Abstract

We investigate the dependence of the spot size in single-emitter confocal imaging on the degree of saturation. We show that single-emitter spots are broadened and flattened significantly already at excitation intensities well below saturation. The resulting single-emitter spot shapes thus deviate significantly from the excitation point spread function. We show and support by Monte Carlo simulations that fitting of a single spot is sufficient to extract the saturation intensity and the maximum emission rate of a single emitter with high accuracy. Our results will be of interest in all areas of single-emitter studies.

© 2006 Optical Society of America

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  1. T. Basché, W. E. Moerner, M. Orrit, and U. P. Wild, eds., Single-Molecule Optical Detection, Imaging and Spectroscopy (VCH Verlagsgesellschaft, Weinheim, 1997).
  2. W. P. Ambrose, T. Basché, and W. E. Moerner, "Detection and spectroscopy of single pentacene molecules in a p-terphenyl crystal by means of fluorescence excitation," J. Chem. Phys. 95, 7150-7163 (1991).
    [CrossRef]
  3. L. Novotny and B. Hecht, Principles of Nano-Optics (Cambridge University Press, 2006).
  4. N. Bobroff, "Position Measurement with a Resolution and Noise-Limited Instrument," Rev. Sci. Instrum. 57, 1152-1157 (1986).
    [CrossRef]
  5. T. Schmidt, G. J. Schütz,W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-2929 (1996).
    [CrossRef] [PubMed]
  6. A. Bloeß, Y. Durand, M. Matsushita, H. van der Meer, G. J. Brakenhoff, and J. Schmidt, "Optical far-field microscopy of single molecules with 3.4 nm lateral resolution," J. Microsc. 205, 76-85 (2002).
    [CrossRef] [PubMed]
  7. A. M. van Oijen, J. K¨ohler, J. Schmidt, M. Müller, and G. J. Brakenhoff, "3-Dimensional super-resolution by spectrally selective imaging," Chem. Phys. Lett. 292, 183-187 (1998).
    [CrossRef]
  8. A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V Walks Hand-Over- Hand: Single Fluorophore Imaging with 1.5-nm Localization," Science 300, 2061-2065 (2003).
    [CrossRef] [PubMed]
  9. J.-M. Segura, A. Renn, and B. Hecht, "A sample-scanning confocal optical microscope for cryogenic operation," Rev. Sci. Instrum. 71, 1706-1711 (2000).
    [CrossRef]
  10. S. Kummer, F. Kulzer, R. Kettner, T. Basché, C. Tietz, C. Glowatz, and C. Kryschi, "Absorption, excitation and emission spectroscopy of terrylene in p-terphenyl: Bulk measurements and single molecule studies," J. Chem. Phys. 107, 7673-7684 (1997).
    [CrossRef]
  11. S. Kummer, T. Basché, and C. Bräuchle, "Terrylene in p-terphenyl: a novel single crystalline system for single molecule spectroscopy at low temperatures," Chem. Phys. Lett. 229, 309-316 (1994).
    [CrossRef]
  12. P. Bordat and R. Brown, "Molecular mechanisms of photo-induced spectral diffusion of single terrylene molecules in p-terphenyl," J. Chem. Phys. 116, 229-236 (2002).
    [CrossRef]
  13. J. Y. P. Butter and B. Hecht, "Aperture scanning near-field optical microscopy and spectroscopy of single terrylene molecules at 1.8 K," Nanotechnology 17, 1547-1550 (2006).
    [CrossRef]
  14. T. Plakhotnik, W. E. Moerner, V. Palm, and U. P. Wild, "Single molecule spectroscopy: maximum emission rate and saturation intensity," Opt. Commun. 114, 83-88 (1995).
    [CrossRef]
  15. J. Bernard, L. Fleury, H. Talon, and M. Orrit, "Photon bunching in the fluorescence from single molecules: A probe for intersystem crossing," J. Chem. Phys. 98, 850-859 (1993).
    [CrossRef]
  16. H. de Vries and D. A. Wiersma, "Photophysical and photochemical molecular hole burning theory," J. Chem. Phys. 72, 1851-1863 (1980).
    [CrossRef]
  17. P. Brémaud, An introdcution to probablistic modeling (Springer-Verlag, 1994).
  18. P. R. Bevington and D. K. Robinson, Data reduction and error analysis for the physical sciences, 2nd ed. (WCB/McGraw-Hill, 1992).

2006

J. Y. P. Butter and B. Hecht, "Aperture scanning near-field optical microscopy and spectroscopy of single terrylene molecules at 1.8 K," Nanotechnology 17, 1547-1550 (2006).
[CrossRef]

2003

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V Walks Hand-Over- Hand: Single Fluorophore Imaging with 1.5-nm Localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

2002

A. Bloeß, Y. Durand, M. Matsushita, H. van der Meer, G. J. Brakenhoff, and J. Schmidt, "Optical far-field microscopy of single molecules with 3.4 nm lateral resolution," J. Microsc. 205, 76-85 (2002).
[CrossRef] [PubMed]

P. Bordat and R. Brown, "Molecular mechanisms of photo-induced spectral diffusion of single terrylene molecules in p-terphenyl," J. Chem. Phys. 116, 229-236 (2002).
[CrossRef]

2000

J.-M. Segura, A. Renn, and B. Hecht, "A sample-scanning confocal optical microscope for cryogenic operation," Rev. Sci. Instrum. 71, 1706-1711 (2000).
[CrossRef]

1998

A. M. van Oijen, J. K¨ohler, J. Schmidt, M. Müller, and G. J. Brakenhoff, "3-Dimensional super-resolution by spectrally selective imaging," Chem. Phys. Lett. 292, 183-187 (1998).
[CrossRef]

1997

S. Kummer, F. Kulzer, R. Kettner, T. Basché, C. Tietz, C. Glowatz, and C. Kryschi, "Absorption, excitation and emission spectroscopy of terrylene in p-terphenyl: Bulk measurements and single molecule studies," J. Chem. Phys. 107, 7673-7684 (1997).
[CrossRef]

1996

T. Schmidt, G. J. Schütz,W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-2929 (1996).
[CrossRef] [PubMed]

1995

T. Plakhotnik, W. E. Moerner, V. Palm, and U. P. Wild, "Single molecule spectroscopy: maximum emission rate and saturation intensity," Opt. Commun. 114, 83-88 (1995).
[CrossRef]

1994

S. Kummer, T. Basché, and C. Bräuchle, "Terrylene in p-terphenyl: a novel single crystalline system for single molecule spectroscopy at low temperatures," Chem. Phys. Lett. 229, 309-316 (1994).
[CrossRef]

1993

J. Bernard, L. Fleury, H. Talon, and M. Orrit, "Photon bunching in the fluorescence from single molecules: A probe for intersystem crossing," J. Chem. Phys. 98, 850-859 (1993).
[CrossRef]

1991

W. P. Ambrose, T. Basché, and W. E. Moerner, "Detection and spectroscopy of single pentacene molecules in a p-terphenyl crystal by means of fluorescence excitation," J. Chem. Phys. 95, 7150-7163 (1991).
[CrossRef]

1986

N. Bobroff, "Position Measurement with a Resolution and Noise-Limited Instrument," Rev. Sci. Instrum. 57, 1152-1157 (1986).
[CrossRef]

1980

H. de Vries and D. A. Wiersma, "Photophysical and photochemical molecular hole burning theory," J. Chem. Phys. 72, 1851-1863 (1980).
[CrossRef]

Ambrose, W. P.

W. P. Ambrose, T. Basché, and W. E. Moerner, "Detection and spectroscopy of single pentacene molecules in a p-terphenyl crystal by means of fluorescence excitation," J. Chem. Phys. 95, 7150-7163 (1991).
[CrossRef]

Basché, T.

S. Kummer, F. Kulzer, R. Kettner, T. Basché, C. Tietz, C. Glowatz, and C. Kryschi, "Absorption, excitation and emission spectroscopy of terrylene in p-terphenyl: Bulk measurements and single molecule studies," J. Chem. Phys. 107, 7673-7684 (1997).
[CrossRef]

S. Kummer, T. Basché, and C. Bräuchle, "Terrylene in p-terphenyl: a novel single crystalline system for single molecule spectroscopy at low temperatures," Chem. Phys. Lett. 229, 309-316 (1994).
[CrossRef]

W. P. Ambrose, T. Basché, and W. E. Moerner, "Detection and spectroscopy of single pentacene molecules in a p-terphenyl crystal by means of fluorescence excitation," J. Chem. Phys. 95, 7150-7163 (1991).
[CrossRef]

Baumgartner, W.

T. Schmidt, G. J. Schütz,W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-2929 (1996).
[CrossRef] [PubMed]

Bernard, J.

J. Bernard, L. Fleury, H. Talon, and M. Orrit, "Photon bunching in the fluorescence from single molecules: A probe for intersystem crossing," J. Chem. Phys. 98, 850-859 (1993).
[CrossRef]

Bloeß, A.

A. Bloeß, Y. Durand, M. Matsushita, H. van der Meer, G. J. Brakenhoff, and J. Schmidt, "Optical far-field microscopy of single molecules with 3.4 nm lateral resolution," J. Microsc. 205, 76-85 (2002).
[CrossRef] [PubMed]

Bobroff, N.

N. Bobroff, "Position Measurement with a Resolution and Noise-Limited Instrument," Rev. Sci. Instrum. 57, 1152-1157 (1986).
[CrossRef]

Bordat, P.

P. Bordat and R. Brown, "Molecular mechanisms of photo-induced spectral diffusion of single terrylene molecules in p-terphenyl," J. Chem. Phys. 116, 229-236 (2002).
[CrossRef]

Brakenhoff, G. J.

A. Bloeß, Y. Durand, M. Matsushita, H. van der Meer, G. J. Brakenhoff, and J. Schmidt, "Optical far-field microscopy of single molecules with 3.4 nm lateral resolution," J. Microsc. 205, 76-85 (2002).
[CrossRef] [PubMed]

Bräuchle, C.

S. Kummer, T. Basché, and C. Bräuchle, "Terrylene in p-terphenyl: a novel single crystalline system for single molecule spectroscopy at low temperatures," Chem. Phys. Lett. 229, 309-316 (1994).
[CrossRef]

Brown, R.

P. Bordat and R. Brown, "Molecular mechanisms of photo-induced spectral diffusion of single terrylene molecules in p-terphenyl," J. Chem. Phys. 116, 229-236 (2002).
[CrossRef]

Butter, J. Y. P.

J. Y. P. Butter and B. Hecht, "Aperture scanning near-field optical microscopy and spectroscopy of single terrylene molecules at 1.8 K," Nanotechnology 17, 1547-1550 (2006).
[CrossRef]

de Vries, H.

H. de Vries and D. A. Wiersma, "Photophysical and photochemical molecular hole burning theory," J. Chem. Phys. 72, 1851-1863 (1980).
[CrossRef]

Durand, Y.

A. Bloeß, Y. Durand, M. Matsushita, H. van der Meer, G. J. Brakenhoff, and J. Schmidt, "Optical far-field microscopy of single molecules with 3.4 nm lateral resolution," J. Microsc. 205, 76-85 (2002).
[CrossRef] [PubMed]

Fleury, L.

J. Bernard, L. Fleury, H. Talon, and M. Orrit, "Photon bunching in the fluorescence from single molecules: A probe for intersystem crossing," J. Chem. Phys. 98, 850-859 (1993).
[CrossRef]

Forkey, J. N.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V Walks Hand-Over- Hand: Single Fluorophore Imaging with 1.5-nm Localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Glowatz, C.

S. Kummer, F. Kulzer, R. Kettner, T. Basché, C. Tietz, C. Glowatz, and C. Kryschi, "Absorption, excitation and emission spectroscopy of terrylene in p-terphenyl: Bulk measurements and single molecule studies," J. Chem. Phys. 107, 7673-7684 (1997).
[CrossRef]

Goldman, Y. E.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V Walks Hand-Over- Hand: Single Fluorophore Imaging with 1.5-nm Localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Gruber, H. J.

T. Schmidt, G. J. Schütz,W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-2929 (1996).
[CrossRef] [PubMed]

Ha, T.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V Walks Hand-Over- Hand: Single Fluorophore Imaging with 1.5-nm Localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Hecht, B.

J. Y. P. Butter and B. Hecht, "Aperture scanning near-field optical microscopy and spectroscopy of single terrylene molecules at 1.8 K," Nanotechnology 17, 1547-1550 (2006).
[CrossRef]

J.-M. Segura, A. Renn, and B. Hecht, "A sample-scanning confocal optical microscope for cryogenic operation," Rev. Sci. Instrum. 71, 1706-1711 (2000).
[CrossRef]

Kettner, R.

S. Kummer, F. Kulzer, R. Kettner, T. Basché, C. Tietz, C. Glowatz, and C. Kryschi, "Absorption, excitation and emission spectroscopy of terrylene in p-terphenyl: Bulk measurements and single molecule studies," J. Chem. Phys. 107, 7673-7684 (1997).
[CrossRef]

Kryschi, C.

S. Kummer, F. Kulzer, R. Kettner, T. Basché, C. Tietz, C. Glowatz, and C. Kryschi, "Absorption, excitation and emission spectroscopy of terrylene in p-terphenyl: Bulk measurements and single molecule studies," J. Chem. Phys. 107, 7673-7684 (1997).
[CrossRef]

Kulzer, F.

S. Kummer, F. Kulzer, R. Kettner, T. Basché, C. Tietz, C. Glowatz, and C. Kryschi, "Absorption, excitation and emission spectroscopy of terrylene in p-terphenyl: Bulk measurements and single molecule studies," J. Chem. Phys. 107, 7673-7684 (1997).
[CrossRef]

Kummer, S.

S. Kummer, F. Kulzer, R. Kettner, T. Basché, C. Tietz, C. Glowatz, and C. Kryschi, "Absorption, excitation and emission spectroscopy of terrylene in p-terphenyl: Bulk measurements and single molecule studies," J. Chem. Phys. 107, 7673-7684 (1997).
[CrossRef]

S. Kummer, T. Basché, and C. Bräuchle, "Terrylene in p-terphenyl: a novel single crystalline system for single molecule spectroscopy at low temperatures," Chem. Phys. Lett. 229, 309-316 (1994).
[CrossRef]

Matsushita, M.

A. Bloeß, Y. Durand, M. Matsushita, H. van der Meer, G. J. Brakenhoff, and J. Schmidt, "Optical far-field microscopy of single molecules with 3.4 nm lateral resolution," J. Microsc. 205, 76-85 (2002).
[CrossRef] [PubMed]

McKinney, S. A.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V Walks Hand-Over- Hand: Single Fluorophore Imaging with 1.5-nm Localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Moerner, W. E.

T. Plakhotnik, W. E. Moerner, V. Palm, and U. P. Wild, "Single molecule spectroscopy: maximum emission rate and saturation intensity," Opt. Commun. 114, 83-88 (1995).
[CrossRef]

W. P. Ambrose, T. Basché, and W. E. Moerner, "Detection and spectroscopy of single pentacene molecules in a p-terphenyl crystal by means of fluorescence excitation," J. Chem. Phys. 95, 7150-7163 (1991).
[CrossRef]

Orrit, M.

J. Bernard, L. Fleury, H. Talon, and M. Orrit, "Photon bunching in the fluorescence from single molecules: A probe for intersystem crossing," J. Chem. Phys. 98, 850-859 (1993).
[CrossRef]

Palm, V.

T. Plakhotnik, W. E. Moerner, V. Palm, and U. P. Wild, "Single molecule spectroscopy: maximum emission rate and saturation intensity," Opt. Commun. 114, 83-88 (1995).
[CrossRef]

Plakhotnik, T.

T. Plakhotnik, W. E. Moerner, V. Palm, and U. P. Wild, "Single molecule spectroscopy: maximum emission rate and saturation intensity," Opt. Commun. 114, 83-88 (1995).
[CrossRef]

Renn, A.

J.-M. Segura, A. Renn, and B. Hecht, "A sample-scanning confocal optical microscope for cryogenic operation," Rev. Sci. Instrum. 71, 1706-1711 (2000).
[CrossRef]

Schindler, H.

T. Schmidt, G. J. Schütz,W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-2929 (1996).
[CrossRef] [PubMed]

Schmidt, J.

A. Bloeß, Y. Durand, M. Matsushita, H. van der Meer, G. J. Brakenhoff, and J. Schmidt, "Optical far-field microscopy of single molecules with 3.4 nm lateral resolution," J. Microsc. 205, 76-85 (2002).
[CrossRef] [PubMed]

Schmidt, T.

T. Schmidt, G. J. Schütz,W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-2929 (1996).
[CrossRef] [PubMed]

Schütz, G. J.

T. Schmidt, G. J. Schütz,W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-2929 (1996).
[CrossRef] [PubMed]

Segura, J.-M.

J.-M. Segura, A. Renn, and B. Hecht, "A sample-scanning confocal optical microscope for cryogenic operation," Rev. Sci. Instrum. 71, 1706-1711 (2000).
[CrossRef]

Selvin, P. R.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V Walks Hand-Over- Hand: Single Fluorophore Imaging with 1.5-nm Localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Talon, H.

J. Bernard, L. Fleury, H. Talon, and M. Orrit, "Photon bunching in the fluorescence from single molecules: A probe for intersystem crossing," J. Chem. Phys. 98, 850-859 (1993).
[CrossRef]

Tietz, C.

S. Kummer, F. Kulzer, R. Kettner, T. Basché, C. Tietz, C. Glowatz, and C. Kryschi, "Absorption, excitation and emission spectroscopy of terrylene in p-terphenyl: Bulk measurements and single molecule studies," J. Chem. Phys. 107, 7673-7684 (1997).
[CrossRef]

van der Meer, H.

A. Bloeß, Y. Durand, M. Matsushita, H. van der Meer, G. J. Brakenhoff, and J. Schmidt, "Optical far-field microscopy of single molecules with 3.4 nm lateral resolution," J. Microsc. 205, 76-85 (2002).
[CrossRef] [PubMed]

van Oijen, A. M.

A. M. van Oijen, J. K¨ohler, J. Schmidt, M. Müller, and G. J. Brakenhoff, "3-Dimensional super-resolution by spectrally selective imaging," Chem. Phys. Lett. 292, 183-187 (1998).
[CrossRef]

Wiersma, D. A.

H. de Vries and D. A. Wiersma, "Photophysical and photochemical molecular hole burning theory," J. Chem. Phys. 72, 1851-1863 (1980).
[CrossRef]

Wild, U. P.

T. Plakhotnik, W. E. Moerner, V. Palm, and U. P. Wild, "Single molecule spectroscopy: maximum emission rate and saturation intensity," Opt. Commun. 114, 83-88 (1995).
[CrossRef]

Yildiz, A.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V Walks Hand-Over- Hand: Single Fluorophore Imaging with 1.5-nm Localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Chem. Phys. Lett.

A. M. van Oijen, J. K¨ohler, J. Schmidt, M. Müller, and G. J. Brakenhoff, "3-Dimensional super-resolution by spectrally selective imaging," Chem. Phys. Lett. 292, 183-187 (1998).
[CrossRef]

S. Kummer, T. Basché, and C. Bräuchle, "Terrylene in p-terphenyl: a novel single crystalline system for single molecule spectroscopy at low temperatures," Chem. Phys. Lett. 229, 309-316 (1994).
[CrossRef]

J. Chem. Phys.

P. Bordat and R. Brown, "Molecular mechanisms of photo-induced spectral diffusion of single terrylene molecules in p-terphenyl," J. Chem. Phys. 116, 229-236 (2002).
[CrossRef]

J. Bernard, L. Fleury, H. Talon, and M. Orrit, "Photon bunching in the fluorescence from single molecules: A probe for intersystem crossing," J. Chem. Phys. 98, 850-859 (1993).
[CrossRef]

H. de Vries and D. A. Wiersma, "Photophysical and photochemical molecular hole burning theory," J. Chem. Phys. 72, 1851-1863 (1980).
[CrossRef]

W. P. Ambrose, T. Basché, and W. E. Moerner, "Detection and spectroscopy of single pentacene molecules in a p-terphenyl crystal by means of fluorescence excitation," J. Chem. Phys. 95, 7150-7163 (1991).
[CrossRef]

S. Kummer, F. Kulzer, R. Kettner, T. Basché, C. Tietz, C. Glowatz, and C. Kryschi, "Absorption, excitation and emission spectroscopy of terrylene in p-terphenyl: Bulk measurements and single molecule studies," J. Chem. Phys. 107, 7673-7684 (1997).
[CrossRef]

J. Microsc.

A. Bloeß, Y. Durand, M. Matsushita, H. van der Meer, G. J. Brakenhoff, and J. Schmidt, "Optical far-field microscopy of single molecules with 3.4 nm lateral resolution," J. Microsc. 205, 76-85 (2002).
[CrossRef] [PubMed]

Nanotechnology

J. Y. P. Butter and B. Hecht, "Aperture scanning near-field optical microscopy and spectroscopy of single terrylene molecules at 1.8 K," Nanotechnology 17, 1547-1550 (2006).
[CrossRef]

Opt. Commun.

T. Plakhotnik, W. E. Moerner, V. Palm, and U. P. Wild, "Single molecule spectroscopy: maximum emission rate and saturation intensity," Opt. Commun. 114, 83-88 (1995).
[CrossRef]

Proc. Natl. Acad. Sci. USA

T. Schmidt, G. J. Schütz,W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-2929 (1996).
[CrossRef] [PubMed]

Rev. Sci. Instrum.

N. Bobroff, "Position Measurement with a Resolution and Noise-Limited Instrument," Rev. Sci. Instrum. 57, 1152-1157 (1986).
[CrossRef]

J.-M. Segura, A. Renn, and B. Hecht, "A sample-scanning confocal optical microscope for cryogenic operation," Rev. Sci. Instrum. 71, 1706-1711 (2000).
[CrossRef]

Science

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V Walks Hand-Over- Hand: Single Fluorophore Imaging with 1.5-nm Localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Other

T. Basché, W. E. Moerner, M. Orrit, and U. P. Wild, eds., Single-Molecule Optical Detection, Imaging and Spectroscopy (VCH Verlagsgesellschaft, Weinheim, 1997).

L. Novotny and B. Hecht, Principles of Nano-Optics (Cambridge University Press, 2006).

P. Brémaud, An introdcution to probablistic modeling (Springer-Verlag, 1994).

P. R. Bevington and D. K. Robinson, Data reduction and error analysis for the physical sciences, 2nd ed. (WCB/McGraw-Hill, 1992).

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Figures (3)

Fig. 1.
Fig. 1.

Classical saturation obtained from a frequency and intensity dependent single-molecule images. (A) Spectral lines of a single molecule at two different intensities. (B) Saturation curve of the same molecule. The lower horizontal axis shows the excitation intensity whereas the upper horizontal axis shows the relative intensities as a function of saturation intensity. (C) Change in the observed HWHMof the imaged spot of the molecule as a function of detuning. The black circles denote the values for I=0.7·I S and the grey triangles for I=6.9·I S . (D) HWHMof the spot in resonance as a function of relative intensity. The spot-size at zero intensity is the measured spot size far out of resonance. The solid grey line denotes the calculated point spread function, which doesn’t take into account any effect due to the presence of superfluid helium. The integration time was 5 ms per pixel.

Fig. 2.
Fig. 2.

Results of the Monte Carlo simulation on the spot-size as a function of detuning below saturation. (A) The average amplitudes with corresponding uncertainties as a function of detuning (squares) and the input values (grey line). (B) The influence of detuning on the HWHM, both absolute in pixels (left axis), with the corresponding input value as a grey line, and in nm assuming a diffraction limited spot size.

Fig. 3.
Fig. 3.

Illustration of the determination of the saturation intensity and the maximum emission rate from 2 spots of a single molecule at the intensity I=0.7·I S . (A) shows the line-profile of the non resonant spot through the center and (B) of the resonant spot. The insets show normalized images, 1.3×1.3 µm in size, of the respective spots. The lines indicate where the line-cut was taken. The solid lines represent Gaussian fits to the data, whereas the dotted line is a fit with Eq. (2).

Equations (5)

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R ( I ) = R · I I S 1 + I I S
R ( x , y , A ) = R · A · e ( x x 0 ) 2 + ( y y 0 ) 2 2 ξ 2 1 + A · e ( x x 0 ) 2 + ( y y 0 ) 2 2 ξ 2
R 1 ( f ) = R 0 + H 4 · ( f f 0 ) 2 W 2 + 1
spot ( x , y , f , ξ ) = R 1 ( f ) · e x 2 + y 2 2 ξ 2
fit ( x , y ) = a + b · e x 2 + y 2 2 c 2

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