005).
[Crossref]

T. Wohland, R. Rigler, and H. Vogel, “The Standard Deviation in Fluorescence Correlation Spectroscopy,” Biophys. J. 80, 2987–2999 (2001).
[Crossref] [PubMed]

E. Bismuto, E. Gratton, and D.C. Lamb, “Dynamics of ANS Binding to Tuna Apomyoglobin Measured with Fluorescence Correlation Spectroscopy,” Biophys. J. 81, 3510–3521 (2001).
[Crossref] [PubMed]

K. Bacia, I.V. Majoul, and P. Schwille, “Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis,” Biophys. J. 83, 1184–1193 (2002).
[Crossref] [PubMed]

Curr. Pharmaceut. Biotech. (1)

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, “Art and artefacts of fluorescence correlation spectroscopy,” Curr. Pharmaceut. Biotech. 5, 155–161 (2004).
[Crossref]

Eur. Biophys. J. Biophys. Lett. (1)

R. Rigler et al., “Fluorescence Correlation Spectroscopy with high Count Rate and low Background - Analysis of Translational Diffusion,” Eur. Biophys. J. Biophys. Lett. 22, 169–175 (1993).
[Crossref]

J. Coll. Interf. Science (1)

K. Starchev, J. Ricka, and J. Buffle, “Noise on Fluorescence Correlation Spectroscopy,” J. Coll. Interf. Science 233, 50–55 (2001).
[Crossref]

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

J. Phys. Chem. (1)

J. Widgengren, Ü. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: A theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995).
[Crossref]

J. Phys. Chem. B (1)

T.A. Laurence, A.N. Kapanidis, X. Kong, D.S. Chemla, and S. Weiss, “Photon Arrival-Time Interval Distribution (PAID): A Novel Tool for Analyzing Molecular Interactions,” J. Phys. Chem. B 108, 3051–3067 (2004).
[Crossref]

J. Phys. Condens. Matter (1)

K. Kneipp, H. Kneipp, I. Itzkan, R.R. Dasari, and M.S. Feld, “Surface-enhanced Raman scattering and biophysics,” J. Phys. Condens. Matter 14, R597–R624 (2002).
[Crossref]

Nanolett. (1)

D. Amarie, N.D. Rawlinson, W.L. Schaich, B. Dragnea, and S.C. Jacobson, “Three-Dimensional Mapping of the Light Intensity Transmitted through Nanoapertures,” Nanolett. 5, 1227–1230 (2005).
[Crossref]

Nanotech. (1)

A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M.P. Bernal, M. Russey, J. Salvi, and D. Van Labeke, “Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies,” Nanotech. 16, S273–S277 (2005).
[Crossref]

Opt. Express (2)

Opt. Lett. (1)

Phys. Rev. A (1)

D.E. Koppel, “Statistical accuracy in fluorescence correlation spectroscopy,” Phys. Rev. A 10, 1938–1945 (1974).
[Crossref]

Phys. Rev. B (1)

G.T. Boyd, Z.H. Yu, and Y.R. Shen, “Photoinduced luminescence from the noble metals and its enhancement on roughned surfaces,” Phys. Rev. B 33, 7923–7936 (1986).
[Crossref]

Phys. Rev. Lett. (1)

H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T.W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005).
[Crossref] [PubMed]

PNAS (1)

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

Rep. Prog. Phys. (1)

O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65, 251–297 (2002).
[Crossref]

Science (2)

M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, and W.W. Webb, “Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations,” Science 299, 682–686 (2003).
[Crossref] [PubMed]

S. Weiss, “Fluorescence Spectroscopy of Single Biomolecules,” Science 283, 1676–1683 (1999).
[Crossref] [PubMed]

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Figures (11)

Fig. 1.
Fig. 1.

Left: SEM image of a 420 nm aperture after evaporation of the 150 nm thick gold film. We noticed small gold particles around the aperture edge. Nanoscale particles and fibers were also found at the border of the gold cap. Right: SEM image of a single 230 nm aperture within a 6×6 array. This aperture was exempt of nearby gold particles.

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Fig. 2.
Fig. 2.

Mask layout with orientation triangles and aperture arrays. Every square represents an array with 6×6 apertures of identical diameter. In each array, the apertures are located on a square grid with 5 μm period. We selected 21 array pairs in order to cover aperture diameters between 115 nm and 520 nm. Inset: Trans-illumination image and scheme of an array pair with 2×6×6 apertures of 300 nm diameter in the 150 nm gold film. The central apertures in the selected arrays were measured with FCS.

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Fig. 3.
Fig. 3.

Confocal trans-illumination setup. The 40x0.9 objective and the multimode fiber were mounted on xyz-translation stages. The aperture mask was aligned with a piezo xyz-translation stage.

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Fig. 4.
Fig. 4.

Simulated excitation fields for aperture diameters of 150 nm (left), 250 nm (center) and 400 nm (right) in a gold film of thickness h = 150 nm. All dimensions are given in nanometers. The coordinate origins are located in the center at the bottom of each aperture. A Gaussian beam with 633 nm wavelength was focused with an opening angle equivalent to a numerical aperture of 0.6 on the apertures. The graphs show three surfaces of equal intensity at e -1 Imax (inner surfaces), e -1.5 Imax (middle surfaces) and e -2 Imax (outer surfaces). Imax is the maximal excitation intensity at z = h. On top of the apertures, the average intensity was reduced to 22%, 73% respectively 87% of the incident intensity at the bottom.

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Fig. 5.
Fig. 5.

Afterpulsing corrected auto-correlations G (τ) versus lag time τ for aperture diameters of 125 nm, 230 nm, 340 nm and 490 nm at a Cy5 concentration of 12 nM. The correlation amplitude for the 490 nm aperture was multiplied by 3. For the 125 nm and the 230 nm aperture, the correlation amplitudes show a different slope for delays between 0.1 ms and 1 ms. We interpret this as constrained diffusion of molecules entering into the 150 nm deep aperture.

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Fig. 6.
Fig. 6.

Auto-correlations and fits G (τ) versus lag time τ for aperture diameters of 125 nm (blue circles), 490 nm (red points) and for free liquid (black dotted) at a Cy5 concentration of 30 nM. Inset: Fit residuals r (τ) = Gfit /G (τ) - 1.

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Fig. 7.
Fig. 7.

Auto-correlations G (τ) versus lag time τ measured on a 125 nm aperture at a Cy5 concentration of 12 nM. The blue dotted line is the measured auto-correlation. The solid line shows the afterpulsing corrected amplitude. The circles trace a second measurement on the same aperture. Inset: intensity trace.

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Fig. 8.
Fig. 8.

Diffusion time τd versus aperture diameter d. The data points show the average and the error bars the standard deviation of 10 measurements per aperture diameter. For clarity, the standard deviation is shown for one case only (12 nM Cy5, 50 μm pinhole). The black dotted line was calculated with Eq. (6) for τ ∞ = 240 μs and dτ = 450 nm. In free liquid, the diffusion time was 160 μs to 170 μs. Inset: concentration of Cy5 and pinhole diameter of all cases.

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Fig. 9.
Fig. 9.

Number of molecules N versus aperture diameter d for a Cy5 concentration of 30 nM and a 50 μm pinhole. The data points show the average and the error bars the standard deviation of 10 measurements per aperture diameter. The dotted line is for guiding the eyes. In free liquid, we measured about 72 molecules in the confocal volume.

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Fig. 10.
Fig. 10.

Background count rates IB , mean intensity 〈I〉 and SNR = 〈I〉/IB - 1 versus aperture diameter d for a Cy5 concentration of 30 nM and a 50 μm pinhole. The data points show the average and the error bars the standard deviation of 10 measurements per aperture diameter. In free liquid, we measured an intensity of 4.7 MHz and a background of 6 kHz.

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Fig. 11.
Fig. 11.

Background corrected count rate per molecule CPM versus aperture diameter d for a Cy5 concentration of 30 nM and a 50 μm pinhole. The data points show the average and the error bars the standard deviation of 10 measurements per aperture diameter. The dotted line is for guiding the eyes. In free liquid, we obtained a CPM ≈ 65 kHz for this experiment.

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Tables (1)

Tables Icon

Table 1. Calculated extensions of the excitation fields along the x,y and z axes, respectively excitation volumes Vex = W 1 + Vap and effective sampling volumes Veff = W 2 1/W 2 + Vap for different aperture diameters d. The extensions are understood as e -2 “half-axes” for comparison with the e -2 xy-waist w 0 = 350 nm of the incident beam.

View Table

Equations (7)

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

G ( τ ) = 〈 I ( t ) I ( t + τ ) 〉 〈 I ( t ) 〉 〈 I ( t + τ ) 〉 = ( T − τ ) ∫ 0 T − τ I ( t ) I ( t + τ ) d t ∫ 0 T − τ I ( t ) d t ∫ τ T I ( t ) d t
W n = I max − n ∫ ∫ ∫ I ( r → ) n d r →
k z 2 = k 2 − k xy 2
G ( τ ) = G ∞ + ( 1 − I B 〈 I 〉 ) 2 γ N { ( 1 + τ τ d ) − 1 ( 1 + τ K 2 τ d ) − 1 2 + P t 1 − P t exp ( − τ τ t ) }
G ap ( τ ) = 〈 ( G uc ( τ ) − 1 ) 〈 I uc ( t ) 〉 1.02 〉
τ d ≈ τ ∞ d 2 d τ 2 + d 2
N = C N A V eff

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