Abstract

We present a polarized goniofluorimeter designed to measure the observation-angle and polarization-dependent intensity emitted by a group of surface-bound fluorescent molecules. We studied two types of surface bonding: In one case, dyes were adsorbed into the surface by spin coating, and in the other, dyes were covalently immobilized to DNA strands. Fluorescent dyes consisted of Cy3 and Alexa546. The substrate was a silicon wafer bearing a silicon dioxide layer. The different samples presented a wide panel of reproducible experimental behavior. By confronting experimental behavior with theory and simulation, we can explain these differences as directly linked to the mean orientation of fluorophores with respect to the surface.

© 2002 Optical Society of America

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References

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  1. E. H. Hellen, D. Axelrod, “Fluorescence emission at dielectric and metal-film interfaces,” J. Opt. Soc. Am. B 4, 337–350 (1987).
    [CrossRef]
  2. R. R. Chance, A. Prock, R. Silbey, “Molecular fluorescence and energy transfer near interfaces,” Adv. Chem. Phys. 37, 1–65 (1978).
    [CrossRef]
  3. W. Lukosz, “Light emission by magnetic and electric dipoles close to a plane dielectric interface. III. Radiation patterns of dipoles with arbitrary orientation,” J. Opt. Soc. Am. 69, 1495–1503 (1979).
    [CrossRef]
  4. J. Enderlein, T. Ruckstuhl, S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt. 38, 724–732 (1999).
    [CrossRef]
  5. P. L. Edminston, J. E. Lee, L. L. Wood, S. S. Saavedra, “Dipole orientation distributions in Langmuir-Blodgett films by planar waveguide linear dichroism and fluorescence anisotropy,” J. Phys. Chem. 100, 775–784 (1996).
    [CrossRef]
  6. A. Lambacher, P. Fromherz, “Orientation of hemicyanine dye in lipid membrane measured by fluorescence interferometry on a silicon chip,” J. Phys. Chem. B 105, 343–346 (2001).
    [CrossRef]
  7. M. Lieberherr, C. Fatinger, W. Lukosz, “Optical environment dependent effects on the fluorescence of submonomolecular dye layers on interfaces,” Surf. Sci. 189/190, 954–959 (1987).
    [CrossRef]
  8. S. Garoff, R. B. Stephens, C. D. Hanson, G. K. Sorenson, “Surface interactions of adsorbed molecules as probed by their optical properties,” Opt. Commun. 41, 257–262 (1982).
    [CrossRef]
  9. N. Thomson, H. McConnell, T. Burghardt, “Order in supported phospholipid monolayers detected by the dichroism of fluorescence excited with polarized evanescent illumination,” Biophys. J. 46, 739–747 (1984).
    [CrossRef]
  10. A. G. T. Ruiter, J. A. Veerman, M. F. Garcia-Parajo, N. F. van Hulst, “Single molecule rotational and translational diffusion observed by near-field scanning optical microscopy,” J. Phys. Chem. A 101, 7318–7323 (1997).
    [CrossRef]
  11. T. Ha, T. A. Laurence, D. S. Chemla, S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B 103, 6839–6850 (1999).
    [CrossRef]
  12. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum, New York, 1999).
    [CrossRef]

2001 (1)

A. Lambacher, P. Fromherz, “Orientation of hemicyanine dye in lipid membrane measured by fluorescence interferometry on a silicon chip,” J. Phys. Chem. B 105, 343–346 (2001).
[CrossRef]

1999 (2)

T. Ha, T. A. Laurence, D. S. Chemla, S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B 103, 6839–6850 (1999).
[CrossRef]

J. Enderlein, T. Ruckstuhl, S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt. 38, 724–732 (1999).
[CrossRef]

1997 (1)

A. G. T. Ruiter, J. A. Veerman, M. F. Garcia-Parajo, N. F. van Hulst, “Single molecule rotational and translational diffusion observed by near-field scanning optical microscopy,” J. Phys. Chem. A 101, 7318–7323 (1997).
[CrossRef]

1996 (1)

P. L. Edminston, J. E. Lee, L. L. Wood, S. S. Saavedra, “Dipole orientation distributions in Langmuir-Blodgett films by planar waveguide linear dichroism and fluorescence anisotropy,” J. Phys. Chem. 100, 775–784 (1996).
[CrossRef]

1987 (2)

M. Lieberherr, C. Fatinger, W. Lukosz, “Optical environment dependent effects on the fluorescence of submonomolecular dye layers on interfaces,” Surf. Sci. 189/190, 954–959 (1987).
[CrossRef]

E. H. Hellen, D. Axelrod, “Fluorescence emission at dielectric and metal-film interfaces,” J. Opt. Soc. Am. B 4, 337–350 (1987).
[CrossRef]

1984 (1)

N. Thomson, H. McConnell, T. Burghardt, “Order in supported phospholipid monolayers detected by the dichroism of fluorescence excited with polarized evanescent illumination,” Biophys. J. 46, 739–747 (1984).
[CrossRef]

1982 (1)

S. Garoff, R. B. Stephens, C. D. Hanson, G. K. Sorenson, “Surface interactions of adsorbed molecules as probed by their optical properties,” Opt. Commun. 41, 257–262 (1982).
[CrossRef]

1979 (1)

1978 (1)

R. R. Chance, A. Prock, R. Silbey, “Molecular fluorescence and energy transfer near interfaces,” Adv. Chem. Phys. 37, 1–65 (1978).
[CrossRef]

Axelrod, D.

Burghardt, T.

N. Thomson, H. McConnell, T. Burghardt, “Order in supported phospholipid monolayers detected by the dichroism of fluorescence excited with polarized evanescent illumination,” Biophys. J. 46, 739–747 (1984).
[CrossRef]

Chance, R. R.

R. R. Chance, A. Prock, R. Silbey, “Molecular fluorescence and energy transfer near interfaces,” Adv. Chem. Phys. 37, 1–65 (1978).
[CrossRef]

Chemla, D. S.

T. Ha, T. A. Laurence, D. S. Chemla, S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B 103, 6839–6850 (1999).
[CrossRef]

Edminston, P. L.

P. L. Edminston, J. E. Lee, L. L. Wood, S. S. Saavedra, “Dipole orientation distributions in Langmuir-Blodgett films by planar waveguide linear dichroism and fluorescence anisotropy,” J. Phys. Chem. 100, 775–784 (1996).
[CrossRef]

Enderlein, J.

Fatinger, C.

M. Lieberherr, C. Fatinger, W. Lukosz, “Optical environment dependent effects on the fluorescence of submonomolecular dye layers on interfaces,” Surf. Sci. 189/190, 954–959 (1987).
[CrossRef]

Fromherz, P.

A. Lambacher, P. Fromherz, “Orientation of hemicyanine dye in lipid membrane measured by fluorescence interferometry on a silicon chip,” J. Phys. Chem. B 105, 343–346 (2001).
[CrossRef]

Garcia-Parajo, M. F.

A. G. T. Ruiter, J. A. Veerman, M. F. Garcia-Parajo, N. F. van Hulst, “Single molecule rotational and translational diffusion observed by near-field scanning optical microscopy,” J. Phys. Chem. A 101, 7318–7323 (1997).
[CrossRef]

Garoff, S.

S. Garoff, R. B. Stephens, C. D. Hanson, G. K. Sorenson, “Surface interactions of adsorbed molecules as probed by their optical properties,” Opt. Commun. 41, 257–262 (1982).
[CrossRef]

Ha, T.

T. Ha, T. A. Laurence, D. S. Chemla, S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B 103, 6839–6850 (1999).
[CrossRef]

Hanson, C. D.

S. Garoff, R. B. Stephens, C. D. Hanson, G. K. Sorenson, “Surface interactions of adsorbed molecules as probed by their optical properties,” Opt. Commun. 41, 257–262 (1982).
[CrossRef]

Hellen, E. H.

Lakowicz, J. R.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum, New York, 1999).
[CrossRef]

Lambacher, A.

A. Lambacher, P. Fromherz, “Orientation of hemicyanine dye in lipid membrane measured by fluorescence interferometry on a silicon chip,” J. Phys. Chem. B 105, 343–346 (2001).
[CrossRef]

Laurence, T. A.

T. Ha, T. A. Laurence, D. S. Chemla, S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B 103, 6839–6850 (1999).
[CrossRef]

Lee, J. E.

P. L. Edminston, J. E. Lee, L. L. Wood, S. S. Saavedra, “Dipole orientation distributions in Langmuir-Blodgett films by planar waveguide linear dichroism and fluorescence anisotropy,” J. Phys. Chem. 100, 775–784 (1996).
[CrossRef]

Lieberherr, M.

M. Lieberherr, C. Fatinger, W. Lukosz, “Optical environment dependent effects on the fluorescence of submonomolecular dye layers on interfaces,” Surf. Sci. 189/190, 954–959 (1987).
[CrossRef]

Lukosz, W.

M. Lieberherr, C. Fatinger, W. Lukosz, “Optical environment dependent effects on the fluorescence of submonomolecular dye layers on interfaces,” Surf. Sci. 189/190, 954–959 (1987).
[CrossRef]

W. Lukosz, “Light emission by magnetic and electric dipoles close to a plane dielectric interface. III. Radiation patterns of dipoles with arbitrary orientation,” J. Opt. Soc. Am. 69, 1495–1503 (1979).
[CrossRef]

McConnell, H.

N. Thomson, H. McConnell, T. Burghardt, “Order in supported phospholipid monolayers detected by the dichroism of fluorescence excited with polarized evanescent illumination,” Biophys. J. 46, 739–747 (1984).
[CrossRef]

Prock, A.

R. R. Chance, A. Prock, R. Silbey, “Molecular fluorescence and energy transfer near interfaces,” Adv. Chem. Phys. 37, 1–65 (1978).
[CrossRef]

Ruckstuhl, T.

Ruiter, A. G. T.

A. G. T. Ruiter, J. A. Veerman, M. F. Garcia-Parajo, N. F. van Hulst, “Single molecule rotational and translational diffusion observed by near-field scanning optical microscopy,” J. Phys. Chem. A 101, 7318–7323 (1997).
[CrossRef]

Saavedra, S. S.

P. L. Edminston, J. E. Lee, L. L. Wood, S. S. Saavedra, “Dipole orientation distributions in Langmuir-Blodgett films by planar waveguide linear dichroism and fluorescence anisotropy,” J. Phys. Chem. 100, 775–784 (1996).
[CrossRef]

Seeger, S.

Silbey, R.

R. R. Chance, A. Prock, R. Silbey, “Molecular fluorescence and energy transfer near interfaces,” Adv. Chem. Phys. 37, 1–65 (1978).
[CrossRef]

Sorenson, G. K.

S. Garoff, R. B. Stephens, C. D. Hanson, G. K. Sorenson, “Surface interactions of adsorbed molecules as probed by their optical properties,” Opt. Commun. 41, 257–262 (1982).
[CrossRef]

Stephens, R. B.

S. Garoff, R. B. Stephens, C. D. Hanson, G. K. Sorenson, “Surface interactions of adsorbed molecules as probed by their optical properties,” Opt. Commun. 41, 257–262 (1982).
[CrossRef]

Thomson, N.

N. Thomson, H. McConnell, T. Burghardt, “Order in supported phospholipid monolayers detected by the dichroism of fluorescence excited with polarized evanescent illumination,” Biophys. J. 46, 739–747 (1984).
[CrossRef]

van Hulst, N. F.

A. G. T. Ruiter, J. A. Veerman, M. F. Garcia-Parajo, N. F. van Hulst, “Single molecule rotational and translational diffusion observed by near-field scanning optical microscopy,” J. Phys. Chem. A 101, 7318–7323 (1997).
[CrossRef]

Veerman, J. A.

A. G. T. Ruiter, J. A. Veerman, M. F. Garcia-Parajo, N. F. van Hulst, “Single molecule rotational and translational diffusion observed by near-field scanning optical microscopy,” J. Phys. Chem. A 101, 7318–7323 (1997).
[CrossRef]

Weiss, S.

T. Ha, T. A. Laurence, D. S. Chemla, S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B 103, 6839–6850 (1999).
[CrossRef]

Wood, L. L.

P. L. Edminston, J. E. Lee, L. L. Wood, S. S. Saavedra, “Dipole orientation distributions in Langmuir-Blodgett films by planar waveguide linear dichroism and fluorescence anisotropy,” J. Phys. Chem. 100, 775–784 (1996).
[CrossRef]

Adv. Chem. Phys. (1)

R. R. Chance, A. Prock, R. Silbey, “Molecular fluorescence and energy transfer near interfaces,” Adv. Chem. Phys. 37, 1–65 (1978).
[CrossRef]

Appl. Opt. (1)

Biophys. J. (1)

N. Thomson, H. McConnell, T. Burghardt, “Order in supported phospholipid monolayers detected by the dichroism of fluorescence excited with polarized evanescent illumination,” Biophys. J. 46, 739–747 (1984).
[CrossRef]

J. Opt. Soc. Am. (1)

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

J. Phys. Chem. (1)

P. L. Edminston, J. E. Lee, L. L. Wood, S. S. Saavedra, “Dipole orientation distributions in Langmuir-Blodgett films by planar waveguide linear dichroism and fluorescence anisotropy,” J. Phys. Chem. 100, 775–784 (1996).
[CrossRef]

J. Phys. Chem. A (1)

A. G. T. Ruiter, J. A. Veerman, M. F. Garcia-Parajo, N. F. van Hulst, “Single molecule rotational and translational diffusion observed by near-field scanning optical microscopy,” J. Phys. Chem. A 101, 7318–7323 (1997).
[CrossRef]

J. Phys. Chem. B (2)

T. Ha, T. A. Laurence, D. S. Chemla, S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B 103, 6839–6850 (1999).
[CrossRef]

A. Lambacher, P. Fromherz, “Orientation of hemicyanine dye in lipid membrane measured by fluorescence interferometry on a silicon chip,” J. Phys. Chem. B 105, 343–346 (2001).
[CrossRef]

Opt. Commun. (1)

S. Garoff, R. B. Stephens, C. D. Hanson, G. K. Sorenson, “Surface interactions of adsorbed molecules as probed by their optical properties,” Opt. Commun. 41, 257–262 (1982).
[CrossRef]

Surf. Sci. (1)

M. Lieberherr, C. Fatinger, W. Lukosz, “Optical environment dependent effects on the fluorescence of submonomolecular dye layers on interfaces,” Surf. Sci. 189/190, 954–959 (1987).
[CrossRef]

Other (1)

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum, New York, 1999).
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

Experimental setup: polarized goniofluorimeter. Gray broken lines, excitation wavelength (543.5 nm); black solid line, fluorescence wavelength (580 nm); E S , E P , directions of the electric field vector for S and P polarization, respectively. Fluorescence detection apparatus: polarizer + interferential filter + photomultiplier + PC connection.

Fig. 2
Fig. 2

Experimental set of fluorescence angular patterns measured for two different samples: gray curve, PVA-Cy3; black curve, PVA-Alexa546. For I αβ, α is the incident polarization, β is the fluorescence polarization; the a* subscript indicates no polarizer.

Fig. 3
Fig. 3

Experimental set of fluorescence angular patterns measured for two different samples: gray curves, DNA-Cy3; black curves, DNA-Alexa546. For I αβ, α is the incident polarization, β is the fluorescence polarization; the a* subscript indicates no polarizer.

Fig. 4
Fig. 4

Experimental results versus simulation obtained for the PVA-Cy3 sample: +, experimental results; gray curve, simulation. For I αβ, α is the incident polarization, β is the fluorescence polarization; the a* subscript indicates no polarizer.

Tables (1)

Tables Icon

Table 1 Fit Parameters and Accuracy for the Four Samples That were Studied

Equations (10)

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

θa,ϕa,z |μa·Eexci|2Sθ, ϕCθa, ϕa, zdθa×sinθadϕadz,
|μa·ESexci|2  sin2θacos2ϕa×|1+rsaθincexpjψa|2,
|μa·EPexci|2  sin2θasin2ϕacos2θinc×|1-rpaθincexpjψa|2+cos2θasin2θinc×|1+rpaθincexpjψa|2+sinθacosθasinϕasin2θinc×1-|rpaθinc|2,
Sθ, ϕ  Ssθ, ϕ+Spθ, ϕ,
Ssθ, ϕ=Ren1sin2θesin2ϕe-ϕ×|1+rseθexpjψe|2,
Spθ, ϕ=Ren1|sinθecosϕe-ϕcosθ×1-rpeθexpjψe-cosθesinθ×1+rpeθexpjψe|2,
Issθ, ϕ  θa,ϕa |μa·Esexci|2Ssθ, ϕCθasinθadθadϕa  θa,ϕaRen1sin2θacos2ϕa|1+rsaθincexpjψa|2×sin2θecos2ϕe|1+rseθexpjψe|2Cθasinθadθadϕa,
Ispθ, ϕ  θa,ϕa |μa · Esexci|2Spθ, ϕCθasinθadθadϕa θa,ϕaRen1sin2θacos2ϕa|1+rsaθincexpjψa|2×|sinθesinϕecosθ1-rpeθexpjψe-cosθesinθ1+rpeθexpjψe|2Cθasinθadθadϕa.
IspIss=ϕa=02π cos2ϕasin2ϕa+Δϕdϕaϕa=02π cos2ϕacos2ϕa+Δϕdϕa,
Issθ  ϕaRen1cos2ϕacos2Δϕ+ϕadϕa×θasin3θasin2θeCθadθa×|1+rsaθincexpjψa|2×|1+rseθexpjψe|2.

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