Abstract

We report a confocal total-internal-reflection fluorescence (TIRF) microscope that generates a detection volume for analyte molecules of less than 5 al 5×10-18 l at a water–glass interface. Compared with conventional confocal microscopy, this represents a reduction of almost 2 orders of magnitude, which is important in isolating individual molecules at high analyte concentrations, where many biologically relevant processes occur. Diffraction-limited supercritical focusing and fluorescence collection is accomplished by a parabolic mirror objective. The system delivers TIRF images with excellent spatial resolution and detects single molecules with a high signal-to-background ratio.

© 2004 Optical Society of America

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  3. M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
    [CrossRef] [PubMed]
  4. A. D. Stout and D. Axelrod, Appl. Opt. 28, 5237 (1989).
    [CrossRef] [PubMed]
  5. T. E. Starr and N. L. Thompson, J. Phys. Chem. B 106, 2365 (2002).
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    [CrossRef] [PubMed]
  7. V. Buschmann, K. D. Weston, and M. Sauer, Bioconjugate Chem. 14, 195 (2003).
    [CrossRef]
  8. M. Rankl, S. Laib, and S. Seeger, Colloids Surf. B 30, 177 (2003).
    [CrossRef]
  9. E. Bismuto, E. Gratton, and D. C. Lamb, Biophys. J. 81, 3510 (2001).
    [CrossRef] [PubMed]
  10. D. A. Koppel, Phys. Rev. A 10, 1938 (1974).
    [CrossRef]
  11. T. E. Starr and N. L. Thompson, Biophys. J. 80, 1575 (2001).
    [CrossRef] [PubMed]

2003 (4)

M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

T. Ruckstuhl and S. Seeger, Appl. Opt. 42, 3277 (2003).
[CrossRef] [PubMed]

V. Buschmann, K. D. Weston, and M. Sauer, Bioconjugate Chem. 14, 195 (2003).
[CrossRef]

M. Rankl, S. Laib, and S. Seeger, Colloids Surf. B 30, 177 (2003).
[CrossRef]

2002 (2)

S. T. Hess, S. Huang, A. A. Heikal, and W. W. Webb, Biochemistry 41, 697 (2002).
[CrossRef] [PubMed]

T. E. Starr and N. L. Thompson, J. Phys. Chem. B 106, 2365 (2002).

2001 (2)

T. E. Starr and N. L. Thompson, Biophys. J. 80, 1575 (2001).
[CrossRef] [PubMed]

E. Bismuto, E. Gratton, and D. C. Lamb, Biophys. J. 81, 3510 (2001).
[CrossRef] [PubMed]

1994 (1)

S. Nie, D. T. Chiu, and R. N. Zare, Science 266, 1018 (1994).
[CrossRef] [PubMed]

1989 (1)

1974 (1)

D. A. Koppel, Phys. Rev. A 10, 1938 (1974).
[CrossRef]

Axelrod, D.

Bismuto, E.

E. Bismuto, E. Gratton, and D. C. Lamb, Biophys. J. 81, 3510 (2001).
[CrossRef] [PubMed]

Buschmann, V.

V. Buschmann, K. D. Weston, and M. Sauer, Bioconjugate Chem. 14, 195 (2003).
[CrossRef]

Chiu, D. T.

S. Nie, D. T. Chiu, and R. N. Zare, Science 266, 1018 (1994).
[CrossRef] [PubMed]

Craighead, H. G.

M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

Foquet, M.

M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

Gratton, E.

E. Bismuto, E. Gratton, and D. C. Lamb, Biophys. J. 81, 3510 (2001).
[CrossRef] [PubMed]

Heikal, A. A.

S. T. Hess, S. Huang, A. A. Heikal, and W. W. Webb, Biochemistry 41, 697 (2002).
[CrossRef] [PubMed]

Hess, S. T.

S. T. Hess, S. Huang, A. A. Heikal, and W. W. Webb, Biochemistry 41, 697 (2002).
[CrossRef] [PubMed]

Huang, S.

S. T. Hess, S. Huang, A. A. Heikal, and W. W. Webb, Biochemistry 41, 697 (2002).
[CrossRef] [PubMed]

Koppel, D. A.

D. A. Koppel, Phys. Rev. A 10, 1938 (1974).
[CrossRef]

Korlach, J.

M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

Laib, S.

M. Rankl, S. Laib, and S. Seeger, Colloids Surf. B 30, 177 (2003).
[CrossRef]

Lamb, D. C.

E. Bismuto, E. Gratton, and D. C. Lamb, Biophys. J. 81, 3510 (2001).
[CrossRef] [PubMed]

Levene, M. J.

M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

Nie, S.

S. Nie, D. T. Chiu, and R. N. Zare, Science 266, 1018 (1994).
[CrossRef] [PubMed]

Rankl, M.

M. Rankl, S. Laib, and S. Seeger, Colloids Surf. B 30, 177 (2003).
[CrossRef]

Ruckstuhl, T.

Sauer, M.

V. Buschmann, K. D. Weston, and M. Sauer, Bioconjugate Chem. 14, 195 (2003).
[CrossRef]

Seeger, S.

M. Rankl, S. Laib, and S. Seeger, Colloids Surf. B 30, 177 (2003).
[CrossRef]

T. Ruckstuhl and S. Seeger, Appl. Opt. 42, 3277 (2003).
[CrossRef] [PubMed]

Starr, T. E.

T. E. Starr and N. L. Thompson, J. Phys. Chem. B 106, 2365 (2002).

T. E. Starr and N. L. Thompson, Biophys. J. 80, 1575 (2001).
[CrossRef] [PubMed]

Stout, A. D.

Thompson, N. L.

T. E. Starr and N. L. Thompson, J. Phys. Chem. B 106, 2365 (2002).

T. E. Starr and N. L. Thompson, Biophys. J. 80, 1575 (2001).
[CrossRef] [PubMed]

Turner, S. W.

M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

Webb, W. W.

M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

S. T. Hess, S. Huang, A. A. Heikal, and W. W. Webb, Biochemistry 41, 697 (2002).
[CrossRef] [PubMed]

Weston, K. D.

V. Buschmann, K. D. Weston, and M. Sauer, Bioconjugate Chem. 14, 195 (2003).
[CrossRef]

Zare, R. N.

S. Nie, D. T. Chiu, and R. N. Zare, Science 266, 1018 (1994).
[CrossRef] [PubMed]

Appl. Opt. (2)

Biochemistry (1)

S. T. Hess, S. Huang, A. A. Heikal, and W. W. Webb, Biochemistry 41, 697 (2002).
[CrossRef] [PubMed]

Bioconjugate Chem. (1)

V. Buschmann, K. D. Weston, and M. Sauer, Bioconjugate Chem. 14, 195 (2003).
[CrossRef]

Biophys. J. (2)

E. Bismuto, E. Gratton, and D. C. Lamb, Biophys. J. 81, 3510 (2001).
[CrossRef] [PubMed]

T. E. Starr and N. L. Thompson, Biophys. J. 80, 1575 (2001).
[CrossRef] [PubMed]

Colloids Surf. B (1)

M. Rankl, S. Laib, and S. Seeger, Colloids Surf. B 30, 177 (2003).
[CrossRef]

J. Phys. Chem. B (1)

T. E. Starr and N. L. Thompson, J. Phys. Chem. B 106, 2365 (2002).

Phys. Rev. A (1)

D. A. Koppel, Phys. Rev. A 10, 1938 (1974).
[CrossRef]

Science (2)

M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

S. Nie, D. T. Chiu, and R. N. Zare, Science 266, 1018 (1994).
[CrossRef] [PubMed]

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Figures (5)

Fig. 1
Fig. 1

Schematic of the confocal TIRF microscope.

Fig. 2
Fig. 2

(a) Calculated focal intensity in the plane of the interface (excitation angles of 62°–75°). (b), (c) Images of fluorescence beads measured without a confocal pinhole. The scan resolution was set to 80 nm, the laser was polarized in the x direction.

Fig. 3
Fig. 3

(a) Calculated point-spread function obtained for a confocal pinhole of 50µm diameter. (b) Confocal image of the beads with a scan resolution of 80 nm. (c) Superposition of the images of four beads.

Fig. 4
Fig. 4

Tracks of the fluorescence intensity of 0.1µmol/l EVOblue 30 on a hydrophilic coverslip showing low nonspecific adsorption (gray curves, 20µs and 1-ms time binning) and 0.5 nmol/l on a less hydrophilic coverslip (black curve, 1-ms time binning).

Fig. 5
Fig. 5

(a) Normalized FCS curves for different dye concentrations measured on a hydrophilic coverslip. From the top, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2 µmol/l. (b) Plot of the average number of molecules in the detection volume against concentration.

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