Abstract

We propose a new type of total internal reflection fluorescence microscopy (TIRFM) called scanning TIRFM (STIRFM) that uses a focused ring-beam illumination and a high-numerical-aperture objective (NA = 1.65). The evanescent field produced by the STIRFM is focused laterally, producing a small excitation volume that can induce a nonlinear effect such as two-photon absorption. Experimental images of CdSe quantum dot nanocrystals and Rhodamine 6G-doped microbeads show that good lateral and axial resolutions are achieved with the current setup. The theoretical simulation of the focal spot produced in STIRFM geometry shows that the focused evanescent field is split into two peaks because of the depolarization effect of a high numerical-aperture objective lens. However, the point-spread function analysis of both one-photon and two-photon excitation cases shows that the detection of the focus-splitting effect is dependent on the detection pinhole size. The effect of pinhole size on image formation is theoretically investigated and confirmed experimentally with the nanocrystal images.

© 2004 Optical Society of America

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  1. H. Watarai, F. Funaki, “Total internal reflection fluorescence measurements of protonation equilibria of Rhodamine B and Octadecylrhodamine B at a toluene/water interface,” Langmuir 12, 6717–6720 (1996).
    [CrossRef]
  2. M. Toriumi, M. Yanagimachi, “Time-resolved total-internal-reflection fluorescence spectroscopy and its applications to solid/polymer interface layers,” in Microchemistry, Spectroscopy and Chemistry in Small Domains, H. Masuhara, F. De Schryver, N. Kitamura, N. Tamai, eds. (Elsevier, New York, 1994), pp. 257–268.
  3. K. Stock, R. Sailer, W. S. L. Strauss, R. Pavesi, M. Lyttek, H. Emmert, H. Schneckenburger, “Total internal reflection fluorescence spectroscopy and microscopy (TIRFS/TIRFM) in cell biology and photobiology,” in Fluorescence Microscopy and Fluorescent Probes, A. Kotyk, ed. (Espero Publishing, Prague, 1999), Vol. 3, pp. 67–79.
  4. R. Sailer, K. Stock, W. S. L. Strauss, M. Lyttek, H. Schneckenburger, “Total internal reflection fluorescence microscopy (TIRFM) of acridine orange in single cells,” Endocytobiosis & Cell Res. 14, 129–136 (2001).
  5. W. P. Ambrose, P. M. Goodwin, J. P. Nolan, “Single-molecule detection with total internal reflection excitation: comparing signal-to-background and total signals in different geometries,” Cytometry 36, 224–231 (1999).
    [CrossRef] [PubMed]
  6. M. F. Paige, E. J. Bjerneld, W. E. Moerner, “A comparison of through-the-objective total internal reflection microscopy and epi-fluorescence microscopy for single-molecule fluorescence imaging,” Single Mol. 2, 191–201 (2001).
    [CrossRef]
  7. M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235, 47–53 (1997).
    [CrossRef] [PubMed]
  8. T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, T. Yanagida, “Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution,” Nature 374, 555–559 (1995).
    [CrossRef] [PubMed]
  9. R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, T. Yanagida, “Direct observation of single kinesin molecules moving along microtubules,” Nature 380, 451–453 (1996).
    [CrossRef] [PubMed]
  10. T. Wazawa, Y. Ishii, T. Funatsu, T. Yanagida, “Spectral fluctuation of a single fluophore conjugated to a protein molecule,” Biophys. J. 78, 1561–1569 (2000).
    [CrossRef] [PubMed]
  11. Y. Sako, S. Minoghchi, T. Yanagida, “Single-molecule imaging of EGFR signaling on the surface of living cells,” Nat. Cell Biol. 2, 168–172 (2000).
    [CrossRef] [PubMed]
  12. R. M. Dickson, D. J. Norris, Y. L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–968 (1996).
    [CrossRef] [PubMed]
  13. B. Sick, B. Hecht, L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85, 4482–4485 (2000).
    [CrossRef] [PubMed]
  14. B. Sick, B. Hecht, U. P. Wild, L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microsc. 202, 365–373 (2000).
    [CrossRef]
  15. L. Novotny, M. R. Beversluis, K. S. Youngworth, T. G. Brown, “Longitudinal field modes probed by single molecules,” Phys. Rev. Lett. 86, 5251–5254 (2001).
    [CrossRef] [PubMed]
  16. P. Török, P. Varga, Z. Laczik, G. R. Booker, “Electromagnetic diffraction of light focused through a planar interface between materials of mismatched refractive indices: an integral representation,” J. Opt. Soc. Am. A 12, 325–332 (1995).
    [CrossRef]
  17. M. Gu, Advanced Optical Imaging Theory (Springer, Heidelberg, 2003).
  18. J. W. M. Chon, X. Gan, M. Gu, “Splitting of the focal spot of a high numerical-aperture objective in free space,” Appl. Phys. Lett. 81, 1576–1578 (2002).
    [CrossRef]
  19. M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopy (World Scientific, Singapore, 1996).
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    [CrossRef]
  21. C. B. Murray, D. J. Norris, M. G. Bawendi, “Synthesis and characterization of nearly monodisperse CdE (E = S, Se, Te) semiconductor nanocrystallites,” J. Am. Chem. Soc. 115, 8706–8715 (1993).
    [CrossRef]
  22. S. A. Empedocles, R. Neuhauser, M. G. Bawendi, “Three-dimensional orientation measurements of symmetric single chromophores using polarization microscopy,” Nature 399, 126–130 (1999).
    [CrossRef]
  23. G. Toraldo di Francia, “Super-gain antennas and optical resolving power,” Nuovo Cimento Suppl. 9, 426–428 (1952).
    [CrossRef]

2002

J. W. M. Chon, X. Gan, M. Gu, “Splitting of the focal spot of a high numerical-aperture objective in free space,” Appl. Phys. Lett. 81, 1576–1578 (2002).
[CrossRef]

2001

L. Novotny, M. R. Beversluis, K. S. Youngworth, T. G. Brown, “Longitudinal field modes probed by single molecules,” Phys. Rev. Lett. 86, 5251–5254 (2001).
[CrossRef] [PubMed]

R. Sailer, K. Stock, W. S. L. Strauss, M. Lyttek, H. Schneckenburger, “Total internal reflection fluorescence microscopy (TIRFM) of acridine orange in single cells,” Endocytobiosis & Cell Res. 14, 129–136 (2001).

M. F. Paige, E. J. Bjerneld, W. E. Moerner, “A comparison of through-the-objective total internal reflection microscopy and epi-fluorescence microscopy for single-molecule fluorescence imaging,” Single Mol. 2, 191–201 (2001).
[CrossRef]

2000

T. Wazawa, Y. Ishii, T. Funatsu, T. Yanagida, “Spectral fluctuation of a single fluophore conjugated to a protein molecule,” Biophys. J. 78, 1561–1569 (2000).
[CrossRef] [PubMed]

Y. Sako, S. Minoghchi, T. Yanagida, “Single-molecule imaging of EGFR signaling on the surface of living cells,” Nat. Cell Biol. 2, 168–172 (2000).
[CrossRef] [PubMed]

B. Sick, B. Hecht, L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85, 4482–4485 (2000).
[CrossRef] [PubMed]

B. Sick, B. Hecht, U. P. Wild, L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microsc. 202, 365–373 (2000).
[CrossRef]

1999

S. A. Empedocles, R. Neuhauser, M. G. Bawendi, “Three-dimensional orientation measurements of symmetric single chromophores using polarization microscopy,” Nature 399, 126–130 (1999).
[CrossRef]

W. P. Ambrose, P. M. Goodwin, J. P. Nolan, “Single-molecule detection with total internal reflection excitation: comparing signal-to-background and total signals in different geometries,” Cytometry 36, 224–231 (1999).
[CrossRef] [PubMed]

1998

1997

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235, 47–53 (1997).
[CrossRef] [PubMed]

1996

R. M. Dickson, D. J. Norris, Y. L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–968 (1996).
[CrossRef] [PubMed]

H. Watarai, F. Funaki, “Total internal reflection fluorescence measurements of protonation equilibria of Rhodamine B and Octadecylrhodamine B at a toluene/water interface,” Langmuir 12, 6717–6720 (1996).
[CrossRef]

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, T. Yanagida, “Direct observation of single kinesin molecules moving along microtubules,” Nature 380, 451–453 (1996).
[CrossRef] [PubMed]

1995

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, T. Yanagida, “Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution,” Nature 374, 555–559 (1995).
[CrossRef] [PubMed]

P. Török, P. Varga, Z. Laczik, G. R. Booker, “Electromagnetic diffraction of light focused through a planar interface between materials of mismatched refractive indices: an integral representation,” J. Opt. Soc. Am. A 12, 325–332 (1995).
[CrossRef]

1993

C. B. Murray, D. J. Norris, M. G. Bawendi, “Synthesis and characterization of nearly monodisperse CdE (E = S, Se, Te) semiconductor nanocrystallites,” J. Am. Chem. Soc. 115, 8706–8715 (1993).
[CrossRef]

1952

G. Toraldo di Francia, “Super-gain antennas and optical resolving power,” Nuovo Cimento Suppl. 9, 426–428 (1952).
[CrossRef]

Ambrose, W. P.

W. P. Ambrose, P. M. Goodwin, J. P. Nolan, “Single-molecule detection with total internal reflection excitation: comparing signal-to-background and total signals in different geometries,” Cytometry 36, 224–231 (1999).
[CrossRef] [PubMed]

Bawendi, M. G.

S. A. Empedocles, R. Neuhauser, M. G. Bawendi, “Three-dimensional orientation measurements of symmetric single chromophores using polarization microscopy,” Nature 399, 126–130 (1999).
[CrossRef]

C. B. Murray, D. J. Norris, M. G. Bawendi, “Synthesis and characterization of nearly monodisperse CdE (E = S, Se, Te) semiconductor nanocrystallites,” J. Am. Chem. Soc. 115, 8706–8715 (1993).
[CrossRef]

Beversluis, M. R.

L. Novotny, M. R. Beversluis, K. S. Youngworth, T. G. Brown, “Longitudinal field modes probed by single molecules,” Phys. Rev. Lett. 86, 5251–5254 (2001).
[CrossRef] [PubMed]

Bjerneld, E. J.

M. F. Paige, E. J. Bjerneld, W. E. Moerner, “A comparison of through-the-objective total internal reflection microscopy and epi-fluorescence microscopy for single-molecule fluorescence imaging,” Single Mol. 2, 191–201 (2001).
[CrossRef]

Booker, G. R.

Brown, T. G.

L. Novotny, M. R. Beversluis, K. S. Youngworth, T. G. Brown, “Longitudinal field modes probed by single molecules,” Phys. Rev. Lett. 86, 5251–5254 (2001).
[CrossRef] [PubMed]

Chon, J. W. M.

J. W. M. Chon, X. Gan, M. Gu, “Splitting of the focal spot of a high numerical-aperture objective in free space,” Appl. Phys. Lett. 81, 1576–1578 (2002).
[CrossRef]

Dickson, R. M.

R. M. Dickson, D. J. Norris, Y. L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–968 (1996).
[CrossRef] [PubMed]

Emmert, H.

K. Stock, R. Sailer, W. S. L. Strauss, R. Pavesi, M. Lyttek, H. Emmert, H. Schneckenburger, “Total internal reflection fluorescence spectroscopy and microscopy (TIRFS/TIRFM) in cell biology and photobiology,” in Fluorescence Microscopy and Fluorescent Probes, A. Kotyk, ed. (Espero Publishing, Prague, 1999), Vol. 3, pp. 67–79.

Empedocles, S. A.

S. A. Empedocles, R. Neuhauser, M. G. Bawendi, “Three-dimensional orientation measurements of symmetric single chromophores using polarization microscopy,” Nature 399, 126–130 (1999).
[CrossRef]

Funaki, F.

H. Watarai, F. Funaki, “Total internal reflection fluorescence measurements of protonation equilibria of Rhodamine B and Octadecylrhodamine B at a toluene/water interface,” Langmuir 12, 6717–6720 (1996).
[CrossRef]

Funatsu, T.

T. Wazawa, Y. Ishii, T. Funatsu, T. Yanagida, “Spectral fluctuation of a single fluophore conjugated to a protein molecule,” Biophys. J. 78, 1561–1569 (2000).
[CrossRef] [PubMed]

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, T. Yanagida, “Direct observation of single kinesin molecules moving along microtubules,” Nature 380, 451–453 (1996).
[CrossRef] [PubMed]

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, T. Yanagida, “Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution,” Nature 374, 555–559 (1995).
[CrossRef] [PubMed]

Gan, X.

J. W. M. Chon, X. Gan, M. Gu, “Splitting of the focal spot of a high numerical-aperture objective in free space,” Appl. Phys. Lett. 81, 1576–1578 (2002).
[CrossRef]

Goodwin, P. M.

W. P. Ambrose, P. M. Goodwin, J. P. Nolan, “Single-molecule detection with total internal reflection excitation: comparing signal-to-background and total signals in different geometries,” Cytometry 36, 224–231 (1999).
[CrossRef] [PubMed]

Gu, M.

J. W. M. Chon, X. Gan, M. Gu, “Splitting of the focal spot of a high numerical-aperture objective in free space,” Appl. Phys. Lett. 81, 1576–1578 (2002).
[CrossRef]

M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopy (World Scientific, Singapore, 1996).

M. Gu, Advanced Optical Imaging Theory (Springer, Heidelberg, 2003).

Harada, Y.

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, T. Yanagida, “Direct observation of single kinesin molecules moving along microtubules,” Nature 380, 451–453 (1996).
[CrossRef] [PubMed]

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, T. Yanagida, “Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution,” Nature 374, 555–559 (1995).
[CrossRef] [PubMed]

Hecht, B.

B. Sick, B. Hecht, L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85, 4482–4485 (2000).
[CrossRef] [PubMed]

B. Sick, B. Hecht, U. P. Wild, L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microsc. 202, 365–373 (2000).
[CrossRef]

Ishii, Y.

T. Wazawa, Y. Ishii, T. Funatsu, T. Yanagida, “Spectral fluctuation of a single fluophore conjugated to a protein molecule,” Biophys. J. 78, 1561–1569 (2000).
[CrossRef] [PubMed]

Iwane, A. H.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235, 47–53 (1997).
[CrossRef] [PubMed]

Kano, H.

Kawata, S.

Kitamura, K.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235, 47–53 (1997).
[CrossRef] [PubMed]

Laczik, Z.

Lyttek, M.

R. Sailer, K. Stock, W. S. L. Strauss, M. Lyttek, H. Schneckenburger, “Total internal reflection fluorescence microscopy (TIRFM) of acridine orange in single cells,” Endocytobiosis & Cell Res. 14, 129–136 (2001).

K. Stock, R. Sailer, W. S. L. Strauss, R. Pavesi, M. Lyttek, H. Emmert, H. Schneckenburger, “Total internal reflection fluorescence spectroscopy and microscopy (TIRFS/TIRFM) in cell biology and photobiology,” in Fluorescence Microscopy and Fluorescent Probes, A. Kotyk, ed. (Espero Publishing, Prague, 1999), Vol. 3, pp. 67–79.

Minoghchi, S.

Y. Sako, S. Minoghchi, T. Yanagida, “Single-molecule imaging of EGFR signaling on the surface of living cells,” Nat. Cell Biol. 2, 168–172 (2000).
[CrossRef] [PubMed]

Mizuguchi, S.

Moerner, W. E.

M. F. Paige, E. J. Bjerneld, W. E. Moerner, “A comparison of through-the-objective total internal reflection microscopy and epi-fluorescence microscopy for single-molecule fluorescence imaging,” Single Mol. 2, 191–201 (2001).
[CrossRef]

R. M. Dickson, D. J. Norris, Y. L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–968 (1996).
[CrossRef] [PubMed]

Murray, C. B.

C. B. Murray, D. J. Norris, M. G. Bawendi, “Synthesis and characterization of nearly monodisperse CdE (E = S, Se, Te) semiconductor nanocrystallites,” J. Am. Chem. Soc. 115, 8706–8715 (1993).
[CrossRef]

Neuhauser, R.

S. A. Empedocles, R. Neuhauser, M. G. Bawendi, “Three-dimensional orientation measurements of symmetric single chromophores using polarization microscopy,” Nature 399, 126–130 (1999).
[CrossRef]

Nolan, J. P.

W. P. Ambrose, P. M. Goodwin, J. P. Nolan, “Single-molecule detection with total internal reflection excitation: comparing signal-to-background and total signals in different geometries,” Cytometry 36, 224–231 (1999).
[CrossRef] [PubMed]

Norris, D. J.

R. M. Dickson, D. J. Norris, Y. L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–968 (1996).
[CrossRef] [PubMed]

C. B. Murray, D. J. Norris, M. G. Bawendi, “Synthesis and characterization of nearly monodisperse CdE (E = S, Se, Te) semiconductor nanocrystallites,” J. Am. Chem. Soc. 115, 8706–8715 (1993).
[CrossRef]

Novotny, L.

L. Novotny, M. R. Beversluis, K. S. Youngworth, T. G. Brown, “Longitudinal field modes probed by single molecules,” Phys. Rev. Lett. 86, 5251–5254 (2001).
[CrossRef] [PubMed]

B. Sick, B. Hecht, U. P. Wild, L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microsc. 202, 365–373 (2000).
[CrossRef]

B. Sick, B. Hecht, L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85, 4482–4485 (2000).
[CrossRef] [PubMed]

Paige, M. F.

M. F. Paige, E. J. Bjerneld, W. E. Moerner, “A comparison of through-the-objective total internal reflection microscopy and epi-fluorescence microscopy for single-molecule fluorescence imaging,” Single Mol. 2, 191–201 (2001).
[CrossRef]

Pavesi, R.

K. Stock, R. Sailer, W. S. L. Strauss, R. Pavesi, M. Lyttek, H. Emmert, H. Schneckenburger, “Total internal reflection fluorescence spectroscopy and microscopy (TIRFS/TIRFM) in cell biology and photobiology,” in Fluorescence Microscopy and Fluorescent Probes, A. Kotyk, ed. (Espero Publishing, Prague, 1999), Vol. 3, pp. 67–79.

Pierce, D. W.

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, T. Yanagida, “Direct observation of single kinesin molecules moving along microtubules,” Nature 380, 451–453 (1996).
[CrossRef] [PubMed]

Romberg, L.

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, T. Yanagida, “Direct observation of single kinesin molecules moving along microtubules,” Nature 380, 451–453 (1996).
[CrossRef] [PubMed]

Sailer, R.

R. Sailer, K. Stock, W. S. L. Strauss, M. Lyttek, H. Schneckenburger, “Total internal reflection fluorescence microscopy (TIRFM) of acridine orange in single cells,” Endocytobiosis & Cell Res. 14, 129–136 (2001).

K. Stock, R. Sailer, W. S. L. Strauss, R. Pavesi, M. Lyttek, H. Emmert, H. Schneckenburger, “Total internal reflection fluorescence spectroscopy and microscopy (TIRFS/TIRFM) in cell biology and photobiology,” in Fluorescence Microscopy and Fluorescent Probes, A. Kotyk, ed. (Espero Publishing, Prague, 1999), Vol. 3, pp. 67–79.

Saito, K.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235, 47–53 (1997).
[CrossRef] [PubMed]

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, T. Yanagida, “Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution,” Nature 374, 555–559 (1995).
[CrossRef] [PubMed]

Sako, Y.

Y. Sako, S. Minoghchi, T. Yanagida, “Single-molecule imaging of EGFR signaling on the surface of living cells,” Nat. Cell Biol. 2, 168–172 (2000).
[CrossRef] [PubMed]

Schneckenburger, H.

R. Sailer, K. Stock, W. S. L. Strauss, M. Lyttek, H. Schneckenburger, “Total internal reflection fluorescence microscopy (TIRFM) of acridine orange in single cells,” Endocytobiosis & Cell Res. 14, 129–136 (2001).

K. Stock, R. Sailer, W. S. L. Strauss, R. Pavesi, M. Lyttek, H. Emmert, H. Schneckenburger, “Total internal reflection fluorescence spectroscopy and microscopy (TIRFS/TIRFM) in cell biology and photobiology,” in Fluorescence Microscopy and Fluorescent Probes, A. Kotyk, ed. (Espero Publishing, Prague, 1999), Vol. 3, pp. 67–79.

Sick, B.

B. Sick, B. Hecht, U. P. Wild, L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microsc. 202, 365–373 (2000).
[CrossRef]

B. Sick, B. Hecht, L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85, 4482–4485 (2000).
[CrossRef] [PubMed]

Stock, K.

R. Sailer, K. Stock, W. S. L. Strauss, M. Lyttek, H. Schneckenburger, “Total internal reflection fluorescence microscopy (TIRFM) of acridine orange in single cells,” Endocytobiosis & Cell Res. 14, 129–136 (2001).

K. Stock, R. Sailer, W. S. L. Strauss, R. Pavesi, M. Lyttek, H. Emmert, H. Schneckenburger, “Total internal reflection fluorescence spectroscopy and microscopy (TIRFS/TIRFM) in cell biology and photobiology,” in Fluorescence Microscopy and Fluorescent Probes, A. Kotyk, ed. (Espero Publishing, Prague, 1999), Vol. 3, pp. 67–79.

Strauss, W. S. L.

R. Sailer, K. Stock, W. S. L. Strauss, M. Lyttek, H. Schneckenburger, “Total internal reflection fluorescence microscopy (TIRFM) of acridine orange in single cells,” Endocytobiosis & Cell Res. 14, 129–136 (2001).

K. Stock, R. Sailer, W. S. L. Strauss, R. Pavesi, M. Lyttek, H. Emmert, H. Schneckenburger, “Total internal reflection fluorescence spectroscopy and microscopy (TIRFS/TIRFM) in cell biology and photobiology,” in Fluorescence Microscopy and Fluorescent Probes, A. Kotyk, ed. (Espero Publishing, Prague, 1999), Vol. 3, pp. 67–79.

Tokunaga, M.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235, 47–53 (1997).
[CrossRef] [PubMed]

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, T. Yanagida, “Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution,” Nature 374, 555–559 (1995).
[CrossRef] [PubMed]

Toraldo di Francia, G.

G. Toraldo di Francia, “Super-gain antennas and optical resolving power,” Nuovo Cimento Suppl. 9, 426–428 (1952).
[CrossRef]

Toriumi, M.

M. Toriumi, M. Yanagimachi, “Time-resolved total-internal-reflection fluorescence spectroscopy and its applications to solid/polymer interface layers,” in Microchemistry, Spectroscopy and Chemistry in Small Domains, H. Masuhara, F. De Schryver, N. Kitamura, N. Tamai, eds. (Elsevier, New York, 1994), pp. 257–268.

Török, P.

Tzeng, Y. L.

R. M. Dickson, D. J. Norris, Y. L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–968 (1996).
[CrossRef] [PubMed]

Vale, R. D.

R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, T. Yanagida, “Direct observation of single kinesin molecules moving along microtubules,” Nature 380, 451–453 (1996).
[CrossRef] [PubMed]

Varga, P.

Watarai, H.

H. Watarai, F. Funaki, “Total internal reflection fluorescence measurements of protonation equilibria of Rhodamine B and Octadecylrhodamine B at a toluene/water interface,” Langmuir 12, 6717–6720 (1996).
[CrossRef]

Wazawa, T.

T. Wazawa, Y. Ishii, T. Funatsu, T. Yanagida, “Spectral fluctuation of a single fluophore conjugated to a protein molecule,” Biophys. J. 78, 1561–1569 (2000).
[CrossRef] [PubMed]

Wild, U. P.

B. Sick, B. Hecht, U. P. Wild, L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microsc. 202, 365–373 (2000).
[CrossRef]

Yanagida, T.

T. Wazawa, Y. Ishii, T. Funatsu, T. Yanagida, “Spectral fluctuation of a single fluophore conjugated to a protein molecule,” Biophys. J. 78, 1561–1569 (2000).
[CrossRef] [PubMed]

Y. Sako, S. Minoghchi, T. Yanagida, “Single-molecule imaging of EGFR signaling on the surface of living cells,” Nat. Cell Biol. 2, 168–172 (2000).
[CrossRef] [PubMed]

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235, 47–53 (1997).
[CrossRef] [PubMed]

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[CrossRef] [PubMed]

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, T. Yanagida, “Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution,” Nature 374, 555–559 (1995).
[CrossRef] [PubMed]

Yanagimachi, M.

M. Toriumi, M. Yanagimachi, “Time-resolved total-internal-reflection fluorescence spectroscopy and its applications to solid/polymer interface layers,” in Microchemistry, Spectroscopy and Chemistry in Small Domains, H. Masuhara, F. De Schryver, N. Kitamura, N. Tamai, eds. (Elsevier, New York, 1994), pp. 257–268.

Youngworth, K. S.

L. Novotny, M. R. Beversluis, K. S. Youngworth, T. G. Brown, “Longitudinal field modes probed by single molecules,” Phys. Rev. Lett. 86, 5251–5254 (2001).
[CrossRef] [PubMed]

Appl. Phys. Lett.

J. W. M. Chon, X. Gan, M. Gu, “Splitting of the focal spot of a high numerical-aperture objective in free space,” Appl. Phys. Lett. 81, 1576–1578 (2002).
[CrossRef]

Biochem. Biophys. Res. Commun.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235, 47–53 (1997).
[CrossRef] [PubMed]

Biophys. J.

T. Wazawa, Y. Ishii, T. Funatsu, T. Yanagida, “Spectral fluctuation of a single fluophore conjugated to a protein molecule,” Biophys. J. 78, 1561–1569 (2000).
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Cytometry

W. P. Ambrose, P. M. Goodwin, J. P. Nolan, “Single-molecule detection with total internal reflection excitation: comparing signal-to-background and total signals in different geometries,” Cytometry 36, 224–231 (1999).
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Y. Sako, S. Minoghchi, T. Yanagida, “Single-molecule imaging of EGFR signaling on the surface of living cells,” Nat. Cell Biol. 2, 168–172 (2000).
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[CrossRef] [PubMed]

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[CrossRef] [PubMed]

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K. Stock, R. Sailer, W. S. L. Strauss, R. Pavesi, M. Lyttek, H. Emmert, H. Schneckenburger, “Total internal reflection fluorescence spectroscopy and microscopy (TIRFS/TIRFM) in cell biology and photobiology,” in Fluorescence Microscopy and Fluorescent Probes, A. Kotyk, ed. (Espero Publishing, Prague, 1999), Vol. 3, pp. 67–79.

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Figures (12)

Fig. 1
Fig. 1

Concept of STIRFM. The focused evanescent field is produced by focusing the ring-beam illumination at the interface between a coverslip glass and a sample medium by a high-NA (1.65) objective. The ring beam is produced by centrally obstructing the beam with a circular disk.

Fig. 2
Fig. 2

Contour plots of intensity near the evanescent focus of an objective (NA = 1.65) illuminated by a ring beam (ε = 0.6): (a) |E x |2, (b) |E y |2, (c) |E z |2, (d) |E| 2. Peak intensities of |E| 2 have been normalized to 100, and the incident polarization is parallel to the x axis. The NA is assumed to be 1.65.

Fig. 3
Fig. 3

Plots of (a) the peak intensity ratio of |E z |2/|E x |2, (b) the peak separation Δν x , and (c) the normalized dip depth η with respect to the normalized obstruction radius ε. The NA of the objective is 1.65. Plots (a), (b), and (c) correspond to the interfaces between immersion oil (n = 1.78) and air, between immersion oil and water, and between immersion oil and glass (n = 1.52), respectively.

Fig. 4
Fig. 4

Plots of (a) the peak intensity ratio of |E z |2/|E x |2, (b) the peak separation Δν x , and (c) the normalized dip depth η with respect to the normalized obstruction radius ε. The normalized transmitted beam intensity through the index-mismatched interface is shown with respect to the obstruction radius.

Fig. 5
Fig. 5

Contour plot of the effective PSF of the STIRFM [Eq. (10)], with a point detector (pinhole) under one-photon excitation. The incident polarization is from parallel to the x axis. The NA is assumed to be 1.65. The excitation wavelength is 532 nm, and the fluorescence wavelength is 600 nm.

Fig. 6
Fig. 6

Plots of the normalized dip depth η with respect to the normalized detector pinhole radius ν d . The actual pinhole sizes used in our experiments are indicated by the two arrows.

Fig. 7
Fig. 7

Contour plot of the effective PSF of the STIRFM under two-photon excitation. The incident polarization is parallel to the x axis. The NA is assumed to be 1.65. The excitation wavelength is 800 nm.

Fig. 8
Fig. 8

Experimental setup. Ring-beam illumination is produced by centrally obstructing the circular beam with an opaque disk just before the dichroic beam splitter. QWP, quarter-wave plate; GTP, Glan-Thompson polarizer; OD, obstruction disk; SS, scanning stage; DB, dichroic beam splitter; NC, nanocrystal sample; OL, objective lens (NA = 1.65); PH, pinhole; PC, personal computer.

Fig. 9
Fig. 9

Cross-section view of the microbeads excited with an evanescent field. ε is the obstruction radius.

Fig. 10
Fig. 10

Images of Rhodamine 6G microbead images (20 μm × 20 μm, excitation at 532 nm): (a) confocal image around the equatorial plane of the beads, (b) confocal image at the interface between coverslip glass and air, (c) STIRFM image with ε = 0.65 (ε c = 0.6), (d) STIRFM image with ε = 0.79.

Fig. 11
Fig. 11

Evanescent field depth as a function of the central obstruction size.

Fig. 12
Fig. 12

(a)–(c) Experimental images (1 μm × 1 μm) of CdSe quantum dot nanocrystals in STIRFM. (d)–(f) Theoretical simulations of the focal spot image in STIRFM. (a) One-photon fluorescence image with 20-μm pinhole detection. (b) One-photon image with 200-μm pinhole detection, with excitation at 532 nm and power of 0.2 kW/cm2. (c) Two-photon fluorescence image with 200-μm pinhole detection, with excitation at 800 nm and power of 0.2 MW/cm2. (d) Theoretical one-photon fluorescence image with a point detector. (e) Theoretical one-photon fluorescence image with an infinitely large area detector. (f) Theoretical two-photon fluorescence image with an infinitely large area detector. Conditions are assumed to be identical to the experimental cases. The arrow indicates the direction of the incident polarization.

Tables (1)

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Table 1 Comparison of the Cutoff Angle for Total Internal Reflection in Various Objectivesa

Equations (14)

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E r 2 ,   ϕ ,   z 2 = π i λ I 0 + cos 2 ϕ I 2 i + sin 2 ϕ I 2 j + 2 i   cos   ϕ I 1 k ,
I 0 = β α cos   θ 1 1 / 2   sin   θ 1 t s + t p   cos   θ 2 exp - ik 0 Φ θ 1 J 0 k 1 r 2   sin   θ 1 exp - ik 2 z 2   cos   θ 2 d θ 1 ,
I 1 = β α cos   θ 1 1 / 2   sin   θ 1 t p   sin   θ 2 exp - ik 0 Φ θ 1 J 1 k 1 r 2   sin   θ 1 exp - ik 2 z 2   cos   θ 2 d θ 1 ,
I 2 = β α cos   θ 1 1 / 2   sin   θ 1 t s - t p   cos   θ 2 exp - ik 0 Φ θ 1 J 2 k 1 r 2   sin   θ 1 exp - ik 2 z 2   cos   θ 2 d θ 1 ,
Φ θ 1 = - d n 1   cos   θ 1 - n 2   cos   θ 2 ,
H 1 p = h ill h det     D ν ,
h ill = | E | 2 = | I 0 | 2 + 4 | I 1 | 2 cos 2   ϕ + | I 2 | 2 + 2   cos   2 ϕ Re I 0 I 2 *
h det = | I 0 | 2 + 2 | I 1 | 2 + | I 2 | 2
D ν = 1 ν < ν d 0 otherwise ,
ν d = 2 π λ f   r d   sin   α d ,
H 1 p = h ill h det .
H 1 p = h ill ,
H 2 p = h ill 2 .
d = r - r 2 - a / 2 2 1 / 2 ,

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