Abstract

We overcame the resolution limit of scanning far-field fluorescence microscopy by disabling the fluorescence from the outer part of the focal spot. Whereas a near-UV pulse generates a diffraction-limited distribution of excited molecules, a spatially offset pulse quenches the excited molecules from the outer part of the focus through stimulated emission. This results in a subdiffraction-sized effective point-spread function. For a 1.4 aperture and a 388-nm excitation wavelength spatial resolution is increased from 150±8 nm to 106±8 nm with a single offset beam. Superior lateral resolution is demonstrated by separation of adjacent Pyridine 2 nanocrystals that are otherwise indiscernible.

© 1999 Optical Society of America

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References

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  1. E. Abbe, Gesammelte Abhandlungen (G. Fischer, Jena, Germany, 1904).
  2. G. Binnig, H. Rohrer, Ch. Gerber, and E. Weibel, Phys. Rev. Lett. 49, 57 (1982).
    [CrossRef]
  3. A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, Ultramicroscopy 13, 227 (1984); D. W. Pohl, W. Denk, and M. Lanz, Appl. Phys. Lett. 44, 651 (1984).
    [CrossRef]
  4. T. Wilson and C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984).
  5. S. W. Hell and J. Wichmann, Opt. Lett. 19, 780 (1994); M. Schrader, F. Meinecke, K. Bahlmann, M. Kroug, C. Cremer, E. Soini, and S. W. Hell, Bioimaging 3, 147 (1995).
    [CrossRef] [PubMed]
  6. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983).
    [CrossRef]
  7. J. R. Lakowicz, I. Gryczynski, V. Bogdanov, and J. Kusba, J. Phys. Chem. 98, 334 (1994).
    [CrossRef]

1994 (2)

1984 (1)

A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, Ultramicroscopy 13, 227 (1984); D. W. Pohl, W. Denk, and M. Lanz, Appl. Phys. Lett. 44, 651 (1984).
[CrossRef]

1982 (1)

G. Binnig, H. Rohrer, Ch. Gerber, and E. Weibel, Phys. Rev. Lett. 49, 57 (1982).
[CrossRef]

Abbe, E.

E. Abbe, Gesammelte Abhandlungen (G. Fischer, Jena, Germany, 1904).

Binnig, G.

G. Binnig, H. Rohrer, Ch. Gerber, and E. Weibel, Phys. Rev. Lett. 49, 57 (1982).
[CrossRef]

Bogdanov, V.

J. R. Lakowicz, I. Gryczynski, V. Bogdanov, and J. Kusba, J. Phys. Chem. 98, 334 (1994).
[CrossRef]

Gerber, Ch.

G. Binnig, H. Rohrer, Ch. Gerber, and E. Weibel, Phys. Rev. Lett. 49, 57 (1982).
[CrossRef]

Gryczynski, I.

J. R. Lakowicz, I. Gryczynski, V. Bogdanov, and J. Kusba, J. Phys. Chem. 98, 334 (1994).
[CrossRef]

Harootunian, A.

A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, Ultramicroscopy 13, 227 (1984); D. W. Pohl, W. Denk, and M. Lanz, Appl. Phys. Lett. 44, 651 (1984).
[CrossRef]

Hell, S. W.

Isaacson, M.

A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, Ultramicroscopy 13, 227 (1984); D. W. Pohl, W. Denk, and M. Lanz, Appl. Phys. Lett. 44, 651 (1984).
[CrossRef]

Kusba, J.

J. R. Lakowicz, I. Gryczynski, V. Bogdanov, and J. Kusba, J. Phys. Chem. 98, 334 (1994).
[CrossRef]

Lakowicz, J. R.

J. R. Lakowicz, I. Gryczynski, V. Bogdanov, and J. Kusba, J. Phys. Chem. 98, 334 (1994).
[CrossRef]

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983).
[CrossRef]

Lewis, A.

A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, Ultramicroscopy 13, 227 (1984); D. W. Pohl, W. Denk, and M. Lanz, Appl. Phys. Lett. 44, 651 (1984).
[CrossRef]

Muray, A.

A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, Ultramicroscopy 13, 227 (1984); D. W. Pohl, W. Denk, and M. Lanz, Appl. Phys. Lett. 44, 651 (1984).
[CrossRef]

Rohrer, H.

G. Binnig, H. Rohrer, Ch. Gerber, and E. Weibel, Phys. Rev. Lett. 49, 57 (1982).
[CrossRef]

Sheppard, C. J. R.

T. Wilson and C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984).

Weibel, E.

G. Binnig, H. Rohrer, Ch. Gerber, and E. Weibel, Phys. Rev. Lett. 49, 57 (1982).
[CrossRef]

Wichmann, J.

Wilson, T.

T. Wilson and C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984).

J. Phys. Chem. (1)

J. R. Lakowicz, I. Gryczynski, V. Bogdanov, and J. Kusba, J. Phys. Chem. 98, 334 (1994).
[CrossRef]

Opt. Lett. (1)

Phys. Rev. Lett. (1)

G. Binnig, H. Rohrer, Ch. Gerber, and E. Weibel, Phys. Rev. Lett. 49, 57 (1982).
[CrossRef]

Ultramicroscopy (1)

A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, Ultramicroscopy 13, 227 (1984); D. W. Pohl, W. Denk, and M. Lanz, Appl. Phys. Lett. 44, 651 (1984).
[CrossRef]

Other (3)

T. Wilson and C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984).

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983).
[CrossRef]

E. Abbe, Gesammelte Abhandlungen (G. Fischer, Jena, Germany, 1904).

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Figures (4)

Fig. 1
Fig. 1

Setup: frequency-doubled (UV) and fundamental (STED) beams from a Ti:sapphire laser are cleaned, passed through pinholes (PH’s), expanded, and combined at a dichroic mirror (HT383/HR766). After the beams are reflected from mirror HT510–710, they overfill the rear aperture of the lens. The STED beam is shifted with respect to the UV beam by a piezo-movable tube lens. APD, avalanche photodiode.

Fig. 2
Fig. 2

(a) Chemical structure and (b) spectra of Pyridine 2 and (c) fluorescence with overlapping UV and STED pulses. Periodic interruption of the STED beam with a chopper leads to quenching and recovery. (d) Fluorescence dynamics on the nanosecond scale: Two UV pulses that are 6 ns apart produce two equal fluorescence decays (solid curve). A STED pulse superimposed upon the first UV pulse quenches the excited molecules, which are, however, fully reexcited 6 ns later by the second pulse. (The solid curve is shifted upward.)

Fig. 3
Fig. 3

Effective PSF’s of (a) standard UV-confocal and (b) corresponding STED-confocal microscopes with the stimulating beam displaced in the y direction. The profiles on the right reveal a narrower PSF in (b) than in (a).

Fig. 4
Fig. 4

Images of adjacent nanocrystals in (a) the standard UV-confocal and (b) the STED-confocal microscopes. Comparison of the images and the profiles reveals significantly improved spatial resolution in the far field.

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