Abstract

Two-photon excitation fluorescence is a powerful technique commonly used for biological imaging. However, the low absorption cross section of this non-linear process is a critical issue for performing biomolecular spectroscopy at the single molecule level. Enhancing the two-photon fluorescence signal would greatly improve the effectiveness of this technique, yet current methods struggle with medium enhancement factors and/or high background noise. Here, we show that the two-photon fluorescence signal from single Alexa Fluor 488 molecules can be enhanced up to 10 times by using a 3 µm diameter latex sphere while adding almost no photoluminescence background. We report a full characterization of the two-photon fluorescence enhancement by a single microsphere using fluorescence correlation spectroscopy. This opens new routes to enhance non-linear optical signals and extend biophotonic applications.

© 2010 OSA

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  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
  2. P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 02, 399–429 (2000).
  3. F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2, 932–940 (2005).
  4. K. M. Berland, P. T. C. So, and E. Gratton, “Two-Photon Fluorescence Correlation Spectroscopy: Method and Application to the Intracellular Environment,” Biophys. J. 68, 694–701 (1995).
  5. L. Brand, C. Eggeling, C. Zander, K. H. Drexhage, and C. A. M. Seidel, “Single-Molecule Identification of Coumarin-120 by Time-Resolved Fluorescence Detection: Comparison of One- and Two-Photon Excitation in Solution,” J. Phys. Chem. A 101, 4313–4321 (1997).
  6. P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77, 2251–2265 (1999).
  7. K. G. Heinze, A. Koltermann, and P. Schwille, “Simultaneous two-photon excitation of distinct labels for dualcolor fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. USA 97, 10377–10382 (2000).
  8. S. A. Kim, K. G. Heinze, K. Bacia, M. N. Waxham, and P. Schwille, “Two-Photon Cross-Correlation Analysis of Intracellular Reactions with Variable Stoichiometry,” Biophys. J. 88, 4319–4336 (2005).
  9. W. W. Webb, “Fluorescence correlation spectroscopy: inception, biophysical experimentations, and prospectus,” Appl. Opt. 40, 3969–3983 (2001).
  10. C. Zander, J. Enderlein, and R. A. Keller, Single-Molecule Detection in Solution - Methods and Applications, (VCH-Wiley, Berlin/New York, 2002).
  11. R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1994).
  12. C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996).
  13. C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).
  14. J. Mertz, C. Xu, and W. W. Webb, “Single-molecule detection by two-photon-excited fluorescence,” Opt. Lett. 20, 2532–2534 (1995).
  15. E. J. Sanchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-Temperature Fluorescence Imaging and Spectroscopy of Single Molecules by Two-Photon Excitation,” J. Phys. Chem. A 101, 7019–7023 (1997).
  16. J. Wenger, D. Gérard, H. Aouani, H. Rigneault, B. Lowder, S. Blair, E. Devaux, and T. W. Ebbesen, “Nanoaperture-Enhanced Signal-to-Noise Ratio in Fluorescence Correlation Spectroscopy,” Anal. Chem. 81, 834–839 (2009).
  17. E. J. Sanchez, L. Novotny, and X. S. Xie, “Near-field fluorescence microscopy based on two-photon excitation with metal tips,” Phys. Rev. Lett. 82, 4014–4017 (1999).
  18. E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D: Appl. Phys. 41, 013001 (2008).
  19. C. Lin, K. Chiu, C. Chang, S. Chang, T. Guo, and S. Chen, “Surface plasmon-enhanced and quenched two-photon excited fluorescence,” Opt. Express 18, 12807–12817 (2010).
  20. P. J. Schuck, D. P. Fromm, A. Sundaramurthy, G. S. Kino, and W. E. Moerner, “Improving the mismatch between light and nanoscale objects with gold bowtie nanoantennas,” Phys. Rev. Lett. 94, 017402 (2005).
  21. Z. Chen, A. Taflove, and V. Backman, “Photonic nanojet enhancement of backscattering of light by nanoparticles: a potential novel visible-light ultramicroscopy technique,” Opt. Express 12, 1214–1220 (2004).
  22. X. Li, Z. Chen, A. Taflove, and V. Backman, “Optical analysis of nanoparticles via enhanced backscattering facilitated by 3-D photonic nanojets,” Opt. Express 13, 526–533 (2005).
  23. P. Ferrand, J. Wenger, M. Pianta, H. Rigneault, A. Devilez, B. Stout, N. Bonod, and E. Popov, “Direct imaging of photonic nanojets,” Opt. Express 16, 6930–6940 (2008).
  24. A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, “Experimental confirmation of backscattering enhancement induced by a photonic jet,” Appl. Phys. Lett. 89, 221118 (2006).
  25. J. Wenger, D. Gérard, H. Aouani, and H. Rigneault, “Disposable microscope objective lenses for fluorescence correlation spectroscopy using latex microspheres,” Anal. Chem. 80, 6800–6804 (2008).
  26. S. Lecler, S. Haacke, N. Lecong, O. Crégut, J.-L. Rehspringer, and C. Hirlimann, “Photonic jet driven non-linear optics: example of two-photon fluorescence enhancement by dielectric microspheres,” Opt. Express 15, 4935–4942 (2007).
  27. A. Devilez, N. Bonod, B. Stout, D. Gérard, J. Wenger, H. Rigneault, and E. Popov, “Three-dimensional subwavelength confinement of photonic nanojets,” Opt. Express 17, 2089–2094 (2009).
  28. D. Gérard, J. Wenger, A. Devilez, D. Gachet, B. Stout, N. Bonod, E. Popov, and H. Rigneault, “Strong electromagnetic confinement near dielectric microspheres to enhance single-molecule fluorescence,” Opt. Express 16, 15297–15303 (2008).
  29. J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008).
  30. D. Gérard, A. Devilez, H. Aouani, B. Stout, N. Bonod, J. Wenger, E. Popov, and H. Rigneault, “Efficient excitation and collection of single-molecule fluorescence close to a dielectric microsphere,” J. Opt. Soc. Am. B 26, 1473–1478 (2009).
  31. J. Wenger and H. Rigneault, “Photonic Methods to Enhance Fluorescence Correlation Spectroscopy and Single Molecule Fluorescence Detection,” Int. J. Mol. Sci. 11, 206–221 (2010).
  32. 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).
  33. H. Aouani, F. Deiss, J. Wenger, P. Ferrand, N. Sojic, and H. Rigneault, “Optical-fiber-microsphere for remote fluorescence correlation spectroscopy,” Opt. Express 17, 19085–19092 (2009).
  34. Y.-C. Chang, J. Y. Ye, T. Thomas, Y. Chen, J. R. Baker, and T. B. Norris, “Two-photon fluorescence correlation spectroscopy through dual-clad optical fiber,” Opt. Express 16, 12640–12649 (2008).

2010 (2)

C. Lin, K. Chiu, C. Chang, S. Chang, T. Guo, and S. Chen, “Surface plasmon-enhanced and quenched two-photon excited fluorescence,” Opt. Express 18, 12807–12817 (2010).

J. Wenger and H. Rigneault, “Photonic Methods to Enhance Fluorescence Correlation Spectroscopy and Single Molecule Fluorescence Detection,” Int. J. Mol. Sci. 11, 206–221 (2010).

2009 (4)

2008 (6)

2007 (1)

2006 (1)

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, “Experimental confirmation of backscattering enhancement induced by a photonic jet,” Appl. Phys. Lett. 89, 221118 (2006).

2005 (4)

X. Li, Z. Chen, A. Taflove, and V. Backman, “Optical analysis of nanoparticles via enhanced backscattering facilitated by 3-D photonic nanojets,” Opt. Express 13, 526–533 (2005).

P. J. Schuck, D. P. Fromm, A. Sundaramurthy, G. S. Kino, and W. E. Moerner, “Improving the mismatch between light and nanoscale objects with gold bowtie nanoantennas,” Phys. Rev. Lett. 94, 017402 (2005).

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2, 932–940 (2005).

S. A. Kim, K. G. Heinze, K. Bacia, M. N. Waxham, and P. Schwille, “Two-Photon Cross-Correlation Analysis of Intracellular Reactions with Variable Stoichiometry,” Biophys. J. 88, 4319–4336 (2005).

2004 (1)

2003 (1)

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).

2001 (1)

2000 (2)

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 02, 399–429 (2000).

K. G. Heinze, A. Koltermann, and P. Schwille, “Simultaneous two-photon excitation of distinct labels for dualcolor fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. USA 97, 10377–10382 (2000).

1999 (2)

E. J. Sanchez, L. Novotny, and X. S. Xie, “Near-field fluorescence microscopy based on two-photon excitation with metal tips,” Phys. Rev. Lett. 82, 4014–4017 (1999).

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77, 2251–2265 (1999).

1997 (2)

L. Brand, C. Eggeling, C. Zander, K. H. Drexhage, and C. A. M. Seidel, “Single-Molecule Identification of Coumarin-120 by Time-Resolved Fluorescence Detection: Comparison of One- and Two-Photon Excitation in Solution,” J. Phys. Chem. A 101, 4313–4321 (1997).

E. J. Sanchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-Temperature Fluorescence Imaging and Spectroscopy of Single Molecules by Two-Photon Excitation,” J. Phys. Chem. A 101, 7019–7023 (1997).

1996 (2)

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996).

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).

1995 (2)

J. Mertz, C. Xu, and W. W. Webb, “Single-molecule detection by two-photon-excited fluorescence,” Opt. Lett. 20, 2532–2534 (1995).

K. M. Berland, P. T. C. So, and E. Gratton, “Two-Photon Fluorescence Correlation Spectroscopy: Method and Application to the Intracellular Environment,” Biophys. J. 68, 694–701 (1995).

1994 (1)

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1994).

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).

Aouani, H.

J. Wenger, D. Gérard, H. Aouani, H. Rigneault, B. Lowder, S. Blair, E. Devaux, and T. W. Ebbesen, “Nanoaperture-Enhanced Signal-to-Noise Ratio in Fluorescence Correlation Spectroscopy,” Anal. Chem. 81, 834–839 (2009).

D. Gérard, A. Devilez, H. Aouani, B. Stout, N. Bonod, J. Wenger, E. Popov, and H. Rigneault, “Efficient excitation and collection of single-molecule fluorescence close to a dielectric microsphere,” J. Opt. Soc. Am. B 26, 1473–1478 (2009).

H. Aouani, F. Deiss, J. Wenger, P. Ferrand, N. Sojic, and H. Rigneault, “Optical-fiber-microsphere for remote fluorescence correlation spectroscopy,” Opt. Express 17, 19085–19092 (2009).

J. Wenger, D. Gérard, H. Aouani, and H. Rigneault, “Disposable microscope objective lenses for fluorescence correlation spectroscopy using latex microspheres,” Anal. Chem. 80, 6800–6804 (2008).

Bacia, K.

S. A. Kim, K. G. Heinze, K. Bacia, M. N. Waxham, and P. Schwille, “Two-Photon Cross-Correlation Analysis of Intracellular Reactions with Variable Stoichiometry,” Biophys. J. 88, 4319–4336 (2005).

Backman, V.

Baker, J. R.

Berland, K. M.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 02, 399–429 (2000).

K. M. Berland, P. T. C. So, and E. Gratton, “Two-Photon Fluorescence Correlation Spectroscopy: Method and Application to the Intracellular Environment,” Biophys. J. 68, 694–701 (1995).

Blair, S.

J. Wenger, D. Gérard, H. Aouani, H. Rigneault, B. Lowder, S. Blair, E. Devaux, and T. W. Ebbesen, “Nanoaperture-Enhanced Signal-to-Noise Ratio in Fluorescence Correlation Spectroscopy,” Anal. Chem. 81, 834–839 (2009).

Bonod, N.

Brand, L.

L. Brand, C. Eggeling, C. Zander, K. H. Drexhage, and C. A. M. Seidel, “Single-Molecule Identification of Coumarin-120 by Time-Resolved Fluorescence Detection: Comparison of One- and Two-Photon Excitation in Solution,” J. Phys. Chem. A 101, 4313–4321 (1997).

Chang, C.

Chang, S.

Chang, Y.-C.

Chen, S.

Chen, Y.

Chen, Z.

Chiu, K.

Craighead, H. G.

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).

Crégut, O.

Deiss, F.

Denk, W.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2, 932–940 (2005).

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).

Devaux, E.

J. Wenger, D. Gérard, H. Aouani, H. Rigneault, B. Lowder, S. Blair, E. Devaux, and T. W. Ebbesen, “Nanoaperture-Enhanced Signal-to-Noise Ratio in Fluorescence Correlation Spectroscopy,” Anal. Chem. 81, 834–839 (2009).

Devilez, A.

Dintinger, J.

Dong, C. Y.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 02, 399–429 (2000).

Drexhage, K. H.

L. Brand, C. Eggeling, C. Zander, K. H. Drexhage, and C. A. M. Seidel, “Single-Molecule Identification of Coumarin-120 by Time-Resolved Fluorescence Detection: Comparison of One- and Two-Photon Excitation in Solution,” J. Phys. Chem. A 101, 4313–4321 (1997).

Ebbesen, T. W.

J. Wenger, D. Gérard, H. Aouani, H. Rigneault, B. Lowder, S. Blair, E. Devaux, and T. W. Ebbesen, “Nanoaperture-Enhanced Signal-to-Noise Ratio in Fluorescence Correlation Spectroscopy,” Anal. Chem. 81, 834–839 (2009).

J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008).

Eggeling, C.

L. Brand, C. Eggeling, C. Zander, K. H. Drexhage, and C. A. M. Seidel, “Single-Molecule Identification of Coumarin-120 by Time-Resolved Fluorescence Detection: Comparison of One- and Two-Photon Excitation in Solution,” J. Phys. Chem. A 101, 4313–4321 (1997).

Enderlein, J.

C. Zander, J. Enderlein, and R. A. Keller, Single-Molecule Detection in Solution - Methods and Applications, (VCH-Wiley, Berlin/New York, 2002).

Ferrand, P.

Foquet, M.

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).

Fort, E.

E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D: Appl. Phys. 41, 013001 (2008).

Fromm, D. P.

P. J. Schuck, D. P. Fromm, A. Sundaramurthy, G. S. Kino, and W. E. Moerner, “Improving the mismatch between light and nanoscale objects with gold bowtie nanoantennas,” Phys. Rev. Lett. 94, 017402 (2005).

Gachet, D.

Gérard, D.

Gratton, E.

K. M. Berland, P. T. C. So, and E. Gratton, “Two-Photon Fluorescence Correlation Spectroscopy: Method and Application to the Intracellular Environment,” Biophys. J. 68, 694–701 (1995).

Grésillon, S.

E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D: Appl. Phys. 41, 013001 (2008).

Guo, T.

Haacke, S.

Haupts, U.

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77, 2251–2265 (1999).

Heifetz, A.

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, “Experimental confirmation of backscattering enhancement induced by a photonic jet,” Appl. Phys. Lett. 89, 221118 (2006).

Heinze, K. G.

S. A. Kim, K. G. Heinze, K. Bacia, M. N. Waxham, and P. Schwille, “Two-Photon Cross-Correlation Analysis of Intracellular Reactions with Variable Stoichiometry,” Biophys. J. 88, 4319–4336 (2005).

K. G. Heinze, A. Koltermann, and P. Schwille, “Simultaneous two-photon excitation of distinct labels for dualcolor fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. USA 97, 10377–10382 (2000).

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2, 932–940 (2005).

Hirlimann, C.

Holtom, G. R.

E. J. Sanchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-Temperature Fluorescence Imaging and Spectroscopy of Single Molecules by Two-Photon Excitation,” J. Phys. Chem. A 101, 7019–7023 (1997).

Huang, K.

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, “Experimental confirmation of backscattering enhancement induced by a photonic jet,” Appl. Phys. Lett. 89, 221118 (2006).

Kask, P.

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1994).

Keller, R. A.

C. Zander, J. Enderlein, and R. A. Keller, Single-Molecule Detection in Solution - Methods and Applications, (VCH-Wiley, Berlin/New York, 2002).

Kim, S. A.

S. A. Kim, K. G. Heinze, K. Bacia, M. N. Waxham, and P. Schwille, “Two-Photon Cross-Correlation Analysis of Intracellular Reactions with Variable Stoichiometry,” Biophys. J. 88, 4319–4336 (2005).

Kino, G. S.

P. J. Schuck, D. P. Fromm, A. Sundaramurthy, G. S. Kino, and W. E. Moerner, “Improving the mismatch between light and nanoscale objects with gold bowtie nanoantennas,” Phys. Rev. Lett. 94, 017402 (2005).

Koltermann, A.

K. G. Heinze, A. Koltermann, and P. Schwille, “Simultaneous two-photon excitation of distinct labels for dualcolor fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. USA 97, 10377–10382 (2000).

Korlach, 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).

Lecler, S.

Lecong, N.

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).

Li, X.

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, “Experimental confirmation of backscattering enhancement induced by a photonic jet,” Appl. Phys. Lett. 89, 221118 (2006).

X. Li, Z. Chen, A. Taflove, and V. Backman, “Optical analysis of nanoparticles via enhanced backscattering facilitated by 3-D photonic nanojets,” Opt. Express 13, 526–533 (2005).

Lin, C.

Lowder, B.

J. Wenger, D. Gérard, H. Aouani, H. Rigneault, B. Lowder, S. Blair, E. Devaux, and T. W. Ebbesen, “Nanoaperture-Enhanced Signal-to-Noise Ratio in Fluorescence Correlation Spectroscopy,” Anal. Chem. 81, 834–839 (2009).

Mahboub, O.

Maiti, S.

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77, 2251–2265 (1999).

Masters, B. R.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 02, 399–429 (2000).

Mertz, J.

Mets, Ü.

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1994).

Moerner, W. E.

P. J. Schuck, D. P. Fromm, A. Sundaramurthy, G. S. Kino, and W. E. Moerner, “Improving the mismatch between light and nanoscale objects with gold bowtie nanoantennas,” Phys. Rev. Lett. 94, 017402 (2005).

Norris, T. B.

Novotny, L.

E. J. Sanchez, L. Novotny, and X. S. Xie, “Near-field fluorescence microscopy based on two-photon excitation with metal tips,” Phys. Rev. Lett. 82, 4014–4017 (1999).

E. J. Sanchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-Temperature Fluorescence Imaging and Spectroscopy of Single Molecules by Two-Photon Excitation,” J. Phys. Chem. A 101, 7019–7023 (1997).

Pianta, M.

Popov, E.

Rehspringer, J.-L.

Rigler, R.

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1994).

Rigneault, H.

J. Wenger and H. Rigneault, “Photonic Methods to Enhance Fluorescence Correlation Spectroscopy and Single Molecule Fluorescence Detection,” Int. J. Mol. Sci. 11, 206–221 (2010).

D. Gérard, A. Devilez, H. Aouani, B. Stout, N. Bonod, J. Wenger, E. Popov, and H. Rigneault, “Efficient excitation and collection of single-molecule fluorescence close to a dielectric microsphere,” J. Opt. Soc. Am. B 26, 1473–1478 (2009).

A. Devilez, N. Bonod, B. Stout, D. Gérard, J. Wenger, H. Rigneault, and E. Popov, “Three-dimensional subwavelength confinement of photonic nanojets,” Opt. Express 17, 2089–2094 (2009).

J. Wenger, D. Gérard, H. Aouani, H. Rigneault, B. Lowder, S. Blair, E. Devaux, and T. W. Ebbesen, “Nanoaperture-Enhanced Signal-to-Noise Ratio in Fluorescence Correlation Spectroscopy,” Anal. Chem. 81, 834–839 (2009).

H. Aouani, F. Deiss, J. Wenger, P. Ferrand, N. Sojic, and H. Rigneault, “Optical-fiber-microsphere for remote fluorescence correlation spectroscopy,” Opt. Express 17, 19085–19092 (2009).

D. Gérard, J. Wenger, A. Devilez, D. Gachet, B. Stout, N. Bonod, E. Popov, and H. Rigneault, “Strong electromagnetic confinement near dielectric microspheres to enhance single-molecule fluorescence,” Opt. Express 16, 15297–15303 (2008).

J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008).

J. Wenger, D. Gérard, H. Aouani, and H. Rigneault, “Disposable microscope objective lenses for fluorescence correlation spectroscopy using latex microspheres,” Anal. Chem. 80, 6800–6804 (2008).

P. Ferrand, J. Wenger, M. Pianta, H. Rigneault, A. Devilez, B. Stout, N. Bonod, and E. Popov, “Direct imaging of photonic nanojets,” Opt. Express 16, 6930–6940 (2008).

Sahakian, A. V.

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, “Experimental confirmation of backscattering enhancement induced by a photonic jet,” Appl. Phys. Lett. 89, 221118 (2006).

Sanchez, E. J.

E. J. Sanchez, L. Novotny, and X. S. Xie, “Near-field fluorescence microscopy based on two-photon excitation with metal tips,” Phys. Rev. Lett. 82, 4014–4017 (1999).

E. J. Sanchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-Temperature Fluorescence Imaging and Spectroscopy of Single Molecules by Two-Photon Excitation,” J. Phys. Chem. A 101, 7019–7023 (1997).

Schuck, P. J.

P. J. Schuck, D. P. Fromm, A. Sundaramurthy, G. S. Kino, and W. E. Moerner, “Improving the mismatch between light and nanoscale objects with gold bowtie nanoantennas,” Phys. Rev. Lett. 94, 017402 (2005).

Schwille, P.

S. A. Kim, K. G. Heinze, K. Bacia, M. N. Waxham, and P. Schwille, “Two-Photon Cross-Correlation Analysis of Intracellular Reactions with Variable Stoichiometry,” Biophys. J. 88, 4319–4336 (2005).

K. G. Heinze, A. Koltermann, and P. Schwille, “Simultaneous two-photon excitation of distinct labels for dualcolor fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. USA 97, 10377–10382 (2000).

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77, 2251–2265 (1999).

Seidel, C. A. M.

L. Brand, C. Eggeling, C. Zander, K. H. Drexhage, and C. A. M. Seidel, “Single-Molecule Identification of Coumarin-120 by Time-Resolved Fluorescence Detection: Comparison of One- and Two-Photon Excitation in Solution,” J. Phys. Chem. A 101, 4313–4321 (1997).

Shear, J. B.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996).

So, P. T. C.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 02, 399–429 (2000).

K. M. Berland, P. T. C. So, and E. Gratton, “Two-Photon Fluorescence Correlation Spectroscopy: Method and Application to the Intracellular Environment,” Biophys. J. 68, 694–701 (1995).

Sojic, N.

Stout, B.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).

Sundaramurthy, A.

P. J. Schuck, D. P. Fromm, A. Sundaramurthy, G. S. Kino, and W. E. Moerner, “Improving the mismatch between light and nanoscale objects with gold bowtie nanoantennas,” Phys. Rev. Lett. 94, 017402 (2005).

Taflove, A.

Thomas, T.

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).

Waxham, M. N.

S. A. Kim, K. G. Heinze, K. Bacia, M. N. Waxham, and P. Schwille, “Two-Photon Cross-Correlation Analysis of Intracellular Reactions with Variable Stoichiometry,” Biophys. J. 88, 4319–4336 (2005).

Webb, W. 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).

W. W. Webb, “Fluorescence correlation spectroscopy: inception, biophysical experimentations, and prospectus,” Appl. Opt. 40, 3969–3983 (2001).

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77, 2251–2265 (1999).

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996).

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).

J. Mertz, C. Xu, and W. W. Webb, “Single-molecule detection by two-photon-excited fluorescence,” Opt. Lett. 20, 2532–2534 (1995).

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).

Wenger, J.

J. Wenger and H. Rigneault, “Photonic Methods to Enhance Fluorescence Correlation Spectroscopy and Single Molecule Fluorescence Detection,” Int. J. Mol. Sci. 11, 206–221 (2010).

D. Gérard, A. Devilez, H. Aouani, B. Stout, N. Bonod, J. Wenger, E. Popov, and H. Rigneault, “Efficient excitation and collection of single-molecule fluorescence close to a dielectric microsphere,” J. Opt. Soc. Am. B 26, 1473–1478 (2009).

A. Devilez, N. Bonod, B. Stout, D. Gérard, J. Wenger, H. Rigneault, and E. Popov, “Three-dimensional subwavelength confinement of photonic nanojets,” Opt. Express 17, 2089–2094 (2009).

J. Wenger, D. Gérard, H. Aouani, H. Rigneault, B. Lowder, S. Blair, E. Devaux, and T. W. Ebbesen, “Nanoaperture-Enhanced Signal-to-Noise Ratio in Fluorescence Correlation Spectroscopy,” Anal. Chem. 81, 834–839 (2009).

H. Aouani, F. Deiss, J. Wenger, P. Ferrand, N. Sojic, and H. Rigneault, “Optical-fiber-microsphere for remote fluorescence correlation spectroscopy,” Opt. Express 17, 19085–19092 (2009).

D. Gérard, J. Wenger, A. Devilez, D. Gachet, B. Stout, N. Bonod, E. Popov, and H. Rigneault, “Strong electromagnetic confinement near dielectric microspheres to enhance single-molecule fluorescence,” Opt. Express 16, 15297–15303 (2008).

J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008).

P. Ferrand, J. Wenger, M. Pianta, H. Rigneault, A. Devilez, B. Stout, N. Bonod, and E. Popov, “Direct imaging of photonic nanojets,” Opt. Express 16, 6930–6940 (2008).

J. Wenger, D. Gérard, H. Aouani, and H. Rigneault, “Disposable microscope objective lenses for fluorescence correlation spectroscopy using latex microspheres,” Anal. Chem. 80, 6800–6804 (2008).

Widengren, J.

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1994).

Williams, R. M.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996).

Xie, X. S.

E. J. Sanchez, L. Novotny, and X. S. Xie, “Near-field fluorescence microscopy based on two-photon excitation with metal tips,” Phys. Rev. Lett. 82, 4014–4017 (1999).

E. J. Sanchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-Temperature Fluorescence Imaging and Spectroscopy of Single Molecules by Two-Photon Excitation,” J. Phys. Chem. A 101, 7019–7023 (1997).

Xu, C.

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996).

J. Mertz, C. Xu, and W. W. Webb, “Single-molecule detection by two-photon-excited fluorescence,” Opt. Lett. 20, 2532–2534 (1995).

Ye, J. Y.

Zander, C.

L. Brand, C. Eggeling, C. Zander, K. H. Drexhage, and C. A. M. Seidel, “Single-Molecule Identification of Coumarin-120 by Time-Resolved Fluorescence Detection: Comparison of One- and Two-Photon Excitation in Solution,” J. Phys. Chem. A 101, 4313–4321 (1997).

C. Zander, J. Enderlein, and R. A. Keller, Single-Molecule Detection in Solution - Methods and Applications, (VCH-Wiley, Berlin/New York, 2002).

Zipfel, W.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996).

Anal. Chem. (2)

J. Wenger, D. Gérard, H. Aouani, H. Rigneault, B. Lowder, S. Blair, E. Devaux, and T. W. Ebbesen, “Nanoaperture-Enhanced Signal-to-Noise Ratio in Fluorescence Correlation Spectroscopy,” Anal. Chem. 81, 834–839 (2009).

J. Wenger, D. Gérard, H. Aouani, and H. Rigneault, “Disposable microscope objective lenses for fluorescence correlation spectroscopy using latex microspheres,” Anal. Chem. 80, 6800–6804 (2008).

Annu. Rev. Biomed. Eng. (1)

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 02, 399–429 (2000).

Appl. Opt. (1)

Appl. Phys. Lett. (1)

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, “Experimental confirmation of backscattering enhancement induced by a photonic jet,” Appl. Phys. Lett. 89, 221118 (2006).

Biophys. J. (3)

K. M. Berland, P. T. C. So, and E. Gratton, “Two-Photon Fluorescence Correlation Spectroscopy: Method and Application to the Intracellular Environment,” Biophys. J. 68, 694–701 (1995).

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77, 2251–2265 (1999).

S. A. Kim, K. G. Heinze, K. Bacia, M. N. Waxham, and P. Schwille, “Two-Photon Cross-Correlation Analysis of Intracellular Reactions with Variable Stoichiometry,” Biophys. J. 88, 4319–4336 (2005).

Eur. Biophys. J. (1)

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, “Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion,” Eur. Biophys. J. 22, 169–175 (1994).

Int. J. Mol. Sci. (1)

J. Wenger and H. Rigneault, “Photonic Methods to Enhance Fluorescence Correlation Spectroscopy and Single Molecule Fluorescence Detection,” Int. J. Mol. Sci. 11, 206–221 (2010).

J. Opt. Soc. Am. B (2)

J. Phys. Chem. A (2)

L. Brand, C. Eggeling, C. Zander, K. H. Drexhage, and C. A. M. Seidel, “Single-Molecule Identification of Coumarin-120 by Time-Resolved Fluorescence Detection: Comparison of One- and Two-Photon Excitation in Solution,” J. Phys. Chem. A 101, 4313–4321 (1997).

E. J. Sanchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-Temperature Fluorescence Imaging and Spectroscopy of Single Molecules by Two-Photon Excitation,” J. Phys. Chem. A 101, 7019–7023 (1997).

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

E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D: Appl. Phys. 41, 013001 (2008).

Nat. Methods (1)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2, 932–940 (2005).

Opt. Express (10)

C. Lin, K. Chiu, C. Chang, S. Chang, T. Guo, and S. Chen, “Surface plasmon-enhanced and quenched two-photon excited fluorescence,” Opt. Express 18, 12807–12817 (2010).

Z. Chen, A. Taflove, and V. Backman, “Photonic nanojet enhancement of backscattering of light by nanoparticles: a potential novel visible-light ultramicroscopy technique,” Opt. Express 12, 1214–1220 (2004).

X. Li, Z. Chen, A. Taflove, and V. Backman, “Optical analysis of nanoparticles via enhanced backscattering facilitated by 3-D photonic nanojets,” Opt. Express 13, 526–533 (2005).

P. Ferrand, J. Wenger, M. Pianta, H. Rigneault, A. Devilez, B. Stout, N. Bonod, and E. Popov, “Direct imaging of photonic nanojets,” Opt. Express 16, 6930–6940 (2008).

S. Lecler, S. Haacke, N. Lecong, O. Crégut, J.-L. Rehspringer, and C. Hirlimann, “Photonic jet driven non-linear optics: example of two-photon fluorescence enhancement by dielectric microspheres,” Opt. Express 15, 4935–4942 (2007).

A. Devilez, N. Bonod, B. Stout, D. Gérard, J. Wenger, H. Rigneault, and E. Popov, “Three-dimensional subwavelength confinement of photonic nanojets,” Opt. Express 17, 2089–2094 (2009).

D. Gérard, J. Wenger, A. Devilez, D. Gachet, B. Stout, N. Bonod, E. Popov, and H. Rigneault, “Strong electromagnetic confinement near dielectric microspheres to enhance single-molecule fluorescence,” Opt. Express 16, 15297–15303 (2008).

J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008).

H. Aouani, F. Deiss, J. Wenger, P. Ferrand, N. Sojic, and H. Rigneault, “Optical-fiber-microsphere for remote fluorescence correlation spectroscopy,” Opt. Express 17, 19085–19092 (2009).

Y.-C. Chang, J. Y. Ye, T. Thomas, Y. Chen, J. R. Baker, and T. B. Norris, “Two-photon fluorescence correlation spectroscopy through dual-clad optical fiber,” Opt. Express 16, 12640–12649 (2008).

Opt. Lett. (1)

Phys. Rev. Lett. (2)

E. J. Sanchez, L. Novotny, and X. S. Xie, “Near-field fluorescence microscopy based on two-photon excitation with metal tips,” Phys. Rev. Lett. 82, 4014–4017 (1999).

P. J. Schuck, D. P. Fromm, A. Sundaramurthy, G. S. Kino, and W. E. Moerner, “Improving the mismatch between light and nanoscale objects with gold bowtie nanoantennas,” Phys. Rev. Lett. 94, 017402 (2005).

Proc. Natl. Acad. Sci. USA (2)

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996).

K. G. Heinze, A. Koltermann, and P. Schwille, “Simultaneous two-photon excitation of distinct labels for dualcolor fluorescence crosscorrelation analysis,” Proc. Natl. Acad. Sci. USA 97, 10377–10382 (2000).

Science (2)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).

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).

Other (1)

C. Zander, J. Enderlein, and R. A. Keller, Single-Molecule Detection in Solution - Methods and Applications, (VCH-Wiley, Berlin/New York, 2002).

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Figures (3)

Fig. 1.
Fig. 1.

(a) and (b) Schematics of the experimental setup. (c) fluorescence intensity autocorrelation functions obtained in solution (blue) and with a 3 µm latex sphere (red). (d) Summary of the reduction in the number of observed molecules N for the different sizes of microspheres as compared to the case with no microsphere.

Fig. 2.
Fig. 2.

(a) Evolution of the CRM versus the excitation intensity (dots) and numerical fit (solid lines) according to Eq. (2) for the different microsphere diameters and in open solution case. (b) Fluorescence enhancement factor ηF in the low excitation regime for the different sphere diameters.

Fig. 3.
Fig. 3.

Excitation (a) and collection (b) contributions to the fluorescence enhancement for the different microsphere diameters.

Equations (2)

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

G ( 2 ) ( τ ) = 1 + 1 N [ 1 + n T exp ( τ τ b T ) ] ( 1 + τ τ d ) 1 + s 2 τ τ d ,
CRM = A I e 2 1 + I e 2 I s 2

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