Abstract

We report the generation of thin (< 50 nm) fluorescence layers on a glass substrate in the focal region of a high-numerical-aperture lens by employing subpicosecond pulses of 0.75-TW/cm2 peak intensity. The conditions for generating the fluorescence layers are described. We find that the fluorescence molecules at the glass interface are less affected by bleaching than those in the surrounding area. The fluorescence layers are suited for measuring and monitoring the axial resolution of two-photon fluorescence microscopes.

© 1995 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon fluorescence scanning microscopy,” Science 248, 73–75 (1990).
    [CrossRef] [PubMed]
  2. E. H. K. Stelzer, S. W. Hell, S. Lindek, R. Pick, C. Storz, R. Stricker, G. Ritter, N. Salmon, “Non-linear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
    [CrossRef]
  3. S. W. Hell, E. H. K. Stelzer, “Properties of a 4Pi confocal microscope,” J. Opt. Soc. Am. A 9, 2159–2166 (1992).
    [CrossRef]
  4. S. W. Hell, E. H. K. Stelzer, “Fundamental improvement of resolution with a 4Pi-confocal microscope using two-photon excitation,” Opt. Commun. 93, 277–282 (1992).
    [CrossRef]
  5. S. W. Hell, S. Lindek, E. H. K. Stelzer, “Enchancing the axial resolution in far-field light microscopy: two-photon excitation 4PI confocal fluorescence microscopy,” J. Mod Opt. 41, 675–681 (1994).
    [CrossRef]
  6. Min Gu, T. Tannous, C. J. R. Sheppard, “Improved axial resolution in a confocal fluorescence microscopy using annular pupils,” Opt. Commun. 110, 533–539 (1994).
    [CrossRef]
  7. P. E. Hänninen, S. W. Hell, “Femtosecond pulse broadening in the focal region of a two-photon excitation microscope,” Bioimaging 2, 117–121 (1994).
    [CrossRef]
  8. T. Wilson, R. Juskaitis, “The axial response of confocal microscopes with high numerical aperture objective lenses,” Bioimaging (to be published).
  9. R. Juskaitis, T. Wilson, Department of Engineering Science, Oxford University, Oxford, OX1 3PJ, UK (personal communication, 1994).
  10. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983) Chap. 5, pp. 112–120.
  11. L. P. Ghislain, W. W. Webb, “Scanning-force microscope based on an optical trap,” Opt. Lett. 18, 1678–1680 (1993).
    [CrossRef] [PubMed]
  12. L. Malmqvist, H. M. Hertz, “Two-color trapped-particle optical microscopy,” Opt. Lett. 19, 853–855 (1994).
    [CrossRef] [PubMed]

1994 (5)

S. W. Hell, S. Lindek, E. H. K. Stelzer, “Enchancing the axial resolution in far-field light microscopy: two-photon excitation 4PI confocal fluorescence microscopy,” J. Mod Opt. 41, 675–681 (1994).
[CrossRef]

Min Gu, T. Tannous, C. J. R. Sheppard, “Improved axial resolution in a confocal fluorescence microscopy using annular pupils,” Opt. Commun. 110, 533–539 (1994).
[CrossRef]

P. E. Hänninen, S. W. Hell, “Femtosecond pulse broadening in the focal region of a two-photon excitation microscope,” Bioimaging 2, 117–121 (1994).
[CrossRef]

E. H. K. Stelzer, S. W. Hell, S. Lindek, R. Pick, C. Storz, R. Stricker, G. Ritter, N. Salmon, “Non-linear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

L. Malmqvist, H. M. Hertz, “Two-color trapped-particle optical microscopy,” Opt. Lett. 19, 853–855 (1994).
[CrossRef] [PubMed]

1993 (1)

1992 (2)

S. W. Hell, E. H. K. Stelzer, “Properties of a 4Pi confocal microscope,” J. Opt. Soc. Am. A 9, 2159–2166 (1992).
[CrossRef]

S. W. Hell, E. H. K. Stelzer, “Fundamental improvement of resolution with a 4Pi-confocal microscope using two-photon excitation,” Opt. Commun. 93, 277–282 (1992).
[CrossRef]

1990 (1)

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon fluorescence scanning microscopy,” Science 248, 73–75 (1990).
[CrossRef] [PubMed]

Denk, W.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon fluorescence scanning microscopy,” Science 248, 73–75 (1990).
[CrossRef] [PubMed]

Ghislain, L. P.

Gu, Min

Min Gu, T. Tannous, C. J. R. Sheppard, “Improved axial resolution in a confocal fluorescence microscopy using annular pupils,” Opt. Commun. 110, 533–539 (1994).
[CrossRef]

Hänninen, P. E.

P. E. Hänninen, S. W. Hell, “Femtosecond pulse broadening in the focal region of a two-photon excitation microscope,” Bioimaging 2, 117–121 (1994).
[CrossRef]

Hell, S. W.

P. E. Hänninen, S. W. Hell, “Femtosecond pulse broadening in the focal region of a two-photon excitation microscope,” Bioimaging 2, 117–121 (1994).
[CrossRef]

E. H. K. Stelzer, S. W. Hell, S. Lindek, R. Pick, C. Storz, R. Stricker, G. Ritter, N. Salmon, “Non-linear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

S. W. Hell, S. Lindek, E. H. K. Stelzer, “Enchancing the axial resolution in far-field light microscopy: two-photon excitation 4PI confocal fluorescence microscopy,” J. Mod Opt. 41, 675–681 (1994).
[CrossRef]

S. W. Hell, E. H. K. Stelzer, “Properties of a 4Pi confocal microscope,” J. Opt. Soc. Am. A 9, 2159–2166 (1992).
[CrossRef]

S. W. Hell, E. H. K. Stelzer, “Fundamental improvement of resolution with a 4Pi-confocal microscope using two-photon excitation,” Opt. Commun. 93, 277–282 (1992).
[CrossRef]

Hertz, H. M.

Juskaitis, R.

R. Juskaitis, T. Wilson, Department of Engineering Science, Oxford University, Oxford, OX1 3PJ, UK (personal communication, 1994).

T. Wilson, R. Juskaitis, “The axial response of confocal microscopes with high numerical aperture objective lenses,” Bioimaging (to be published).

Lakowicz, J. R.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983) Chap. 5, pp. 112–120.

Lindek, S.

E. H. K. Stelzer, S. W. Hell, S. Lindek, R. Pick, C. Storz, R. Stricker, G. Ritter, N. Salmon, “Non-linear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

S. W. Hell, S. Lindek, E. H. K. Stelzer, “Enchancing the axial resolution in far-field light microscopy: two-photon excitation 4PI confocal fluorescence microscopy,” J. Mod Opt. 41, 675–681 (1994).
[CrossRef]

Malmqvist, L.

Pick, R.

E. H. K. Stelzer, S. W. Hell, S. Lindek, R. Pick, C. Storz, R. Stricker, G. Ritter, N. Salmon, “Non-linear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

Ritter, G.

E. H. K. Stelzer, S. W. Hell, S. Lindek, R. Pick, C. Storz, R. Stricker, G. Ritter, N. Salmon, “Non-linear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

Salmon, N.

E. H. K. Stelzer, S. W. Hell, S. Lindek, R. Pick, C. Storz, R. Stricker, G. Ritter, N. Salmon, “Non-linear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

Sheppard, C. J. R.

Min Gu, T. Tannous, C. J. R. Sheppard, “Improved axial resolution in a confocal fluorescence microscopy using annular pupils,” Opt. Commun. 110, 533–539 (1994).
[CrossRef]

Stelzer, E. H. K.

E. H. K. Stelzer, S. W. Hell, S. Lindek, R. Pick, C. Storz, R. Stricker, G. Ritter, N. Salmon, “Non-linear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

S. W. Hell, S. Lindek, E. H. K. Stelzer, “Enchancing the axial resolution in far-field light microscopy: two-photon excitation 4PI confocal fluorescence microscopy,” J. Mod Opt. 41, 675–681 (1994).
[CrossRef]

S. W. Hell, E. H. K. Stelzer, “Properties of a 4Pi confocal microscope,” J. Opt. Soc. Am. A 9, 2159–2166 (1992).
[CrossRef]

S. W. Hell, E. H. K. Stelzer, “Fundamental improvement of resolution with a 4Pi-confocal microscope using two-photon excitation,” Opt. Commun. 93, 277–282 (1992).
[CrossRef]

Storz, C.

E. H. K. Stelzer, S. W. Hell, S. Lindek, R. Pick, C. Storz, R. Stricker, G. Ritter, N. Salmon, “Non-linear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

Stricker, R.

E. H. K. Stelzer, S. W. Hell, S. Lindek, R. Pick, C. Storz, R. Stricker, G. Ritter, N. Salmon, “Non-linear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon fluorescence scanning microscopy,” Science 248, 73–75 (1990).
[CrossRef] [PubMed]

Tannous, T.

Min Gu, T. Tannous, C. J. R. Sheppard, “Improved axial resolution in a confocal fluorescence microscopy using annular pupils,” Opt. Commun. 110, 533–539 (1994).
[CrossRef]

Webb, W. W.

L. P. Ghislain, W. W. Webb, “Scanning-force microscope based on an optical trap,” Opt. Lett. 18, 1678–1680 (1993).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon fluorescence scanning microscopy,” Science 248, 73–75 (1990).
[CrossRef] [PubMed]

Wilson, T.

R. Juskaitis, T. Wilson, Department of Engineering Science, Oxford University, Oxford, OX1 3PJ, UK (personal communication, 1994).

T. Wilson, R. Juskaitis, “The axial response of confocal microscopes with high numerical aperture objective lenses,” Bioimaging (to be published).

Bioimaging (1)

P. E. Hänninen, S. W. Hell, “Femtosecond pulse broadening in the focal region of a two-photon excitation microscope,” Bioimaging 2, 117–121 (1994).
[CrossRef]

J. Mod Opt. (1)

S. W. Hell, S. Lindek, E. H. K. Stelzer, “Enchancing the axial resolution in far-field light microscopy: two-photon excitation 4PI confocal fluorescence microscopy,” J. Mod Opt. 41, 675–681 (1994).
[CrossRef]

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

Opt. Commun. (3)

S. W. Hell, E. H. K. Stelzer, “Fundamental improvement of resolution with a 4Pi-confocal microscope using two-photon excitation,” Opt. Commun. 93, 277–282 (1992).
[CrossRef]

E. H. K. Stelzer, S. W. Hell, S. Lindek, R. Pick, C. Storz, R. Stricker, G. Ritter, N. Salmon, “Non-linear absorption extends confocal fluorescence microscopy into the ultraviolet regime and confines the illumination volume,” Opt. Commun. 104, 223–228 (1994).
[CrossRef]

Min Gu, T. Tannous, C. J. R. Sheppard, “Improved axial resolution in a confocal fluorescence microscopy using annular pupils,” Opt. Commun. 110, 533–539 (1994).
[CrossRef]

Opt. Lett. (2)

Science (1)

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon fluorescence scanning microscopy,” Science 248, 73–75 (1990).
[CrossRef] [PubMed]

Other (3)

T. Wilson, R. Juskaitis, “The axial response of confocal microscopes with high numerical aperture objective lenses,” Bioimaging (to be published).

R. Juskaitis, T. Wilson, Department of Engineering Science, Oxford University, Oxford, OX1 3PJ, UK (personal communication, 1994).

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983) Chap. 5, pp. 112–120.

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (3)

Fig. 1
Fig. 1

(a) Response to an axially scanned fluorescent layer–glass interface as a measure of axial resolution of a two-photon fluorescence microscope recorded with pulses of peak intensities of 0.15 TW/cm2, (b) the peak of fourfold increased intensity at the interface after exposure to pulses of 0.74 TW/cm2, (c) the fluorescence signal of a 7.5-μm-thick layer when the layer is scanned through the focus. The curve shows successive recordings of the layer, each square corresponding to an axial image of the layer. The signal is recorded over 30 s. The first three scans were performed with the lower peak intensity, the succeeding two scans with the high intensity. After 10 s the low intensity was used again to image the distribution of the fluorophore after exposure to 0.74 TW/cm2 peak intensities.

Fig. 2
Fig. 2

(a) Lateral and (b) axial image of the fluorescence spot generated through exposure of the layer to the high peak intensity.

Fig. 3
Fig. 3

Axial response of a two-photon fluorescence confocal microscope. (a) Axial response of a two-photon-excitation confocal microscope measured with a PLFD-generated layer of Rhodamine 6G (solid curve), by differentiation of an edge response (thin dashed curve), along with the theoretically calculated curve (bold, dashed curve), for an aberration-free system with an effective numerical aperture of 1.35; (b) axial response of a 4Pi confocal microscope by a PLFD-generated signal.

Equations (3)

Equations on this page are rendered with MathJax. Learn more.

I ( z ) = h exc ( x , y , z ) 2 h det ( x , y , z ) d x d y .
h exc , det ( x , y , z ) = ( e x , e y , e z ) 2
( e x , e y , e z ) = - i [ I 0 + I 2 cos ( 2 φ ) , I 2 sin ( 2 φ ) , - 2 i I 1 cos φ ] ,

Metrics