Abstract

We introduce a method for detecting and tracking small particles in a solution near a surface. The method is based on blocking the backreflected illumination beam in an objective-type total internal reflection microscope, leaving unhindered the light scattered by the particles and resulting in dark-field illumination. Using this method, we tracked the motion of 60-nm polystyrene beads with a signal-to-noise ratio of 6 and detected 20-nm gold particles with a signal-to-noise ratio of 5. We illustrate the method’s use by following the Brownian motion of small beads attached by short DNA tethers to a substrate.

© 2001 Optical Society of America

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  1. D. C. Prieve, N. A. Frej, “Total internal reflection microscopy: a quantitative tool for the measurement of colloidal forces,” Langmuir 6, 396–403 (1990).
    [CrossRef]
  2. T. R. Strick, J-F. Allemand, A. Bensimon, D. Bensimon, V. Croquette, “The elasticity of a single supercoiled DNA molecule,” Science 271, 1835–1837 (1996).
    [CrossRef] [PubMed]
  3. G. V. Shivashankar, M. Feingold, O. Krichevsky, A. Libchaber, “RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis,” Proc. Natl. Acad. Sci. USA 96, 7916–7921 (1999).
    [CrossRef] [PubMed]
  4. T. R. Strick, T. V. Croquette, D. Bensimon, “Single-molecule analysis of DNA uncoiling by a type II topoisomerase,” Nature (London) 404, 901–904 (2000).
  5. H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, J. Gelles, “Transcription against an applied force,” Science 270, 1653–1657 (1995).
    [CrossRef] [PubMed]
  6. G. Zocchi, “Proteins unfold in steps,” Proc. Natl. Acad. Sci. USA 94, 10647–10651 (1997).
    [CrossRef] [PubMed]
  7. L. Finzi, J. Gelles, “Measurement of lactose repressor-mediated loop formation and breakdown in single DNA molecules,” Science 267, 378–380 (1995).
    [CrossRef] [PubMed]
  8. H. C. van de Hulst, Light Scattering by Small Particles (Dover, New York, 1981).
  9. A. L. Stout, D. Axelrod, “Evanescent field excitation of fluorescence by epi-illumination microscopy,” Appl. Opt. 28, 5237–5242 (1989).
    [CrossRef] [PubMed]
  10. D. Axelrod, “Zero-cost modification of bright-field microscopes for imaging phase gradient on cells: Schlieren optics,” Cell Biophys. 3, 167–173 (1981).
    [PubMed]
  11. W. B. Piekos, “Diffracted-light contrast enhancement: a re-examination of oblique illumination,” Microsc. Res. Tech. 46, 334–337 (1999), and references therein.
    [CrossRef]
  12. R. Ozeri, L. Khaykovich, N. Friedman, N. Davidson, “Large volume single-beam dark optical trap for atoms using binary phase elements,” J. Opt. Soc. Am. B 17, 1113–1116 (2000).
    [CrossRef]
  13. J. C. Crocker, D. G. Grier, “Methods of digital video microscopy for colloidal studies,” J. Colloid Interface Sci. 179, 298–310 (1996).
    [CrossRef]
  14. H. Qian, E. L. Elson, “Quantitative study of polymer conformation and dynamics by single-particle tracking,” Biophys. J. 76, 1598–1605 (1999).
    [CrossRef] [PubMed]
  15. H. Yin, R. Landick, J. Gelles, “Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule,” Biophys. J. 67, 2468–2478 (1994).
    [CrossRef] [PubMed]
  16. S. G. Lipson, H. Lipson, Optical Physics (Cambridge University, Cambridge, UK, 1995).
    [CrossRef]
  17. B. M. J. Ali, R. Amit, I. Braslavsky, A. B. Oppenheim, O. Gileadi, J. Stavans, “Compaction of single DNA molecules induced by binding of integration host factor (IHF),” Proc. Natl. Acad. Sci. USA 98, 10658–10663 (2001).
    [CrossRef] [PubMed]
  18. M. Nishiyama, E. Muto, Y. Inoue, T. Yanagida, H. Higuchi, “Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules,” Nature Cell Biol. 3, 425–428 (2001).
    [CrossRef] [PubMed]
  19. R. Bar-Ziv, A. Meller, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: probing single particle dynamics at the nanoscale,” Phys. Rev. Lett. 78, 154–157 (1997).
    [CrossRef]
  20. A. Meller, R. Bar-Ziv, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy,” Biophys. J. 74, 1541–1548 (1998).
    [CrossRef] [PubMed]
  21. G. V. Shivashankar, G. Stolovitzky, A. Libchaber, “Backscattering from a tethered bead as a probe of DNA flexibility,” Appl. Phys. Lett. 73, 291–293 (1998).
    [CrossRef]

2001 (2)

B. M. J. Ali, R. Amit, I. Braslavsky, A. B. Oppenheim, O. Gileadi, J. Stavans, “Compaction of single DNA molecules induced by binding of integration host factor (IHF),” Proc. Natl. Acad. Sci. USA 98, 10658–10663 (2001).
[CrossRef] [PubMed]

M. Nishiyama, E. Muto, Y. Inoue, T. Yanagida, H. Higuchi, “Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules,” Nature Cell Biol. 3, 425–428 (2001).
[CrossRef] [PubMed]

2000 (2)

T. R. Strick, T. V. Croquette, D. Bensimon, “Single-molecule analysis of DNA uncoiling by a type II topoisomerase,” Nature (London) 404, 901–904 (2000).

R. Ozeri, L. Khaykovich, N. Friedman, N. Davidson, “Large volume single-beam dark optical trap for atoms using binary phase elements,” J. Opt. Soc. Am. B 17, 1113–1116 (2000).
[CrossRef]

1999 (3)

G. V. Shivashankar, M. Feingold, O. Krichevsky, A. Libchaber, “RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis,” Proc. Natl. Acad. Sci. USA 96, 7916–7921 (1999).
[CrossRef] [PubMed]

H. Qian, E. L. Elson, “Quantitative study of polymer conformation and dynamics by single-particle tracking,” Biophys. J. 76, 1598–1605 (1999).
[CrossRef] [PubMed]

W. B. Piekos, “Diffracted-light contrast enhancement: a re-examination of oblique illumination,” Microsc. Res. Tech. 46, 334–337 (1999), and references therein.
[CrossRef]

1998 (2)

A. Meller, R. Bar-Ziv, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy,” Biophys. J. 74, 1541–1548 (1998).
[CrossRef] [PubMed]

G. V. Shivashankar, G. Stolovitzky, A. Libchaber, “Backscattering from a tethered bead as a probe of DNA flexibility,” Appl. Phys. Lett. 73, 291–293 (1998).
[CrossRef]

1997 (2)

R. Bar-Ziv, A. Meller, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: probing single particle dynamics at the nanoscale,” Phys. Rev. Lett. 78, 154–157 (1997).
[CrossRef]

G. Zocchi, “Proteins unfold in steps,” Proc. Natl. Acad. Sci. USA 94, 10647–10651 (1997).
[CrossRef] [PubMed]

1996 (2)

J. C. Crocker, D. G. Grier, “Methods of digital video microscopy for colloidal studies,” J. Colloid Interface Sci. 179, 298–310 (1996).
[CrossRef]

T. R. Strick, J-F. Allemand, A. Bensimon, D. Bensimon, V. Croquette, “The elasticity of a single supercoiled DNA molecule,” Science 271, 1835–1837 (1996).
[CrossRef] [PubMed]

1995 (2)

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, J. Gelles, “Transcription against an applied force,” Science 270, 1653–1657 (1995).
[CrossRef] [PubMed]

L. Finzi, J. Gelles, “Measurement of lactose repressor-mediated loop formation and breakdown in single DNA molecules,” Science 267, 378–380 (1995).
[CrossRef] [PubMed]

1994 (1)

H. Yin, R. Landick, J. Gelles, “Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule,” Biophys. J. 67, 2468–2478 (1994).
[CrossRef] [PubMed]

1990 (1)

D. C. Prieve, N. A. Frej, “Total internal reflection microscopy: a quantitative tool for the measurement of colloidal forces,” Langmuir 6, 396–403 (1990).
[CrossRef]

1989 (1)

1981 (1)

D. Axelrod, “Zero-cost modification of bright-field microscopes for imaging phase gradient on cells: Schlieren optics,” Cell Biophys. 3, 167–173 (1981).
[PubMed]

Ali, B. M. J.

B. M. J. Ali, R. Amit, I. Braslavsky, A. B. Oppenheim, O. Gileadi, J. Stavans, “Compaction of single DNA molecules induced by binding of integration host factor (IHF),” Proc. Natl. Acad. Sci. USA 98, 10658–10663 (2001).
[CrossRef] [PubMed]

Allemand, J-F.

T. R. Strick, J-F. Allemand, A. Bensimon, D. Bensimon, V. Croquette, “The elasticity of a single supercoiled DNA molecule,” Science 271, 1835–1837 (1996).
[CrossRef] [PubMed]

Amit, R.

B. M. J. Ali, R. Amit, I. Braslavsky, A. B. Oppenheim, O. Gileadi, J. Stavans, “Compaction of single DNA molecules induced by binding of integration host factor (IHF),” Proc. Natl. Acad. Sci. USA 98, 10658–10663 (2001).
[CrossRef] [PubMed]

Axelrod, D.

A. L. Stout, D. Axelrod, “Evanescent field excitation of fluorescence by epi-illumination microscopy,” Appl. Opt. 28, 5237–5242 (1989).
[CrossRef] [PubMed]

D. Axelrod, “Zero-cost modification of bright-field microscopes for imaging phase gradient on cells: Schlieren optics,” Cell Biophys. 3, 167–173 (1981).
[PubMed]

Bar-Ziv, R.

A. Meller, R. Bar-Ziv, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy,” Biophys. J. 74, 1541–1548 (1998).
[CrossRef] [PubMed]

R. Bar-Ziv, A. Meller, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: probing single particle dynamics at the nanoscale,” Phys. Rev. Lett. 78, 154–157 (1997).
[CrossRef]

Bensimon, A.

T. R. Strick, J-F. Allemand, A. Bensimon, D. Bensimon, V. Croquette, “The elasticity of a single supercoiled DNA molecule,” Science 271, 1835–1837 (1996).
[CrossRef] [PubMed]

Bensimon, D.

T. R. Strick, T. V. Croquette, D. Bensimon, “Single-molecule analysis of DNA uncoiling by a type II topoisomerase,” Nature (London) 404, 901–904 (2000).

T. R. Strick, J-F. Allemand, A. Bensimon, D. Bensimon, V. Croquette, “The elasticity of a single supercoiled DNA molecule,” Science 271, 1835–1837 (1996).
[CrossRef] [PubMed]

Block, S. M.

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, J. Gelles, “Transcription against an applied force,” Science 270, 1653–1657 (1995).
[CrossRef] [PubMed]

Braslavsky, I.

B. M. J. Ali, R. Amit, I. Braslavsky, A. B. Oppenheim, O. Gileadi, J. Stavans, “Compaction of single DNA molecules induced by binding of integration host factor (IHF),” Proc. Natl. Acad. Sci. USA 98, 10658–10663 (2001).
[CrossRef] [PubMed]

Crocker, J. C.

J. C. Crocker, D. G. Grier, “Methods of digital video microscopy for colloidal studies,” J. Colloid Interface Sci. 179, 298–310 (1996).
[CrossRef]

Croquette, T. V.

T. R. Strick, T. V. Croquette, D. Bensimon, “Single-molecule analysis of DNA uncoiling by a type II topoisomerase,” Nature (London) 404, 901–904 (2000).

Croquette, V.

T. R. Strick, J-F. Allemand, A. Bensimon, D. Bensimon, V. Croquette, “The elasticity of a single supercoiled DNA molecule,” Science 271, 1835–1837 (1996).
[CrossRef] [PubMed]

Davidson, N.

Elson, E. L.

H. Qian, E. L. Elson, “Quantitative study of polymer conformation and dynamics by single-particle tracking,” Biophys. J. 76, 1598–1605 (1999).
[CrossRef] [PubMed]

Feingold, M.

G. V. Shivashankar, M. Feingold, O. Krichevsky, A. Libchaber, “RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis,” Proc. Natl. Acad. Sci. USA 96, 7916–7921 (1999).
[CrossRef] [PubMed]

Finzi, L.

L. Finzi, J. Gelles, “Measurement of lactose repressor-mediated loop formation and breakdown in single DNA molecules,” Science 267, 378–380 (1995).
[CrossRef] [PubMed]

Frej, N. A.

D. C. Prieve, N. A. Frej, “Total internal reflection microscopy: a quantitative tool for the measurement of colloidal forces,” Langmuir 6, 396–403 (1990).
[CrossRef]

Friedman, N.

Gelles, J.

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, J. Gelles, “Transcription against an applied force,” Science 270, 1653–1657 (1995).
[CrossRef] [PubMed]

L. Finzi, J. Gelles, “Measurement of lactose repressor-mediated loop formation and breakdown in single DNA molecules,” Science 267, 378–380 (1995).
[CrossRef] [PubMed]

H. Yin, R. Landick, J. Gelles, “Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule,” Biophys. J. 67, 2468–2478 (1994).
[CrossRef] [PubMed]

Gileadi, O.

B. M. J. Ali, R. Amit, I. Braslavsky, A. B. Oppenheim, O. Gileadi, J. Stavans, “Compaction of single DNA molecules induced by binding of integration host factor (IHF),” Proc. Natl. Acad. Sci. USA 98, 10658–10663 (2001).
[CrossRef] [PubMed]

Grier, D. G.

J. C. Crocker, D. G. Grier, “Methods of digital video microscopy for colloidal studies,” J. Colloid Interface Sci. 179, 298–310 (1996).
[CrossRef]

Higuchi, H.

M. Nishiyama, E. Muto, Y. Inoue, T. Yanagida, H. Higuchi, “Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules,” Nature Cell Biol. 3, 425–428 (2001).
[CrossRef] [PubMed]

Inoue, Y.

M. Nishiyama, E. Muto, Y. Inoue, T. Yanagida, H. Higuchi, “Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules,” Nature Cell Biol. 3, 425–428 (2001).
[CrossRef] [PubMed]

Khaykovich, L.

Krichevsky, O.

G. V. Shivashankar, M. Feingold, O. Krichevsky, A. Libchaber, “RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis,” Proc. Natl. Acad. Sci. USA 96, 7916–7921 (1999).
[CrossRef] [PubMed]

Landick, R.

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, J. Gelles, “Transcription against an applied force,” Science 270, 1653–1657 (1995).
[CrossRef] [PubMed]

H. Yin, R. Landick, J. Gelles, “Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule,” Biophys. J. 67, 2468–2478 (1994).
[CrossRef] [PubMed]

Libchaber, A.

G. V. Shivashankar, M. Feingold, O. Krichevsky, A. Libchaber, “RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis,” Proc. Natl. Acad. Sci. USA 96, 7916–7921 (1999).
[CrossRef] [PubMed]

G. V. Shivashankar, G. Stolovitzky, A. Libchaber, “Backscattering from a tethered bead as a probe of DNA flexibility,” Appl. Phys. Lett. 73, 291–293 (1998).
[CrossRef]

Lipson, H.

S. G. Lipson, H. Lipson, Optical Physics (Cambridge University, Cambridge, UK, 1995).
[CrossRef]

Lipson, S. G.

S. G. Lipson, H. Lipson, Optical Physics (Cambridge University, Cambridge, UK, 1995).
[CrossRef]

Meller, A.

A. Meller, R. Bar-Ziv, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy,” Biophys. J. 74, 1541–1548 (1998).
[CrossRef] [PubMed]

R. Bar-Ziv, A. Meller, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: probing single particle dynamics at the nanoscale,” Phys. Rev. Lett. 78, 154–157 (1997).
[CrossRef]

Moses, E.

A. Meller, R. Bar-Ziv, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy,” Biophys. J. 74, 1541–1548 (1998).
[CrossRef] [PubMed]

R. Bar-Ziv, A. Meller, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: probing single particle dynamics at the nanoscale,” Phys. Rev. Lett. 78, 154–157 (1997).
[CrossRef]

Muto, E.

M. Nishiyama, E. Muto, Y. Inoue, T. Yanagida, H. Higuchi, “Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules,” Nature Cell Biol. 3, 425–428 (2001).
[CrossRef] [PubMed]

Nishiyama, M.

M. Nishiyama, E. Muto, Y. Inoue, T. Yanagida, H. Higuchi, “Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules,” Nature Cell Biol. 3, 425–428 (2001).
[CrossRef] [PubMed]

Oppenheim, A. B.

B. M. J. Ali, R. Amit, I. Braslavsky, A. B. Oppenheim, O. Gileadi, J. Stavans, “Compaction of single DNA molecules induced by binding of integration host factor (IHF),” Proc. Natl. Acad. Sci. USA 98, 10658–10663 (2001).
[CrossRef] [PubMed]

Ozeri, R.

Piekos, W. B.

W. B. Piekos, “Diffracted-light contrast enhancement: a re-examination of oblique illumination,” Microsc. Res. Tech. 46, 334–337 (1999), and references therein.
[CrossRef]

Prieve, D. C.

D. C. Prieve, N. A. Frej, “Total internal reflection microscopy: a quantitative tool for the measurement of colloidal forces,” Langmuir 6, 396–403 (1990).
[CrossRef]

Qian, H.

H. Qian, E. L. Elson, “Quantitative study of polymer conformation and dynamics by single-particle tracking,” Biophys. J. 76, 1598–1605 (1999).
[CrossRef] [PubMed]

Safran, S. A.

A. Meller, R. Bar-Ziv, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy,” Biophys. J. 74, 1541–1548 (1998).
[CrossRef] [PubMed]

R. Bar-Ziv, A. Meller, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: probing single particle dynamics at the nanoscale,” Phys. Rev. Lett. 78, 154–157 (1997).
[CrossRef]

Shivashankar, G. V.

G. V. Shivashankar, M. Feingold, O. Krichevsky, A. Libchaber, “RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis,” Proc. Natl. Acad. Sci. USA 96, 7916–7921 (1999).
[CrossRef] [PubMed]

G. V. Shivashankar, G. Stolovitzky, A. Libchaber, “Backscattering from a tethered bead as a probe of DNA flexibility,” Appl. Phys. Lett. 73, 291–293 (1998).
[CrossRef]

Stavans, J.

B. M. J. Ali, R. Amit, I. Braslavsky, A. B. Oppenheim, O. Gileadi, J. Stavans, “Compaction of single DNA molecules induced by binding of integration host factor (IHF),” Proc. Natl. Acad. Sci. USA 98, 10658–10663 (2001).
[CrossRef] [PubMed]

A. Meller, R. Bar-Ziv, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy,” Biophys. J. 74, 1541–1548 (1998).
[CrossRef] [PubMed]

R. Bar-Ziv, A. Meller, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: probing single particle dynamics at the nanoscale,” Phys. Rev. Lett. 78, 154–157 (1997).
[CrossRef]

Stolovitzky, G.

G. V. Shivashankar, G. Stolovitzky, A. Libchaber, “Backscattering from a tethered bead as a probe of DNA flexibility,” Appl. Phys. Lett. 73, 291–293 (1998).
[CrossRef]

Stout, A. L.

Strick, T. R.

T. R. Strick, T. V. Croquette, D. Bensimon, “Single-molecule analysis of DNA uncoiling by a type II topoisomerase,” Nature (London) 404, 901–904 (2000).

T. R. Strick, J-F. Allemand, A. Bensimon, D. Bensimon, V. Croquette, “The elasticity of a single supercoiled DNA molecule,” Science 271, 1835–1837 (1996).
[CrossRef] [PubMed]

Svoboda, K.

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, J. Gelles, “Transcription against an applied force,” Science 270, 1653–1657 (1995).
[CrossRef] [PubMed]

Tlusty, T.

A. Meller, R. Bar-Ziv, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy,” Biophys. J. 74, 1541–1548 (1998).
[CrossRef] [PubMed]

R. Bar-Ziv, A. Meller, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: probing single particle dynamics at the nanoscale,” Phys. Rev. Lett. 78, 154–157 (1997).
[CrossRef]

van de Hulst, H. C.

H. C. van de Hulst, Light Scattering by Small Particles (Dover, New York, 1981).

Wang, M. D.

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, J. Gelles, “Transcription against an applied force,” Science 270, 1653–1657 (1995).
[CrossRef] [PubMed]

Yanagida, T.

M. Nishiyama, E. Muto, Y. Inoue, T. Yanagida, H. Higuchi, “Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules,” Nature Cell Biol. 3, 425–428 (2001).
[CrossRef] [PubMed]

Yin, H.

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, J. Gelles, “Transcription against an applied force,” Science 270, 1653–1657 (1995).
[CrossRef] [PubMed]

H. Yin, R. Landick, J. Gelles, “Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule,” Biophys. J. 67, 2468–2478 (1994).
[CrossRef] [PubMed]

Zocchi, G.

G. Zocchi, “Proteins unfold in steps,” Proc. Natl. Acad. Sci. USA 94, 10647–10651 (1997).
[CrossRef] [PubMed]

Appl. Opt. (1)

Appl. Phys. Lett. (1)

G. V. Shivashankar, G. Stolovitzky, A. Libchaber, “Backscattering from a tethered bead as a probe of DNA flexibility,” Appl. Phys. Lett. 73, 291–293 (1998).
[CrossRef]

Biophys. J. (3)

A. Meller, R. Bar-Ziv, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy,” Biophys. J. 74, 1541–1548 (1998).
[CrossRef] [PubMed]

H. Qian, E. L. Elson, “Quantitative study of polymer conformation and dynamics by single-particle tracking,” Biophys. J. 76, 1598–1605 (1999).
[CrossRef] [PubMed]

H. Yin, R. Landick, J. Gelles, “Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule,” Biophys. J. 67, 2468–2478 (1994).
[CrossRef] [PubMed]

Cell Biophys. (1)

D. Axelrod, “Zero-cost modification of bright-field microscopes for imaging phase gradient on cells: Schlieren optics,” Cell Biophys. 3, 167–173 (1981).
[PubMed]

J. Colloid Interface Sci. (1)

J. C. Crocker, D. G. Grier, “Methods of digital video microscopy for colloidal studies,” J. Colloid Interface Sci. 179, 298–310 (1996).
[CrossRef]

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

Langmuir (1)

D. C. Prieve, N. A. Frej, “Total internal reflection microscopy: a quantitative tool for the measurement of colloidal forces,” Langmuir 6, 396–403 (1990).
[CrossRef]

Microsc. Res. Tech. (1)

W. B. Piekos, “Diffracted-light contrast enhancement: a re-examination of oblique illumination,” Microsc. Res. Tech. 46, 334–337 (1999), and references therein.
[CrossRef]

Nature (London) (1)

T. R. Strick, T. V. Croquette, D. Bensimon, “Single-molecule analysis of DNA uncoiling by a type II topoisomerase,” Nature (London) 404, 901–904 (2000).

Nature Cell Biol. (1)

M. Nishiyama, E. Muto, Y. Inoue, T. Yanagida, H. Higuchi, “Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules,” Nature Cell Biol. 3, 425–428 (2001).
[CrossRef] [PubMed]

Phys. Rev. Lett. (1)

R. Bar-Ziv, A. Meller, T. Tlusty, E. Moses, J. Stavans, S. A. Safran, “Localized dynamic light scattering: probing single particle dynamics at the nanoscale,” Phys. Rev. Lett. 78, 154–157 (1997).
[CrossRef]

Proc. Natl. Acad. Sci. USA (3)

B. M. J. Ali, R. Amit, I. Braslavsky, A. B. Oppenheim, O. Gileadi, J. Stavans, “Compaction of single DNA molecules induced by binding of integration host factor (IHF),” Proc. Natl. Acad. Sci. USA 98, 10658–10663 (2001).
[CrossRef] [PubMed]

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[CrossRef]

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Figures (9)

Fig. 1
Fig. 1

Configuration for implementation of ODFSM: the diffraction element method: solid lines, the laser beam passing through a diffractive element that concentrates 80% of the beam into a diverging ring. Lenses L1a, L2a, and the 50% transmission neutral density beam splitter, BS, steer the beam into a Zeiss NA 1.4, 63× objective to form nearly evanescent illumination. Dashed lines, the illumination beam reflected by the coverslip–water interface back into the objective. Lens L3a forms an image of the backreflected beam on the annular block placed at the equivalent back focal plane, EBFP2, thereby filtering out the nonscattered light. L4a images the scattered light from the beads unto the CCD device. For clarity, incoming rays impinging on the BFP from only one side of the objective and their associated reflected outcoming rays are shown passing through the objective. With this method the illumination area in the sample is a spot with a size of ∼30 µm in our setup. The pair of focal spots on both sides of each lens (of focal distance f i ) are shown as full circles and have been joined by dotted–dashed lines.

Fig. 2
Fig. 2

Configuration for implementation of ODFSM: The blocking disk method. This method can produce either spotlike illumination in which a focused beam is conjugated to the image plane or large-field illumination. In the latter case a rotating diffusing screen is placed at a plane conjugate to the sample plane at the focus of lens L1b. The screen converts the laser beam into an extended source. A large block placed at the equivalent back focal plane, EBFP1, blocks everything aside for a small crescent-shaped cross section. The arc is incident on one edge of the back aperture of the objective, and the backreflected light is blocked on the diametrically opposed side at an equivalent back focal plane, EBFP2, created by lens L4b. The scattered light is imaged by lens L5b on the CCD.

Fig. 3
Fig. 3

Modifications in the detection path for the implementation of ODFSM in a Zeiss Axiovert 135 microscope. Lenses L2c and L3c and the beam block are added between the microscope’s outport and a CCD camera.

Fig. 4
Fig. 4

Images of beads of diameters of 290, 490, and 810 nm (top to bottom) with, left, bright-field illumination and, right, ODFSM. The scale bar corresponds to 1 µm.

Fig. 5
Fig. 5

Images of beads of different sizes obtained with ODFSM with the configuration shown in Fig. 1: a, 60 nm; b, 72 nm; c, 86 nm; d, 110 nm; e, 160 nm; f, 200 nm; g, 290 nm; h, 490 nm; i 810 nm. The scale bar corresponds to 1 µm.

Fig. 6
Fig. 6

Signal-to-noise ratio as a function of the polystyrene bead diameter by using, ○, bright-field illumination; ●, ODFSM; ▲, S/N for a gold particle of 20 nm. For polystyrene beads observed with ODFSM, S/N has been calculated only for diameters below 110 nm for which the images do not show saturation. These data correspond to the images shown in Figs. 4 and 5. Note that beads with a diameter of 60 nm are observed with a value of S/N ∼ 6.

Fig. 7
Fig. 7

The xy position of the center of a bead tethered to a glass slide by a short DNA molecule (1073 base pairs) at different times. Data were accumulated for 400 s.

Fig. 8
Fig. 8

Radial distribution function f(r) corresponding to the data shown in Fig. 7.

Fig. 9
Fig. 9

Measured amplitude of Brownian motion A BM of 290-nm beads tethered to a glass surface by DNA molecules as a function of, ●, DNA length. The solid line denotes the calculated amplitude of Brownian motion A BM as described in the text.

Equations (2)

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SN=Ibead-Ibackgroundσbackground.
ABM=ρ2+D21/2.

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