Abstract

The interactions between single molecules and three-dimensional donut modes in fluorescence microscopy are discussed based on the vector diffraction theory of light. We find that the use of donut modes generated from a linearly polarized laser beam can yield information about the orientation of immobilized single molecules, allowing for their use in orientational imaging. While fairly insensitive over a range of orientations, this technique is seen to be very sensitive for the subset of orientations where the transition dipole of the molecule is oriented close to the optical axis of the microscope and perpendicular to the input polarization. In a second part of the paper we discuss the impact of the molecular orientation on the resolution improvement in STED microscopy. We find that, even for circularly polarized excitation light, the expected resolution improvement depends on the orientation of the molecule relative to the optical axis of the microscope.

© 2007 Optical Society of America

Full Article  |  PDF Article
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References

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  1. T. Klar, S. Jakobs, M. Dyba, A. Egner, and S. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97,8206–8210 (2000).
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  2. A. Mair, A. Vaziri, G. Weihs, and A. Zeilinger, “Entanglement of the orbital angular momentum states of photons,” Nature 412,313–316 (2001).
    [Crossref] [PubMed]
  3. D. Zhang and X. Yuan, “Optical doughnut for optical tweezers,” Opt. Lett. 28,740–742 (2003).
    [Crossref] [PubMed]
  4. P. Rodrigo, V. Daria, and J. Gluckstad, “Real-time interactive optical micromanipulation of a mixture of high-and low-index particles,” Opt. Express. 12,1417–1425 (2004).
    [Crossref] [PubMed]
  5. J. Hotta, H. Uji-i, and J. Hofkens, “The fabrication of a thin, circular polymer film based phase shaper for generating doughnut modes,” Opt. Express 14,6273–6278 (2006).
    [Crossref] [PubMed]
  6. D. Ganic, X. Gan, and M. Gu, “Focusing of doughnut laser beams by a high numerical-aperture objective in free space,” Opt. Express. 11,2747–2752 (2003).
    [Crossref] [PubMed]
  7. K. Youngworth and T. Brown, “Focusing of high numerical aperture cylindrical-vector beams,” Opt. Express. 7,77–87 (2000).
    [Crossref] [PubMed]
  8. R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91,233,901 (2003).
    [Crossref]
  9. T. Hirayama, Y. Kozawa, T. Nakamura, and S. Shunichi, “Generation of a cylindrically symmetric, polarized laser beam with narrow linewidth and fine tunability,” Opt. Express. 14,12,839–12,845 (2006).
    [Crossref]
  10. S. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21,1347–1355 (2003).
    [Crossref] [PubMed]
  11. M. Hofmann, C. Eggeling, S. Jakobs, and S. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U. S. A. 102,17,565–17,569 (2005).
    [Crossref]
  12. B. Sick, B. Hecht, U. Wild, and L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microscop.-Oxf. 202,365–373 (2001).
    [Crossref]
  13. E. Wolf, “Electromagnetic diffraction in optical systems I. An integral representation of the image field,” Proc. R. Soc. Lond. A 253,349–357 (1959).
    [Crossref]
  14. B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A 253,358–379 (1959).
    [Crossref]
  15. C. Sheppard and T. Wilson, “The image of a single point in microscopes of large numerical aperture,” Proc. R. Soc. A 379,145–158 (1982).
    [Crossref]
  16. J. Enderlein, T. Ruckstuhl, and S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt. 38,724–732 (1999).
    [Crossref]
  17. A. Bartko and R. Dickson, “Imaging three-dimensional single molecule orientations,” J. Phys. Chem. B. 103,11,237–11,241 (1999).
  18. T. Ha, T. Enderle, D. Chemla, P. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77,3979–3982 (1996).
    [Crossref] [PubMed]
  19. T. Ha, T. Laurence, D. Chemla, and S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B. 103,6839–6850 (1999).
    [Crossref]
  20. J. Jasny and J. Sepiol, “Single molecules observed by immersion mirror objective. A novel method of finding the orientation of a radiating dipole,” Chem. Phys. Lett. 273,439–443 (1997).
    [Crossref]
  21. J. Sepiol, J. Jasny, J. Keller, and U. Wild, “Single molecules observed by immersion mirror objective. The orientation of terrylene molecules via the direction of its transition dipole moment,” Chem. Phys. Lett. 273,444–448 (1997).
    [Crossref]
  22. M. Böhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” J. Opt. Soc. Am. B. 20,554–559 (2003).
    [Crossref]
  23. D. Patra, I. Gregor, and J. Enderlein, “Image analysis of defocused single-molecule images for three-dimensional molecule orientation studies,” J. Phys. Chem. A. 108,6836–6841 (2004).
    [Crossref]
  24. M. Lieb, J. Zavislan, and L. Novotny, “Single-molecule orientations determined by direct emission pattern imaging,” J. Opt. Soc. Am. B. 21,1210–1215 (2004).
    [Crossref]
  25. B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85,4482–4485 (2000).
    [Crossref] [PubMed]
  26. L. Novotny, M. Beversluis, K. Youngworth, and T. Brown, “Longitudinal field modes probed by single molecules,” Phys. Rev. Lett. 86,5251–5254 (2001).
    [Crossref] [PubMed]
  27. S. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19,780–782 (1994).
    [Crossref] [PubMed]
  28. G. Donnert, J. Keller, R. Medda, M. Andrei, S. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103,11,440–11,445 (2006).
    [Crossref]
  29. G. Donnert, C. Eggeling, and S. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods. 4,81–86 (2007).
    [Crossref]
  30. V. Westphal, L. Kastrup, and S. Hell, “Lateral resolution of 28 nm (lambda/25) in far-field fluorescence microscopy,” Appl. Phys. B. 77,377–380 (2003).
    [Crossref]
  31. P. Török and P. Munro, “The use of Gauss-Laguerre vector beams in STED microscopy,” Opt. Express. 12,3605–3617 (2004).
    [Crossref] [PubMed]
  32. M. Dyba, J. Keller, and S. Hell, “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment,” New. J. Phys. 7,134 (2005).
    [Crossref]
  33. J. Enderlein, E. Toprak, and P. Selvin, “Polarization effect on position accuracy of fluorophore localization,” Opt. Express. 14,8111–8120 (2006).
    [Crossref] [PubMed]

2007 (1)

G. Donnert, C. Eggeling, and S. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods. 4,81–86 (2007).
[Crossref]

2006 (4)

G. Donnert, J. Keller, R. Medda, M. Andrei, S. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103,11,440–11,445 (2006).
[Crossref]

J. Enderlein, E. Toprak, and P. Selvin, “Polarization effect on position accuracy of fluorophore localization,” Opt. Express. 14,8111–8120 (2006).
[Crossref] [PubMed]

J. Hotta, H. Uji-i, and J. Hofkens, “The fabrication of a thin, circular polymer film based phase shaper for generating doughnut modes,” Opt. Express 14,6273–6278 (2006).
[Crossref] [PubMed]

T. Hirayama, Y. Kozawa, T. Nakamura, and S. Shunichi, “Generation of a cylindrically symmetric, polarized laser beam with narrow linewidth and fine tunability,” Opt. Express. 14,12,839–12,845 (2006).
[Crossref]

2005 (2)

M. Hofmann, C. Eggeling, S. Jakobs, and S. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U. S. A. 102,17,565–17,569 (2005).
[Crossref]

M. Dyba, J. Keller, and S. Hell, “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment,” New. J. Phys. 7,134 (2005).
[Crossref]

2004 (4)

P. Török and P. Munro, “The use of Gauss-Laguerre vector beams in STED microscopy,” Opt. Express. 12,3605–3617 (2004).
[Crossref] [PubMed]

P. Rodrigo, V. Daria, and J. Gluckstad, “Real-time interactive optical micromanipulation of a mixture of high-and low-index particles,” Opt. Express. 12,1417–1425 (2004).
[Crossref] [PubMed]

D. Patra, I. Gregor, and J. Enderlein, “Image analysis of defocused single-molecule images for three-dimensional molecule orientation studies,” J. Phys. Chem. A. 108,6836–6841 (2004).
[Crossref]

M. Lieb, J. Zavislan, and L. Novotny, “Single-molecule orientations determined by direct emission pattern imaging,” J. Opt. Soc. Am. B. 21,1210–1215 (2004).
[Crossref]

2003 (6)

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91,233,901 (2003).
[Crossref]

S. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21,1347–1355 (2003).
[Crossref] [PubMed]

D. Ganic, X. Gan, and M. Gu, “Focusing of doughnut laser beams by a high numerical-aperture objective in free space,” Opt. Express. 11,2747–2752 (2003).
[Crossref] [PubMed]

V. Westphal, L. Kastrup, and S. Hell, “Lateral resolution of 28 nm (lambda/25) in far-field fluorescence microscopy,” Appl. Phys. B. 77,377–380 (2003).
[Crossref]

M. Böhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” J. Opt. Soc. Am. B. 20,554–559 (2003).
[Crossref]

D. Zhang and X. Yuan, “Optical doughnut for optical tweezers,” Opt. Lett. 28,740–742 (2003).
[Crossref] [PubMed]

2001 (3)

A. Mair, A. Vaziri, G. Weihs, and A. Zeilinger, “Entanglement of the orbital angular momentum states of photons,” Nature 412,313–316 (2001).
[Crossref] [PubMed]

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

B. Sick, B. Hecht, U. Wild, and L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microscop.-Oxf. 202,365–373 (2001).
[Crossref]

2000 (3)

K. Youngworth and T. Brown, “Focusing of high numerical aperture cylindrical-vector beams,” Opt. Express. 7,77–87 (2000).
[Crossref] [PubMed]

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

T. Klar, S. Jakobs, M. Dyba, A. Egner, and S. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97,8206–8210 (2000).
[Crossref] [PubMed]

1999 (3)

J. Enderlein, T. Ruckstuhl, and S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt. 38,724–732 (1999).
[Crossref]

T. Ha, T. Laurence, D. Chemla, and S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B. 103,6839–6850 (1999).
[Crossref]

A. Bartko and R. Dickson, “Imaging three-dimensional single molecule orientations,” J. Phys. Chem. B. 103,11,237–11,241 (1999).

1997 (2)

J. Jasny and J. Sepiol, “Single molecules observed by immersion mirror objective. A novel method of finding the orientation of a radiating dipole,” Chem. Phys. Lett. 273,439–443 (1997).
[Crossref]

J. Sepiol, J. Jasny, J. Keller, and U. Wild, “Single molecules observed by immersion mirror objective. The orientation of terrylene molecules via the direction of its transition dipole moment,” Chem. Phys. Lett. 273,444–448 (1997).
[Crossref]

1996 (1)

T. Ha, T. Enderle, D. Chemla, P. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77,3979–3982 (1996).
[Crossref] [PubMed]

1994 (1)

1982 (1)

C. Sheppard and T. Wilson, “The image of a single point in microscopes of large numerical aperture,” Proc. R. Soc. A 379,145–158 (1982).
[Crossref]

1959 (2)

E. Wolf, “Electromagnetic diffraction in optical systems I. An integral representation of the image field,” Proc. R. Soc. Lond. A 253,349–357 (1959).
[Crossref]

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A 253,358–379 (1959).
[Crossref]

Andrei, M.

G. Donnert, J. Keller, R. Medda, M. Andrei, S. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103,11,440–11,445 (2006).
[Crossref]

Bartko, A.

A. Bartko and R. Dickson, “Imaging three-dimensional single molecule orientations,” J. Phys. Chem. B. 103,11,237–11,241 (1999).

Beversluis, M.

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

Böhmer, M.

M. Böhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” J. Opt. Soc. Am. B. 20,554–559 (2003).
[Crossref]

Brown, T.

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

K. Youngworth and T. Brown, “Focusing of high numerical aperture cylindrical-vector beams,” Opt. Express. 7,77–87 (2000).
[Crossref] [PubMed]

Chemla, D.

T. Ha, T. Laurence, D. Chemla, and S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B. 103,6839–6850 (1999).
[Crossref]

T. Ha, T. Enderle, D. Chemla, P. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77,3979–3982 (1996).
[Crossref] [PubMed]

Daria, V.

P. Rodrigo, V. Daria, and J. Gluckstad, “Real-time interactive optical micromanipulation of a mixture of high-and low-index particles,” Opt. Express. 12,1417–1425 (2004).
[Crossref] [PubMed]

Dickson, R.

A. Bartko and R. Dickson, “Imaging three-dimensional single molecule orientations,” J. Phys. Chem. B. 103,11,237–11,241 (1999).

Donnert, G.

G. Donnert, C. Eggeling, and S. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods. 4,81–86 (2007).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. Andrei, S. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103,11,440–11,445 (2006).
[Crossref]

Dorn, R.

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91,233,901 (2003).
[Crossref]

Dyba, M.

M. Dyba, J. Keller, and S. Hell, “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment,” New. J. Phys. 7,134 (2005).
[Crossref]

T. Klar, S. Jakobs, M. Dyba, A. Egner, and S. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97,8206–8210 (2000).
[Crossref] [PubMed]

Eggeling, C.

G. Donnert, C. Eggeling, and S. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods. 4,81–86 (2007).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. Andrei, S. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103,11,440–11,445 (2006).
[Crossref]

M. Hofmann, C. Eggeling, S. Jakobs, and S. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U. S. A. 102,17,565–17,569 (2005).
[Crossref]

Egner, A.

T. Klar, S. Jakobs, M. Dyba, A. Egner, and S. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97,8206–8210 (2000).
[Crossref] [PubMed]

Enderle, T.

T. Ha, T. Enderle, D. Chemla, P. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77,3979–3982 (1996).
[Crossref] [PubMed]

Enderlein, J.

J. Enderlein, E. Toprak, and P. Selvin, “Polarization effect on position accuracy of fluorophore localization,” Opt. Express. 14,8111–8120 (2006).
[Crossref] [PubMed]

D. Patra, I. Gregor, and J. Enderlein, “Image analysis of defocused single-molecule images for three-dimensional molecule orientation studies,” J. Phys. Chem. A. 108,6836–6841 (2004).
[Crossref]

M. Böhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” J. Opt. Soc. Am. B. 20,554–559 (2003).
[Crossref]

J. Enderlein, T. Ruckstuhl, and S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt. 38,724–732 (1999).
[Crossref]

Gan, X.

D. Ganic, X. Gan, and M. Gu, “Focusing of doughnut laser beams by a high numerical-aperture objective in free space,” Opt. Express. 11,2747–2752 (2003).
[Crossref] [PubMed]

Ganic, D.

D. Ganic, X. Gan, and M. Gu, “Focusing of doughnut laser beams by a high numerical-aperture objective in free space,” Opt. Express. 11,2747–2752 (2003).
[Crossref] [PubMed]

Gluckstad, J.

P. Rodrigo, V. Daria, and J. Gluckstad, “Real-time interactive optical micromanipulation of a mixture of high-and low-index particles,” Opt. Express. 12,1417–1425 (2004).
[Crossref] [PubMed]

Gregor, I.

D. Patra, I. Gregor, and J. Enderlein, “Image analysis of defocused single-molecule images for three-dimensional molecule orientation studies,” J. Phys. Chem. A. 108,6836–6841 (2004).
[Crossref]

Gu, M.

D. Ganic, X. Gan, and M. Gu, “Focusing of doughnut laser beams by a high numerical-aperture objective in free space,” Opt. Express. 11,2747–2752 (2003).
[Crossref] [PubMed]

Ha, T.

T. Ha, T. Laurence, D. Chemla, and S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B. 103,6839–6850 (1999).
[Crossref]

T. Ha, T. Enderle, D. Chemla, P. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77,3979–3982 (1996).
[Crossref] [PubMed]

Hecht, B.

B. Sick, B. Hecht, U. Wild, and L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microscop.-Oxf. 202,365–373 (2001).
[Crossref]

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

Hell, S.

G. Donnert, C. Eggeling, and S. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods. 4,81–86 (2007).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. Andrei, S. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103,11,440–11,445 (2006).
[Crossref]

M. Dyba, J. Keller, and S. Hell, “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment,” New. J. Phys. 7,134 (2005).
[Crossref]

M. Hofmann, C. Eggeling, S. Jakobs, and S. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U. S. A. 102,17,565–17,569 (2005).
[Crossref]

S. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21,1347–1355 (2003).
[Crossref] [PubMed]

V. Westphal, L. Kastrup, and S. Hell, “Lateral resolution of 28 nm (lambda/25) in far-field fluorescence microscopy,” Appl. Phys. B. 77,377–380 (2003).
[Crossref]

T. Klar, S. Jakobs, M. Dyba, A. Egner, and S. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97,8206–8210 (2000).
[Crossref] [PubMed]

S. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19,780–782 (1994).
[Crossref] [PubMed]

Hirayama, T.

T. Hirayama, Y. Kozawa, T. Nakamura, and S. Shunichi, “Generation of a cylindrically symmetric, polarized laser beam with narrow linewidth and fine tunability,” Opt. Express. 14,12,839–12,845 (2006).
[Crossref]

Hofkens, J.

Hofmann, M.

M. Hofmann, C. Eggeling, S. Jakobs, and S. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U. S. A. 102,17,565–17,569 (2005).
[Crossref]

Hotta, J.

Jahn, R.

G. Donnert, J. Keller, R. Medda, M. Andrei, S. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103,11,440–11,445 (2006).
[Crossref]

Jakobs, S.

M. Hofmann, C. Eggeling, S. Jakobs, and S. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U. S. A. 102,17,565–17,569 (2005).
[Crossref]

T. Klar, S. Jakobs, M. Dyba, A. Egner, and S. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97,8206–8210 (2000).
[Crossref] [PubMed]

Jasny, J.

J. Jasny and J. Sepiol, “Single molecules observed by immersion mirror objective. A novel method of finding the orientation of a radiating dipole,” Chem. Phys. Lett. 273,439–443 (1997).
[Crossref]

J. Sepiol, J. Jasny, J. Keller, and U. Wild, “Single molecules observed by immersion mirror objective. The orientation of terrylene molecules via the direction of its transition dipole moment,” Chem. Phys. Lett. 273,444–448 (1997).
[Crossref]

Kastrup, L.

V. Westphal, L. Kastrup, and S. Hell, “Lateral resolution of 28 nm (lambda/25) in far-field fluorescence microscopy,” Appl. Phys. B. 77,377–380 (2003).
[Crossref]

Keller, J.

G. Donnert, J. Keller, R. Medda, M. Andrei, S. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103,11,440–11,445 (2006).
[Crossref]

M. Dyba, J. Keller, and S. Hell, “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment,” New. J. Phys. 7,134 (2005).
[Crossref]

J. Sepiol, J. Jasny, J. Keller, and U. Wild, “Single molecules observed by immersion mirror objective. The orientation of terrylene molecules via the direction of its transition dipole moment,” Chem. Phys. Lett. 273,444–448 (1997).
[Crossref]

Klar, T.

T. Klar, S. Jakobs, M. Dyba, A. Egner, and S. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97,8206–8210 (2000).
[Crossref] [PubMed]

Kozawa, Y.

T. Hirayama, Y. Kozawa, T. Nakamura, and S. Shunichi, “Generation of a cylindrically symmetric, polarized laser beam with narrow linewidth and fine tunability,” Opt. Express. 14,12,839–12,845 (2006).
[Crossref]

Laurence, T.

T. Ha, T. Laurence, D. Chemla, and S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B. 103,6839–6850 (1999).
[Crossref]

Leuchs, G.

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91,233,901 (2003).
[Crossref]

Lieb, M.

M. Lieb, J. Zavislan, and L. Novotny, “Single-molecule orientations determined by direct emission pattern imaging,” J. Opt. Soc. Am. B. 21,1210–1215 (2004).
[Crossref]

Lührmann, R.

G. Donnert, J. Keller, R. Medda, M. Andrei, S. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103,11,440–11,445 (2006).
[Crossref]

Mair, A.

A. Mair, A. Vaziri, G. Weihs, and A. Zeilinger, “Entanglement of the orbital angular momentum states of photons,” Nature 412,313–316 (2001).
[Crossref] [PubMed]

Medda, R.

G. Donnert, J. Keller, R. Medda, M. Andrei, S. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103,11,440–11,445 (2006).
[Crossref]

Munro, P.

P. Török and P. Munro, “The use of Gauss-Laguerre vector beams in STED microscopy,” Opt. Express. 12,3605–3617 (2004).
[Crossref] [PubMed]

Nakamura, T.

T. Hirayama, Y. Kozawa, T. Nakamura, and S. Shunichi, “Generation of a cylindrically symmetric, polarized laser beam with narrow linewidth and fine tunability,” Opt. Express. 14,12,839–12,845 (2006).
[Crossref]

Novotny, L.

M. Lieb, J. Zavislan, and L. Novotny, “Single-molecule orientations determined by direct emission pattern imaging,” J. Opt. Soc. Am. B. 21,1210–1215 (2004).
[Crossref]

B. Sick, B. Hecht, U. Wild, and L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microscop.-Oxf. 202,365–373 (2001).
[Crossref]

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

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

Patra, D.

D. Patra, I. Gregor, and J. Enderlein, “Image analysis of defocused single-molecule images for three-dimensional molecule orientation studies,” J. Phys. Chem. A. 108,6836–6841 (2004).
[Crossref]

Quabis, S.

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91,233,901 (2003).
[Crossref]

Richards, B.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A 253,358–379 (1959).
[Crossref]

Rizzoli, S.

G. Donnert, J. Keller, R. Medda, M. Andrei, S. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103,11,440–11,445 (2006).
[Crossref]

Rodrigo, P.

P. Rodrigo, V. Daria, and J. Gluckstad, “Real-time interactive optical micromanipulation of a mixture of high-and low-index particles,” Opt. Express. 12,1417–1425 (2004).
[Crossref] [PubMed]

Ruckstuhl, T.

Seeger, S.

Selvin, P.

J. Enderlein, E. Toprak, and P. Selvin, “Polarization effect on position accuracy of fluorophore localization,” Opt. Express. 14,8111–8120 (2006).
[Crossref] [PubMed]

T. Ha, T. Enderle, D. Chemla, P. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77,3979–3982 (1996).
[Crossref] [PubMed]

Sepiol, J.

J. Jasny and J. Sepiol, “Single molecules observed by immersion mirror objective. A novel method of finding the orientation of a radiating dipole,” Chem. Phys. Lett. 273,439–443 (1997).
[Crossref]

J. Sepiol, J. Jasny, J. Keller, and U. Wild, “Single molecules observed by immersion mirror objective. The orientation of terrylene molecules via the direction of its transition dipole moment,” Chem. Phys. Lett. 273,444–448 (1997).
[Crossref]

Sheppard, C.

C. Sheppard and T. Wilson, “The image of a single point in microscopes of large numerical aperture,” Proc. R. Soc. A 379,145–158 (1982).
[Crossref]

Shunichi, S.

T. Hirayama, Y. Kozawa, T. Nakamura, and S. Shunichi, “Generation of a cylindrically symmetric, polarized laser beam with narrow linewidth and fine tunability,” Opt. Express. 14,12,839–12,845 (2006).
[Crossref]

Sick, B.

B. Sick, B. Hecht, U. Wild, and L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microscop.-Oxf. 202,365–373 (2001).
[Crossref]

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

Toprak, E.

J. Enderlein, E. Toprak, and P. Selvin, “Polarization effect on position accuracy of fluorophore localization,” Opt. Express. 14,8111–8120 (2006).
[Crossref] [PubMed]

Török, P.

P. Török and P. Munro, “The use of Gauss-Laguerre vector beams in STED microscopy,” Opt. Express. 12,3605–3617 (2004).
[Crossref] [PubMed]

Uji-i, H.

Vaziri, A.

A. Mair, A. Vaziri, G. Weihs, and A. Zeilinger, “Entanglement of the orbital angular momentum states of photons,” Nature 412,313–316 (2001).
[Crossref] [PubMed]

Weihs, G.

A. Mair, A. Vaziri, G. Weihs, and A. Zeilinger, “Entanglement of the orbital angular momentum states of photons,” Nature 412,313–316 (2001).
[Crossref] [PubMed]

Weiss, S.

T. Ha, T. Laurence, D. Chemla, and S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B. 103,6839–6850 (1999).
[Crossref]

T. Ha, T. Enderle, D. Chemla, P. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77,3979–3982 (1996).
[Crossref] [PubMed]

Westphal, V.

V. Westphal, L. Kastrup, and S. Hell, “Lateral resolution of 28 nm (lambda/25) in far-field fluorescence microscopy,” Appl. Phys. B. 77,377–380 (2003).
[Crossref]

Wichmann, J.

Wild, U.

B. Sick, B. Hecht, U. Wild, and L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microscop.-Oxf. 202,365–373 (2001).
[Crossref]

J. Sepiol, J. Jasny, J. Keller, and U. Wild, “Single molecules observed by immersion mirror objective. The orientation of terrylene molecules via the direction of its transition dipole moment,” Chem. Phys. Lett. 273,444–448 (1997).
[Crossref]

Wilson, T.

C. Sheppard and T. Wilson, “The image of a single point in microscopes of large numerical aperture,” Proc. R. Soc. A 379,145–158 (1982).
[Crossref]

Wolf, E.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A 253,358–379 (1959).
[Crossref]

E. Wolf, “Electromagnetic diffraction in optical systems I. An integral representation of the image field,” Proc. R. Soc. Lond. A 253,349–357 (1959).
[Crossref]

Youngworth, K.

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

K. Youngworth and T. Brown, “Focusing of high numerical aperture cylindrical-vector beams,” Opt. Express. 7,77–87 (2000).
[Crossref] [PubMed]

Yuan, X.

Zavislan, J.

M. Lieb, J. Zavislan, and L. Novotny, “Single-molecule orientations determined by direct emission pattern imaging,” J. Opt. Soc. Am. B. 21,1210–1215 (2004).
[Crossref]

Zeilinger, A.

A. Mair, A. Vaziri, G. Weihs, and A. Zeilinger, “Entanglement of the orbital angular momentum states of photons,” Nature 412,313–316 (2001).
[Crossref] [PubMed]

Zhang, D.

Appl. Opt. (1)

Appl. Phys. B. (1)

V. Westphal, L. Kastrup, and S. Hell, “Lateral resolution of 28 nm (lambda/25) in far-field fluorescence microscopy,” Appl. Phys. B. 77,377–380 (2003).
[Crossref]

Chem. Phys. Lett. (2)

J. Jasny and J. Sepiol, “Single molecules observed by immersion mirror objective. A novel method of finding the orientation of a radiating dipole,” Chem. Phys. Lett. 273,439–443 (1997).
[Crossref]

J. Sepiol, J. Jasny, J. Keller, and U. Wild, “Single molecules observed by immersion mirror objective. The orientation of terrylene molecules via the direction of its transition dipole moment,” Chem. Phys. Lett. 273,444–448 (1997).
[Crossref]

J. Microscop.-Oxf. (1)

B. Sick, B. Hecht, U. Wild, and L. Novotny, “Probing confined fields with single molecules and vice versa,” J. Microscop.-Oxf. 202,365–373 (2001).
[Crossref]

J. Opt. Soc. Am. B. (2)

M. Böhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” J. Opt. Soc. Am. B. 20,554–559 (2003).
[Crossref]

M. Lieb, J. Zavislan, and L. Novotny, “Single-molecule orientations determined by direct emission pattern imaging,” J. Opt. Soc. Am. B. 21,1210–1215 (2004).
[Crossref]

J. Phys. Chem. A. (1)

D. Patra, I. Gregor, and J. Enderlein, “Image analysis of defocused single-molecule images for three-dimensional molecule orientation studies,” J. Phys. Chem. A. 108,6836–6841 (2004).
[Crossref]

J. Phys. Chem. B. (2)

T. Ha, T. Laurence, D. Chemla, and S. Weiss, “Polarization spectroscopy of single fluorescent molecules,” J. Phys. Chem. B. 103,6839–6850 (1999).
[Crossref]

A. Bartko and R. Dickson, “Imaging three-dimensional single molecule orientations,” J. Phys. Chem. B. 103,11,237–11,241 (1999).

Nat. Biotechnol. (1)

S. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21,1347–1355 (2003).
[Crossref] [PubMed]

Nat. Methods. (1)

G. Donnert, C. Eggeling, and S. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods. 4,81–86 (2007).
[Crossref]

Nature (1)

A. Mair, A. Vaziri, G. Weihs, and A. Zeilinger, “Entanglement of the orbital angular momentum states of photons,” Nature 412,313–316 (2001).
[Crossref] [PubMed]

New. J. Phys. (1)

M. Dyba, J. Keller, and S. Hell, “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment,” New. J. Phys. 7,134 (2005).
[Crossref]

Opt. Express (1)

Opt. Express. (6)

D. Ganic, X. Gan, and M. Gu, “Focusing of doughnut laser beams by a high numerical-aperture objective in free space,” Opt. Express. 11,2747–2752 (2003).
[Crossref] [PubMed]

K. Youngworth and T. Brown, “Focusing of high numerical aperture cylindrical-vector beams,” Opt. Express. 7,77–87 (2000).
[Crossref] [PubMed]

P. Rodrigo, V. Daria, and J. Gluckstad, “Real-time interactive optical micromanipulation of a mixture of high-and low-index particles,” Opt. Express. 12,1417–1425 (2004).
[Crossref] [PubMed]

T. Hirayama, Y. Kozawa, T. Nakamura, and S. Shunichi, “Generation of a cylindrically symmetric, polarized laser beam with narrow linewidth and fine tunability,” Opt. Express. 14,12,839–12,845 (2006).
[Crossref]

J. Enderlein, E. Toprak, and P. Selvin, “Polarization effect on position accuracy of fluorophore localization,” Opt. Express. 14,8111–8120 (2006).
[Crossref] [PubMed]

P. Török and P. Munro, “The use of Gauss-Laguerre vector beams in STED microscopy,” Opt. Express. 12,3605–3617 (2004).
[Crossref] [PubMed]

Opt. Lett. (2)

Phys. Rev. Lett. (4)

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91,233,901 (2003).
[Crossref]

T. Ha, T. Enderle, D. Chemla, P. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77,3979–3982 (1996).
[Crossref] [PubMed]

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

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

Proc. Natl. Acad. Sci. U. S. A. (3)

G. Donnert, J. Keller, R. Medda, M. Andrei, S. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103,11,440–11,445 (2006).
[Crossref]

M. Hofmann, C. Eggeling, S. Jakobs, and S. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U. S. A. 102,17,565–17,569 (2005).
[Crossref]

T. Klar, S. Jakobs, M. Dyba, A. Egner, and S. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97,8206–8210 (2000).
[Crossref] [PubMed]

Proc. R. Soc. A (1)

C. Sheppard and T. Wilson, “The image of a single point in microscopes of large numerical aperture,” Proc. R. Soc. A 379,145–158 (1982).
[Crossref]

Proc. R. Soc. Lond. A (2)

E. Wolf, “Electromagnetic diffraction in optical systems I. An integral representation of the image field,” Proc. R. Soc. Lond. A 253,349–357 (1959).
[Crossref]

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A 253,358–379 (1959).
[Crossref]

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Figures (7)

Fig. 1.
Fig. 1.

An experimental fluorescence scanning image for rotationally immobilized single TDI molecules in a PMMA matrix, with two different color scalings. Patterns of differing shapes and intensities are clearly visible.

Fig. 2.
Fig. 2.

(a) The coordinate system used for defining the molecular orientation, where the shaded double arrow indicates the molecular transition dipole moment. (b-d) Experimentally obtained scanning images of single TDI molecules with different orientations, and (e-g) comparison with theoretically calculated patterns.

Fig. 3.
Fig. 3.

Simulated fluorescence distributions for single molecules at different molecular orientations, using a linearly polarized donut-mode laser beam as the excitation source. The bright parts of the image are the positions with the most intense dipole interaction (where the molecule is most likely to absorb a photon). The values listed next to the images refer to the maximum observable fluorescence emission for that orientation, relative to that of an x-oriented molecule.

Fig. 4.
Fig. 4.

Expansion of the fluorescence distributions for molecular orientations close to the optical axis of the microscope and perpendicular to the polarization of the input laser beam (θ ≈ 90°, ϕ ≈ 90°) The values listed next to the images refer to the maximum observable fluorescence emission for that orientation, relative to that of an x-oriented molecule.

Fig. 5.
Fig. 5.

(a) Simulated dipole interactions for a linearly polarized normal-mode laser beam (‘excitation’) and donut-mode beam (‘dump’) for different molecular orientations. (b) Simulated fluorescence distributions obtained for STED measurements at different powers for the dump beam. The molecular orientations corresponds to those in (a). For comparison purposes the relative fluorescence maximum of each image F r max is given relative to the maximum for normal-mode excitation of an x-oriented molecule. Note that the values for F r max are not corrected for the different collection and detection efficiencies associated with a particular experimental geometry and dipole orientation.

Fig. 6.
Fig. 6.

(Top and Middle) Simulated dipole interactions for molecules at different out-of-plane orientations, using circularly polarized input beams. (Bottom) The resulting simulated fluorescence images. In these calculations the ratio of kSTED to k STED sat was 100.

Fig. 7.
Fig. 7.

(a) The maximum fluorescence emission calculated for each simulated STED image as a function of the out-of-plane angle θ, with (red) and without (blue) the dump beam. (b) Expected FWHM of the fluorescence images as a function of θ for measurements with (red) and without (blue) the dump beam. In both of these plots k STED k STED sat = 100. (c) The probability distribution to obtain a certain FWHM (with arbitrary scaling), determined by the probability for a randomly oriented molecule to have its transition dipole moment between θ and θ + . The values of k STED k STED sat for each distribution are given in the figure legend.

Equations (1)

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I = I 0 1 k de act + k STED { k de act + k STED exp [ ( k de act + k STED ) τ ] }

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