Abstract

We describe a new evanescent-wave fluorescence excitation method, ideally suited for imaging of biological samples. The excitation light propagates in a planar optical waveguide, consisting of a thin waveguide core sandwiched between a sample in an aqueous solution and a polymer with a matching refractive index, forming a symmetric cladding environment. This configuration offers clear advantages over other waveguide-excitation methods, such as superior image quality, wide tunability of the evanescent field penetration depth and compatibility with optical fibers. The method is well suited for cell membrane imaging on cells in culture, including cell-cell and cell-matrix interaction, monitoring of surface binding events and similar applications involving aqueous solutions.

© 2009 Optical Society of America

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References

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  1. For a recent review on fluorescence imaging, see Nature Methods, 2 (2005).
  2. D. Axelrod, E.H. Hellen, and R.M. Fulbright, “Total internal reflection fluorescence,” in Topics in Fluorescence Spectroscopy, Volume 3: Biochemical Applications, J.R. Lakowicz, ed. (Plenum Press, New York, 1992), pp. 289–343
  3. K. Kawano and T. Kitoh, Optical waveguide analysis (Wiley, Chichester, 2001).
    [Crossref]
  4. H.M. Grandin, B. Städler, M. Textor, and J. Vörös, “Waveguide excitation fluorescence microscopy: A new tool for sensing and imaging the biointerface,” Biosens. Bioelectron. 21, 1476–1482 (2006).
    [Crossref]
  5. K. Schmitt, K. Oehse, G. Sulz, and C. Hoffmann, “Evanescent field Sensors Based on Tantalum Pentoxide Waveguides-A Review,” Sensors 8, 711–738 (2008).
    [Crossref]
  6. R. Horvath, H.C. Pedersen, N. Skivesen, C. Svanberg, and N.B. Larsen, “Fabrication of reverse symmetry polymer waveguide sensor chips on nanoporous substrates using dip-floating,” J. Micromech. Microeng. 15, 1260–1264 (2005).
    [Crossref]
  7. R. Horvath, K. Cottier, H.C. Pedersen, and J.J. Ramsden, “Multidepth screening of living cells using optical waveguides,” Biosens. Bioelectron. 24, 799–804 (2008).
    [Crossref]
  8. H. Shadpour, H. Musyimi, J. Chen, and S.A. Soper, “Physiochemical properties of various polymer substrates and their effects on microchip electrophoresis performance,” J. Chromatogr. A,  1111, 238–251 (2006).
    [Crossref] [PubMed]
  9. S.C. Brooks, E.R. Locke, and H.D. Soule, “Estrogen Receptor in a Human Cell Line (MCF-7) from Breast Carcinoma,” J. Biol. Chem. 248, 6251–6253 (1973).
    [PubMed]
  10. Release on the Refractive Index of Ordinary Water Substance as a Function of Wavelength, Temperature and Pressure, The International Association for the Properties of Water and Steam (Erlangen, Germany, 1997).
    [PubMed]
  11. R. Bernini, N. Cennamo, A. Minardo, and L. Zeni, “Planar Waveguides for Fluorescence-Based Biosensing: Optimization and Analysis,” IEEE Sens. J. 6, 1218–1226 (2006).
    [Crossref]
  12. W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R.R. Dasari, and M.S. Feld, “Tomographic phase microscopy,” Nat. Methods 4, 717–719 (2007).
    [Crossref] [PubMed]
  13. R. Fiolka, M. Beck, and A. Stemmer, “Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator,” Opt. Lett. 33, 1629–1631 (2008).
    [Crossref] [PubMed]

2008 (3)

K. Schmitt, K. Oehse, G. Sulz, and C. Hoffmann, “Evanescent field Sensors Based on Tantalum Pentoxide Waveguides-A Review,” Sensors 8, 711–738 (2008).
[Crossref]

R. Horvath, K. Cottier, H.C. Pedersen, and J.J. Ramsden, “Multidepth screening of living cells using optical waveguides,” Biosens. Bioelectron. 24, 799–804 (2008).
[Crossref]

R. Fiolka, M. Beck, and A. Stemmer, “Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator,” Opt. Lett. 33, 1629–1631 (2008).
[Crossref] [PubMed]

2007 (1)

W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R.R. Dasari, and M.S. Feld, “Tomographic phase microscopy,” Nat. Methods 4, 717–719 (2007).
[Crossref] [PubMed]

2006 (3)

H.M. Grandin, B. Städler, M. Textor, and J. Vörös, “Waveguide excitation fluorescence microscopy: A new tool for sensing and imaging the biointerface,” Biosens. Bioelectron. 21, 1476–1482 (2006).
[Crossref]

R. Bernini, N. Cennamo, A. Minardo, and L. Zeni, “Planar Waveguides for Fluorescence-Based Biosensing: Optimization and Analysis,” IEEE Sens. J. 6, 1218–1226 (2006).
[Crossref]

H. Shadpour, H. Musyimi, J. Chen, and S.A. Soper, “Physiochemical properties of various polymer substrates and their effects on microchip electrophoresis performance,” J. Chromatogr. A,  1111, 238–251 (2006).
[Crossref] [PubMed]

2005 (1)

R. Horvath, H.C. Pedersen, N. Skivesen, C. Svanberg, and N.B. Larsen, “Fabrication of reverse symmetry polymer waveguide sensor chips on nanoporous substrates using dip-floating,” J. Micromech. Microeng. 15, 1260–1264 (2005).
[Crossref]

1973 (1)

S.C. Brooks, E.R. Locke, and H.D. Soule, “Estrogen Receptor in a Human Cell Line (MCF-7) from Breast Carcinoma,” J. Biol. Chem. 248, 6251–6253 (1973).
[PubMed]

Axelrod, D.

D. Axelrod, E.H. Hellen, and R.M. Fulbright, “Total internal reflection fluorescence,” in Topics in Fluorescence Spectroscopy, Volume 3: Biochemical Applications, J.R. Lakowicz, ed. (Plenum Press, New York, 1992), pp. 289–343

Badizadegan, K.

W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R.R. Dasari, and M.S. Feld, “Tomographic phase microscopy,” Nat. Methods 4, 717–719 (2007).
[Crossref] [PubMed]

Beck, M.

Bernini, R.

R. Bernini, N. Cennamo, A. Minardo, and L. Zeni, “Planar Waveguides for Fluorescence-Based Biosensing: Optimization and Analysis,” IEEE Sens. J. 6, 1218–1226 (2006).
[Crossref]

Brooks, S.C.

S.C. Brooks, E.R. Locke, and H.D. Soule, “Estrogen Receptor in a Human Cell Line (MCF-7) from Breast Carcinoma,” J. Biol. Chem. 248, 6251–6253 (1973).
[PubMed]

Cennamo, N.

R. Bernini, N. Cennamo, A. Minardo, and L. Zeni, “Planar Waveguides for Fluorescence-Based Biosensing: Optimization and Analysis,” IEEE Sens. J. 6, 1218–1226 (2006).
[Crossref]

Chen, J.

H. Shadpour, H. Musyimi, J. Chen, and S.A. Soper, “Physiochemical properties of various polymer substrates and their effects on microchip electrophoresis performance,” J. Chromatogr. A,  1111, 238–251 (2006).
[Crossref] [PubMed]

Choi, W.

W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R.R. Dasari, and M.S. Feld, “Tomographic phase microscopy,” Nat. Methods 4, 717–719 (2007).
[Crossref] [PubMed]

Cottier, K.

R. Horvath, K. Cottier, H.C. Pedersen, and J.J. Ramsden, “Multidepth screening of living cells using optical waveguides,” Biosens. Bioelectron. 24, 799–804 (2008).
[Crossref]

Dasari, R.R.

W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R.R. Dasari, and M.S. Feld, “Tomographic phase microscopy,” Nat. Methods 4, 717–719 (2007).
[Crossref] [PubMed]

Fang-Yen, C.

W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R.R. Dasari, and M.S. Feld, “Tomographic phase microscopy,” Nat. Methods 4, 717–719 (2007).
[Crossref] [PubMed]

Feld, M.S.

W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R.R. Dasari, and M.S. Feld, “Tomographic phase microscopy,” Nat. Methods 4, 717–719 (2007).
[Crossref] [PubMed]

Fiolka, R.

Fulbright, R.M.

D. Axelrod, E.H. Hellen, and R.M. Fulbright, “Total internal reflection fluorescence,” in Topics in Fluorescence Spectroscopy, Volume 3: Biochemical Applications, J.R. Lakowicz, ed. (Plenum Press, New York, 1992), pp. 289–343

Grandin, H.M.

H.M. Grandin, B. Städler, M. Textor, and J. Vörös, “Waveguide excitation fluorescence microscopy: A new tool for sensing and imaging the biointerface,” Biosens. Bioelectron. 21, 1476–1482 (2006).
[Crossref]

Hellen, E.H.

D. Axelrod, E.H. Hellen, and R.M. Fulbright, “Total internal reflection fluorescence,” in Topics in Fluorescence Spectroscopy, Volume 3: Biochemical Applications, J.R. Lakowicz, ed. (Plenum Press, New York, 1992), pp. 289–343

Hoffmann, C.

K. Schmitt, K. Oehse, G. Sulz, and C. Hoffmann, “Evanescent field Sensors Based on Tantalum Pentoxide Waveguides-A Review,” Sensors 8, 711–738 (2008).
[Crossref]

Horvath, R.

R. Horvath, K. Cottier, H.C. Pedersen, and J.J. Ramsden, “Multidepth screening of living cells using optical waveguides,” Biosens. Bioelectron. 24, 799–804 (2008).
[Crossref]

R. Horvath, H.C. Pedersen, N. Skivesen, C. Svanberg, and N.B. Larsen, “Fabrication of reverse symmetry polymer waveguide sensor chips on nanoporous substrates using dip-floating,” J. Micromech. Microeng. 15, 1260–1264 (2005).
[Crossref]

Kawano, K.

K. Kawano and T. Kitoh, Optical waveguide analysis (Wiley, Chichester, 2001).
[Crossref]

Kitoh, T.

K. Kawano and T. Kitoh, Optical waveguide analysis (Wiley, Chichester, 2001).
[Crossref]

Larsen, N.B.

R. Horvath, H.C. Pedersen, N. Skivesen, C. Svanberg, and N.B. Larsen, “Fabrication of reverse symmetry polymer waveguide sensor chips on nanoporous substrates using dip-floating,” J. Micromech. Microeng. 15, 1260–1264 (2005).
[Crossref]

Locke, E.R.

S.C. Brooks, E.R. Locke, and H.D. Soule, “Estrogen Receptor in a Human Cell Line (MCF-7) from Breast Carcinoma,” J. Biol. Chem. 248, 6251–6253 (1973).
[PubMed]

Lue, N.

W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R.R. Dasari, and M.S. Feld, “Tomographic phase microscopy,” Nat. Methods 4, 717–719 (2007).
[Crossref] [PubMed]

Minardo, A.

R. Bernini, N. Cennamo, A. Minardo, and L. Zeni, “Planar Waveguides for Fluorescence-Based Biosensing: Optimization and Analysis,” IEEE Sens. J. 6, 1218–1226 (2006).
[Crossref]

Musyimi, H.

H. Shadpour, H. Musyimi, J. Chen, and S.A. Soper, “Physiochemical properties of various polymer substrates and their effects on microchip electrophoresis performance,” J. Chromatogr. A,  1111, 238–251 (2006).
[Crossref] [PubMed]

Oehse, K.

K. Schmitt, K. Oehse, G. Sulz, and C. Hoffmann, “Evanescent field Sensors Based on Tantalum Pentoxide Waveguides-A Review,” Sensors 8, 711–738 (2008).
[Crossref]

Oh, S.

W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R.R. Dasari, and M.S. Feld, “Tomographic phase microscopy,” Nat. Methods 4, 717–719 (2007).
[Crossref] [PubMed]

Pedersen, H.C.

R. Horvath, K. Cottier, H.C. Pedersen, and J.J. Ramsden, “Multidepth screening of living cells using optical waveguides,” Biosens. Bioelectron. 24, 799–804 (2008).
[Crossref]

R. Horvath, H.C. Pedersen, N. Skivesen, C. Svanberg, and N.B. Larsen, “Fabrication of reverse symmetry polymer waveguide sensor chips on nanoporous substrates using dip-floating,” J. Micromech. Microeng. 15, 1260–1264 (2005).
[Crossref]

Ramsden, J.J.

R. Horvath, K. Cottier, H.C. Pedersen, and J.J. Ramsden, “Multidepth screening of living cells using optical waveguides,” Biosens. Bioelectron. 24, 799–804 (2008).
[Crossref]

Schmitt, K.

K. Schmitt, K. Oehse, G. Sulz, and C. Hoffmann, “Evanescent field Sensors Based on Tantalum Pentoxide Waveguides-A Review,” Sensors 8, 711–738 (2008).
[Crossref]

Shadpour, H.

H. Shadpour, H. Musyimi, J. Chen, and S.A. Soper, “Physiochemical properties of various polymer substrates and their effects on microchip electrophoresis performance,” J. Chromatogr. A,  1111, 238–251 (2006).
[Crossref] [PubMed]

Skivesen, N.

R. Horvath, H.C. Pedersen, N. Skivesen, C. Svanberg, and N.B. Larsen, “Fabrication of reverse symmetry polymer waveguide sensor chips on nanoporous substrates using dip-floating,” J. Micromech. Microeng. 15, 1260–1264 (2005).
[Crossref]

Soper, S.A.

H. Shadpour, H. Musyimi, J. Chen, and S.A. Soper, “Physiochemical properties of various polymer substrates and their effects on microchip electrophoresis performance,” J. Chromatogr. A,  1111, 238–251 (2006).
[Crossref] [PubMed]

Soule, H.D.

S.C. Brooks, E.R. Locke, and H.D. Soule, “Estrogen Receptor in a Human Cell Line (MCF-7) from Breast Carcinoma,” J. Biol. Chem. 248, 6251–6253 (1973).
[PubMed]

Städler, B.

H.M. Grandin, B. Städler, M. Textor, and J. Vörös, “Waveguide excitation fluorescence microscopy: A new tool for sensing and imaging the biointerface,” Biosens. Bioelectron. 21, 1476–1482 (2006).
[Crossref]

Stemmer, A.

Sulz, G.

K. Schmitt, K. Oehse, G. Sulz, and C. Hoffmann, “Evanescent field Sensors Based on Tantalum Pentoxide Waveguides-A Review,” Sensors 8, 711–738 (2008).
[Crossref]

Svanberg, C.

R. Horvath, H.C. Pedersen, N. Skivesen, C. Svanberg, and N.B. Larsen, “Fabrication of reverse symmetry polymer waveguide sensor chips on nanoporous substrates using dip-floating,” J. Micromech. Microeng. 15, 1260–1264 (2005).
[Crossref]

Textor, M.

H.M. Grandin, B. Städler, M. Textor, and J. Vörös, “Waveguide excitation fluorescence microscopy: A new tool for sensing and imaging the biointerface,” Biosens. Bioelectron. 21, 1476–1482 (2006).
[Crossref]

Vörös, J.

H.M. Grandin, B. Städler, M. Textor, and J. Vörös, “Waveguide excitation fluorescence microscopy: A new tool for sensing and imaging the biointerface,” Biosens. Bioelectron. 21, 1476–1482 (2006).
[Crossref]

Zeni, L.

R. Bernini, N. Cennamo, A. Minardo, and L. Zeni, “Planar Waveguides for Fluorescence-Based Biosensing: Optimization and Analysis,” IEEE Sens. J. 6, 1218–1226 (2006).
[Crossref]

Biosens. Bioelectron. (2)

H.M. Grandin, B. Städler, M. Textor, and J. Vörös, “Waveguide excitation fluorescence microscopy: A new tool for sensing and imaging the biointerface,” Biosens. Bioelectron. 21, 1476–1482 (2006).
[Crossref]

R. Horvath, K. Cottier, H.C. Pedersen, and J.J. Ramsden, “Multidepth screening of living cells using optical waveguides,” Biosens. Bioelectron. 24, 799–804 (2008).
[Crossref]

IEEE Sens. J. (1)

R. Bernini, N. Cennamo, A. Minardo, and L. Zeni, “Planar Waveguides for Fluorescence-Based Biosensing: Optimization and Analysis,” IEEE Sens. J. 6, 1218–1226 (2006).
[Crossref]

J. Biol. Chem. (1)

S.C. Brooks, E.R. Locke, and H.D. Soule, “Estrogen Receptor in a Human Cell Line (MCF-7) from Breast Carcinoma,” J. Biol. Chem. 248, 6251–6253 (1973).
[PubMed]

J. Chromatogr. A (1)

H. Shadpour, H. Musyimi, J. Chen, and S.A. Soper, “Physiochemical properties of various polymer substrates and their effects on microchip electrophoresis performance,” J. Chromatogr. A,  1111, 238–251 (2006).
[Crossref] [PubMed]

J. Micromech. Microeng. (1)

R. Horvath, H.C. Pedersen, N. Skivesen, C. Svanberg, and N.B. Larsen, “Fabrication of reverse symmetry polymer waveguide sensor chips on nanoporous substrates using dip-floating,” J. Micromech. Microeng. 15, 1260–1264 (2005).
[Crossref]

Nat. Methods (1)

W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R.R. Dasari, and M.S. Feld, “Tomographic phase microscopy,” Nat. Methods 4, 717–719 (2007).
[Crossref] [PubMed]

Opt. Lett. (1)

Sensors (1)

K. Schmitt, K. Oehse, G. Sulz, and C. Hoffmann, “Evanescent field Sensors Based on Tantalum Pentoxide Waveguides-A Review,” Sensors 8, 711–738 (2008).
[Crossref]

Other (4)

For a recent review on fluorescence imaging, see Nature Methods, 2 (2005).

D. Axelrod, E.H. Hellen, and R.M. Fulbright, “Total internal reflection fluorescence,” in Topics in Fluorescence Spectroscopy, Volume 3: Biochemical Applications, J.R. Lakowicz, ed. (Plenum Press, New York, 1992), pp. 289–343

K. Kawano and T. Kitoh, Optical waveguide analysis (Wiley, Chichester, 2001).
[Crossref]

Release on the Refractive Index of Ordinary Water Substance as a Function of Wavelength, Temperature and Pressure, The International Association for the Properties of Water and Steam (Erlangen, Germany, 1997).
[PubMed]

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Figures (5)

Fig. 1.
Fig. 1.

Methods of evanescent-wave excitation. (a) Total internal reflection and (b-d) waveguide-excitation using a substrate with a refractive index that is (b) higher, (c) lower and (d) approximately equal to the sample solution. Arrows indicate the preferred method of coupling of excitation light to the evanescent mode, being either incident from within the substrate, onto a surface grating or end-fire coupled directly into the waveguide.

Fig. 2.
Fig. 2.

Schematic illustration of the experimental configuration. Excitation light was provided by a supercontinuum source (SuperK) and filtered using an acousto-optic tunable filter (AOTF). The light was coupled through a single-mode optical fiber into the planar waveguide. Fluorescence excited by the evanescent tail of the bound mode was picked up by the microscope objective. Transmission through the waveguide was monitored using a second objective to ensure optimum coupling.

Fig. 3.
Fig. 3.

Calculated parameters for waveguide chips. Effective index of the waveguide modes (bottom) and the corresponding penetration depths into pure water (top) for common fluorophore excitation wavelength. The limit for live-cell imaging and the onset of multimode behavior for the Cytop-PMMA chip structure are indicated.

Fig. 4.
Fig. 4.

Comparison of waveguide-excitation and epi-fluorescence imaging. a) Cluster of MFC7 breast cancer cells labeled with antibody against the transmembrane protein E-cadherin imaged using symmetric-waveguide-excitation fluorescence microscopy. The estimated penetration depth of the excitation light is about 185 nm into the cells. Arrows indicate dark bands caused by scattering from particles in the waveguide film. b) Same cluster imaged using a standard fluorescence microscope (dashed line indicates field of view). c) Detail of the evanescent-wave image (pixel size corresponds to 200 nm = 0.7r). d) Detail of the epi-fluorescence image (pixel size corresponds to 50 nm = 0.18r after downsampling). Scale bar in all images represents 10 μm.

Fig. 5.
Fig. 5.

Comparison of images obtained using waveguide-excitation and laser scanning microscopy. (a) Symmetric waveguide excitation with a calculated penetration depth of 185 nm for the excitation light. (b) Same cell cluster imaged using a laser scanning microscope with a large field of view. In the former case, the signal in the near-surface region is significantly enhanced and imaging time is reduced by an order of magnitude. Scale bars represent 10 μm in both images.

Equations (1)

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d = λ 2 π 1 n eff 2 n sample 2

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