Abstract

Fluorescence correlation spectroscopy (FCS) is a versatile method that would greatly benefit to remote optical-fiber fluorescence sensors. However, the current state-of-the-art struggles with high background and low detection sensitivities that prevent the extension of fiber-based FCS down to the single-molecule level. Here we report the use of an optical fiber combined with a latex microsphere to perform FCS analysis. The sensitivity of the technique is demonstrated at the single molecule level thanks to a photonic nanojet effect. This offers new opportunities for reducing the bulky microscope setup and extending FCS to remote or in vivo applications.

© 2009 Optical Society of America

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  1. S. Maiti, U. Haupts, andW.W.Webb, "Fluorescence correlation spectroscopy: diagnostics for sparse molecules," Proc. Natl. Acad. Sci. U.S.A. 94, 11753-11757 (1997).
    [CrossRef] [PubMed]
  2. W. W. Webb, "Fluorescence correlation spectroscopy: inception, biophysical experimentations, and prospectus," Appl. Opt. 40, 3969-3983 (2001).
    [CrossRef]
  3. J. Wenger, D. Gerard, H. Aouani, H. Rigneault, B. Lowder, S. Blair, E. Devaux, T. W. Ebbesen, "Nanoaperture-Enhanced Signal-to-Noise Ratio in Fluorescence Correlation Spectroscopy," Anal. Chem. 81, 834-839 (2009).
    [CrossRef]
  4. F. Helmchen, "Miniaturization of fluorescence microscopes using fibre optics," Exp. Physiol. 87, 737-745 (2002).
    [CrossRef] [PubMed]
  5. J. R. Epstein and D. R. Walt, "Fluorescence-based fibre optic arrays: a universal platform for sensing," Chem. Soc. Rev. 32, 203-214 (2003).
    [CrossRef] [PubMed]
  6. O. S. Wolfbeis, "Fiber-optic chemical sensors and biosensors," Anal. Chem. 78, 3859-3873 (2006).
    [CrossRef] [PubMed]
  7. K. Garai, M. Muralidhar, and S. Maiti, "Fiber-optic fluorescence correlation spectrometer," Appl. Opt. 45, 7538-7542 (2006).
    [CrossRef] [PubMed]
  8. K. Garai, R. Sureka, and S. Maiti, "Detecting amyloid-beta aggregation with fiber-based fluorescence correlation spectroscopy," Biophys. J. 92, L55-L57 (2007).
    [CrossRef] [PubMed]
  9. 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).
    [PubMed]
  10. 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).
    [CrossRef] [PubMed]
  11. 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).
    [CrossRef] [PubMed]
  12. 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).
    [CrossRef] [PubMed]
  13. A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, V. Backman, "Experimental confirmation of backscattering enhancement induced by a photonic jet," Appl. Phys. Lett. 89, 221118 (2006).
    [CrossRef]
  14. D. Gerard, J. Wenger, A. Devilez, D. Gachet, B. Stout, N. Bonod, E. Popov, H. Rigneault, "Strong electromagnetic confinement near dielectric microspheres to enhance single-molecule fluorescence," Opt. Express 16, 15297-15303 (2008).
    [CrossRef] [PubMed]
  15. D. Gerard, 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).
    [CrossRef]
  16. A. Devilez, N. Bonod, B. Stout, D. G’erard, J.Wenger, H. Rigneault, and E. Popov, "Three-dimensional subwavelength confinement of photonic nanojets," Opt. Express 17, 2089-2094 (2009).
    [CrossRef] [PubMed]
  17. J. Wenger, D. Gerard, H. Aouani, and H. Rigneault, "Disposable microscope objective lenses for fluorescence correlation spectroscopy using latex microspheres," Anal. Chem. 80, 6800-6804 (2008).
    [CrossRef] [PubMed]
  18. A. Gennerich and D. Schild, "Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model used," Biophys. J. 79, 3294-3306 (2000).
    [CrossRef] [PubMed]
  19. J. Wenger, D. Gerard, N. Bonod, E. Popov, H. Rigneault, J. Dintinger, O. Mahboub and T.W. Ebbesen,"Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures," Opt. Express 16, 3008-3020 (2008).
    [CrossRef] [PubMed]
  20. M. Pitschke, R. Prior, M. Haupt, and D. Riesner, "Detection of single amyloid b -protein aggregates in the cerebrospinal fluid of Alzheimer’s patients by fluorescence correlation spectroscopy," Nature Medicine 4, 832-834 (1998).
    [CrossRef] [PubMed]
  21. N. Opitz, P. J. Rothwell, B. Oeke, and P. Schwille, "Single molecule FCS-based oxygen sensor (O2-FCSensor): a new intrinsically calibrated oxygen sensor utilizing fluorescence correlation spectroscopy (FCS) with single fluorescent molecule detection sensitivity," Sens. Actuators B 96, 460-467 (2003).
    [CrossRef]
  22. F. H. C. Wong, D. S. Banks, A. Abu-Arish, and C. Fradin, "A Molecular Thermometer Based on Fluorescent Protein Blinking," J. Am. Chem. Soc. 129, 10302-10303 (2007).
    [CrossRef] [PubMed]

2009 (3)

2008 (5)

2007 (2)

F. H. C. Wong, D. S. Banks, A. Abu-Arish, and C. Fradin, "A Molecular Thermometer Based on Fluorescent Protein Blinking," J. Am. Chem. Soc. 129, 10302-10303 (2007).
[CrossRef] [PubMed]

K. Garai, R. Sureka, and S. Maiti, "Detecting amyloid-beta aggregation with fiber-based fluorescence correlation spectroscopy," Biophys. J. 92, L55-L57 (2007).
[CrossRef] [PubMed]

2006 (3)

O. S. Wolfbeis, "Fiber-optic chemical sensors and biosensors," Anal. Chem. 78, 3859-3873 (2006).
[CrossRef] [PubMed]

K. Garai, M. Muralidhar, and S. Maiti, "Fiber-optic fluorescence correlation spectrometer," Appl. Opt. 45, 7538-7542 (2006).
[CrossRef] [PubMed]

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

2005 (1)

2004 (1)

2003 (2)

J. R. Epstein and D. R. Walt, "Fluorescence-based fibre optic arrays: a universal platform for sensing," Chem. Soc. Rev. 32, 203-214 (2003).
[CrossRef] [PubMed]

N. Opitz, P. J. Rothwell, B. Oeke, and P. Schwille, "Single molecule FCS-based oxygen sensor (O2-FCSensor): a new intrinsically calibrated oxygen sensor utilizing fluorescence correlation spectroscopy (FCS) with single fluorescent molecule detection sensitivity," Sens. Actuators B 96, 460-467 (2003).
[CrossRef]

2002 (1)

F. Helmchen, "Miniaturization of fluorescence microscopes using fibre optics," Exp. Physiol. 87, 737-745 (2002).
[CrossRef] [PubMed]

2001 (1)

2000 (1)

A. Gennerich and D. Schild, "Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model used," Biophys. J. 79, 3294-3306 (2000).
[CrossRef] [PubMed]

1998 (1)

M. Pitschke, R. Prior, M. Haupt, and D. Riesner, "Detection of single amyloid b -protein aggregates in the cerebrospinal fluid of Alzheimer’s patients by fluorescence correlation spectroscopy," Nature Medicine 4, 832-834 (1998).
[CrossRef] [PubMed]

1997 (1)

S. Maiti, U. Haupts, andW.W.Webb, "Fluorescence correlation spectroscopy: diagnostics for sparse molecules," Proc. Natl. Acad. Sci. U.S.A. 94, 11753-11757 (1997).
[CrossRef] [PubMed]

Abu-Arish, A.

F. H. C. Wong, D. S. Banks, A. Abu-Arish, and C. Fradin, "A Molecular Thermometer Based on Fluorescent Protein Blinking," J. Am. Chem. Soc. 129, 10302-10303 (2007).
[CrossRef] [PubMed]

Aouani, H.

J. Wenger, D. Gerard, H. Aouani, and H. Rigneault, "Disposable microscope objective lenses for fluorescence correlation spectroscopy using latex microspheres," Anal. Chem. 80, 6800-6804 (2008).
[CrossRef] [PubMed]

Backman, V.

Baker, J. R.

Banks, D. S.

F. H. C. Wong, D. S. Banks, A. Abu-Arish, and C. Fradin, "A Molecular Thermometer Based on Fluorescent Protein Blinking," J. Am. Chem. Soc. 129, 10302-10303 (2007).
[CrossRef] [PubMed]

Bonod, N.

Chang, Y.-C.

Chen, Y.

Chen, Z.

Devilez, A.

Epstein, J. R.

J. R. Epstein and D. R. Walt, "Fluorescence-based fibre optic arrays: a universal platform for sensing," Chem. Soc. Rev. 32, 203-214 (2003).
[CrossRef] [PubMed]

Ferrand, P.

Fradin, C.

F. H. C. Wong, D. S. Banks, A. Abu-Arish, and C. Fradin, "A Molecular Thermometer Based on Fluorescent Protein Blinking," J. Am. Chem. Soc. 129, 10302-10303 (2007).
[CrossRef] [PubMed]

Gachet, D.

Garai, K.

K. Garai, R. Sureka, and S. Maiti, "Detecting amyloid-beta aggregation with fiber-based fluorescence correlation spectroscopy," Biophys. J. 92, L55-L57 (2007).
[CrossRef] [PubMed]

K. Garai, M. Muralidhar, and S. Maiti, "Fiber-optic fluorescence correlation spectrometer," Appl. Opt. 45, 7538-7542 (2006).
[CrossRef] [PubMed]

Gennerich, A.

A. Gennerich and D. Schild, "Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model used," Biophys. J. 79, 3294-3306 (2000).
[CrossRef] [PubMed]

Gerard, D.

D. Gerard, J. Wenger, A. Devilez, D. Gachet, B. Stout, N. Bonod, E. Popov, H. Rigneault, "Strong electromagnetic confinement near dielectric microspheres to enhance single-molecule fluorescence," Opt. Express 16, 15297-15303 (2008).
[CrossRef] [PubMed]

J. Wenger, D. Gerard, H. Aouani, and H. Rigneault, "Disposable microscope objective lenses for fluorescence correlation spectroscopy using latex microspheres," Anal. Chem. 80, 6800-6804 (2008).
[CrossRef] [PubMed]

Haupt, M.

M. Pitschke, R. Prior, M. Haupt, and D. Riesner, "Detection of single amyloid b -protein aggregates in the cerebrospinal fluid of Alzheimer’s patients by fluorescence correlation spectroscopy," Nature Medicine 4, 832-834 (1998).
[CrossRef] [PubMed]

Haupts, U.

S. Maiti, U. Haupts, andW.W.Webb, "Fluorescence correlation spectroscopy: diagnostics for sparse molecules," Proc. Natl. Acad. Sci. U.S.A. 94, 11753-11757 (1997).
[CrossRef] [PubMed]

Heifetz, A.

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

Helmchen, F.

F. Helmchen, "Miniaturization of fluorescence microscopes using fibre optics," Exp. Physiol. 87, 737-745 (2002).
[CrossRef] [PubMed]

Huang, K.

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

Li, X.

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

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).
[CrossRef] [PubMed]

Maiti, S.

K. Garai, R. Sureka, and S. Maiti, "Detecting amyloid-beta aggregation with fiber-based fluorescence correlation spectroscopy," Biophys. J. 92, L55-L57 (2007).
[CrossRef] [PubMed]

K. Garai, M. Muralidhar, and S. Maiti, "Fiber-optic fluorescence correlation spectrometer," Appl. Opt. 45, 7538-7542 (2006).
[CrossRef] [PubMed]

S. Maiti, U. Haupts, andW.W.Webb, "Fluorescence correlation spectroscopy: diagnostics for sparse molecules," Proc. Natl. Acad. Sci. U.S.A. 94, 11753-11757 (1997).
[CrossRef] [PubMed]

Muralidhar, M.

Norris, T. B.

Oeke, B.

N. Opitz, P. J. Rothwell, B. Oeke, and P. Schwille, "Single molecule FCS-based oxygen sensor (O2-FCSensor): a new intrinsically calibrated oxygen sensor utilizing fluorescence correlation spectroscopy (FCS) with single fluorescent molecule detection sensitivity," Sens. Actuators B 96, 460-467 (2003).
[CrossRef]

Opitz, N.

N. Opitz, P. J. Rothwell, B. Oeke, and P. Schwille, "Single molecule FCS-based oxygen sensor (O2-FCSensor): a new intrinsically calibrated oxygen sensor utilizing fluorescence correlation spectroscopy (FCS) with single fluorescent molecule detection sensitivity," Sens. Actuators B 96, 460-467 (2003).
[CrossRef]

Pianta, M.

Pitschke, M.

M. Pitschke, R. Prior, M. Haupt, and D. Riesner, "Detection of single amyloid b -protein aggregates in the cerebrospinal fluid of Alzheimer’s patients by fluorescence correlation spectroscopy," Nature Medicine 4, 832-834 (1998).
[CrossRef] [PubMed]

Popov, E.

Prior, R.

M. Pitschke, R. Prior, M. Haupt, and D. Riesner, "Detection of single amyloid b -protein aggregates in the cerebrospinal fluid of Alzheimer’s patients by fluorescence correlation spectroscopy," Nature Medicine 4, 832-834 (1998).
[CrossRef] [PubMed]

Riesner, D.

M. Pitschke, R. Prior, M. Haupt, and D. Riesner, "Detection of single amyloid b -protein aggregates in the cerebrospinal fluid of Alzheimer’s patients by fluorescence correlation spectroscopy," Nature Medicine 4, 832-834 (1998).
[CrossRef] [PubMed]

Rigneault, H.

Rothwell, P. J.

N. Opitz, P. J. Rothwell, B. Oeke, and P. Schwille, "Single molecule FCS-based oxygen sensor (O2-FCSensor): a new intrinsically calibrated oxygen sensor utilizing fluorescence correlation spectroscopy (FCS) with single fluorescent molecule detection sensitivity," Sens. Actuators B 96, 460-467 (2003).
[CrossRef]

Sahakian, A. V.

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

Schild, D.

A. Gennerich and D. Schild, "Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model used," Biophys. J. 79, 3294-3306 (2000).
[CrossRef] [PubMed]

Schwille, P.

N. Opitz, P. J. Rothwell, B. Oeke, and P. Schwille, "Single molecule FCS-based oxygen sensor (O2-FCSensor): a new intrinsically calibrated oxygen sensor utilizing fluorescence correlation spectroscopy (FCS) with single fluorescent molecule detection sensitivity," Sens. Actuators B 96, 460-467 (2003).
[CrossRef]

Stout, B.

Sureka, R.

K. Garai, R. Sureka, and S. Maiti, "Detecting amyloid-beta aggregation with fiber-based fluorescence correlation spectroscopy," Biophys. J. 92, L55-L57 (2007).
[CrossRef] [PubMed]

Taflove, A.

Thomas, T.

Walt, D. R.

J. R. Epstein and D. R. Walt, "Fluorescence-based fibre optic arrays: a universal platform for sensing," Chem. Soc. Rev. 32, 203-214 (2003).
[CrossRef] [PubMed]

Webb, W. W.

Wenger, J.

Wolfbeis, O. S.

O. S. Wolfbeis, "Fiber-optic chemical sensors and biosensors," Anal. Chem. 78, 3859-3873 (2006).
[CrossRef] [PubMed]

Wong, F. H. C.

F. H. C. Wong, D. S. Banks, A. Abu-Arish, and C. Fradin, "A Molecular Thermometer Based on Fluorescent Protein Blinking," J. Am. Chem. Soc. 129, 10302-10303 (2007).
[CrossRef] [PubMed]

Ye, J. Y.

Anal. Chem. (3)

J. Wenger, D. Gerard, H. Aouani, H. Rigneault, B. Lowder, S. Blair, E. Devaux, T. W. Ebbesen, "Nanoaperture-Enhanced Signal-to-Noise Ratio in Fluorescence Correlation Spectroscopy," Anal. Chem. 81, 834-839 (2009).
[CrossRef]

O. S. Wolfbeis, "Fiber-optic chemical sensors and biosensors," Anal. Chem. 78, 3859-3873 (2006).
[CrossRef] [PubMed]

J. Wenger, D. Gerard, H. Aouani, and H. Rigneault, "Disposable microscope objective lenses for fluorescence correlation spectroscopy using latex microspheres," Anal. Chem. 80, 6800-6804 (2008).
[CrossRef] [PubMed]

Appl. Opt. (2)

Appl. Phys. Lett. (1)

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

Biophys. J. (2)

A. Gennerich and D. Schild, "Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model used," Biophys. J. 79, 3294-3306 (2000).
[CrossRef] [PubMed]

K. Garai, R. Sureka, and S. Maiti, "Detecting amyloid-beta aggregation with fiber-based fluorescence correlation spectroscopy," Biophys. J. 92, L55-L57 (2007).
[CrossRef] [PubMed]

Chem. Soc. Rev. (1)

J. R. Epstein and D. R. Walt, "Fluorescence-based fibre optic arrays: a universal platform for sensing," Chem. Soc. Rev. 32, 203-214 (2003).
[CrossRef] [PubMed]

Exp. Physiol. (1)

F. Helmchen, "Miniaturization of fluorescence microscopes using fibre optics," Exp. Physiol. 87, 737-745 (2002).
[CrossRef] [PubMed]

J. Am. Chem. Soc. (1)

F. H. C. Wong, D. S. Banks, A. Abu-Arish, and C. Fradin, "A Molecular Thermometer Based on Fluorescent Protein Blinking," J. Am. Chem. Soc. 129, 10302-10303 (2007).
[CrossRef] [PubMed]

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

Nature Medicine (1)

M. Pitschke, R. Prior, M. Haupt, and D. Riesner, "Detection of single amyloid b -protein aggregates in the cerebrospinal fluid of Alzheimer’s patients by fluorescence correlation spectroscopy," Nature Medicine 4, 832-834 (1998).
[CrossRef] [PubMed]

Opt. Express (7)

J. Wenger, D. Gerard, N. Bonod, E. Popov, H. Rigneault, J. Dintinger, O. Mahboub and T.W. Ebbesen,"Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures," Opt. Express 16, 3008-3020 (2008).
[CrossRef] [PubMed]

A. Devilez, N. Bonod, B. Stout, D. G’erard, J.Wenger, H. Rigneault, and E. Popov, "Three-dimensional subwavelength confinement of photonic nanojets," Opt. Express 17, 2089-2094 (2009).
[CrossRef] [PubMed]

D. Gerard, J. Wenger, A. Devilez, D. Gachet, B. Stout, N. Bonod, E. Popov, H. Rigneault, "Strong electromagnetic confinement near dielectric microspheres to enhance single-molecule fluorescence," Opt. Express 16, 15297-15303 (2008).
[CrossRef] [PubMed]

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).
[PubMed]

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).
[CrossRef] [PubMed]

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).
[CrossRef] [PubMed]

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).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (1)

S. Maiti, U. Haupts, andW.W.Webb, "Fluorescence correlation spectroscopy: diagnostics for sparse molecules," Proc. Natl. Acad. Sci. U.S.A. 94, 11753-11757 (1997).
[CrossRef] [PubMed]

Sens. Actuators B (1)

N. Opitz, P. J. Rothwell, B. Oeke, and P. Schwille, "Single molecule FCS-based oxygen sensor (O2-FCSensor): a new intrinsically calibrated oxygen sensor utilizing fluorescence correlation spectroscopy (FCS) with single fluorescent molecule detection sensitivity," Sens. Actuators B 96, 460-467 (2003).
[CrossRef]

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Figures (4)

Fig. 1.
Fig. 1.

(a) Schematic view of the experimental setup. (b) shows a close-up view of the end of the optical fiber core covered with the microsphere. (c) Electron microscope image of the optical fiber bundle output partly etched with polystyrene microspheres.

Fig. 2.
Fig. 2.

(a) Experimental configuration of the fiber core-clad and microsphere configuration. FDTD-calculated electric field intensity with (b) and without (c) a 2 µm cylinder illuminated by the fundamental fiber mode at λ=633 nm (see text for structure details).

Fig. 3.
Fig. 3.

(a) Scanning image of the bundle: bright areas correspond to the presence of micro-sphere, where autocorrelation function is obtained (b). Analysis of the correlation function in (b) yield: N=284, τd =123µs, s=0.2, nT =0.39, τT =2µs, CRM=1.9kHz, the laser power being set to 0.6 mW at the entrance of the fiber. Without microsphere (dark areas), FCS measurements cannot be performed (c). (d) and (e) are the fluorescence signal traces corresponding respectively to (b) and (c).

Fig. 4.
Fig. 4.

(a) Evolution of the detected number of A647 molecules versus calibrated molecular concentration. (b) Count rate per molecule plotted versus excitation power (circle) and numerical fit (solid line).

Tables (1)

Tables Icon

Table 1. Results of the numerical fits of the FCS data measured with the Zeiss objective and OFM. The observation volume Veff is inferred from the number of molecules N and the dye concentration.

Equations (1)

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g(2)(τ)=1+1N(1BF)2 [1+nTexp(ττT)] 1(1+τ/τd)1+s2ττd

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