Abstract

We propose a new type of scanning fluorescence microscope capable of resolving 35 nm in the far field. We overcome the diffraction resolution limit by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point-spread function. In contrast to near-field scanning optical microscopy, this method can produce three-dimensional images of translucent specimens.

© 1994 Optical Society of America

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References

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  1. J. Darnell, H. Lodish, D. Baltimore, Molecular Cell Biology (Freeman, New York, 1990), Chap. 4.
  2. R. Kopelman, W. Tan, Science 262, 1382 (1993).
    [CrossRef] [PubMed]
  3. S. W. Hell, Opt. Commun. 106, 19 (1994).
    [CrossRef]
  4. K. H. Drexhage, in Dye Lasers, F. P. Schäfer, ed. (Springer-Verlag, Berlin, 1977), p. 144.
  5. T. Wilson, C. J. R. Sheppard, Theory and Practice of Optical Scanning Microscopy (Academic, London, 1984), p. 47.

1994 (1)

S. W. Hell, Opt. Commun. 106, 19 (1994).
[CrossRef]

1993 (1)

R. Kopelman, W. Tan, Science 262, 1382 (1993).
[CrossRef] [PubMed]

Baltimore, D.

J. Darnell, H. Lodish, D. Baltimore, Molecular Cell Biology (Freeman, New York, 1990), Chap. 4.

Darnell, J.

J. Darnell, H. Lodish, D. Baltimore, Molecular Cell Biology (Freeman, New York, 1990), Chap. 4.

Drexhage, K. H.

K. H. Drexhage, in Dye Lasers, F. P. Schäfer, ed. (Springer-Verlag, Berlin, 1977), p. 144.

Hell, S. W.

S. W. Hell, Opt. Commun. 106, 19 (1994).
[CrossRef]

Kopelman, R.

R. Kopelman, W. Tan, Science 262, 1382 (1993).
[CrossRef] [PubMed]

Lodish, H.

J. Darnell, H. Lodish, D. Baltimore, Molecular Cell Biology (Freeman, New York, 1990), Chap. 4.

Sheppard, C. J. R.

T. Wilson, C. J. R. Sheppard, Theory and Practice of Optical Scanning Microscopy (Academic, London, 1984), p. 47.

Tan, W.

R. Kopelman, W. Tan, Science 262, 1382 (1993).
[CrossRef] [PubMed]

Wilson, T.

T. Wilson, C. J. R. Sheppard, Theory and Practice of Optical Scanning Microscopy (Academic, London, 1984), p. 47.

Opt. Commun. (1)

S. W. Hell, Opt. Commun. 106, 19 (1994).
[CrossRef]

Science (1)

R. Kopelman, W. Tan, Science 262, 1382 (1993).
[CrossRef] [PubMed]

Other (3)

J. Darnell, H. Lodish, D. Baltimore, Molecular Cell Biology (Freeman, New York, 1990), Chap. 4.

K. H. Drexhage, in Dye Lasers, F. P. Schäfer, ed. (Springer-Verlag, Berlin, 1977), p. 144.

T. Wilson, C. J. R. Sheppard, Theory and Practice of Optical Scanning Microscopy (Academic, London, 1984), p. 47.

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Figures (5)

Fig. 1
Fig. 1

Energy levels of a typical fluorophore.

Fig. 2
Fig. 2

Principles of a STED fluorescence scanning microscope. An excitation beam and two offset STED beams are focused into the object for excitation and stimulated emission, respectively. The spontaneously emitted light is recorded in a (point) detector. We accomplish imaging by scanning the beams with respect to the object. Two additional STED beams are used for enhancing the lateral resolution in the direction perpendicular to the plane of the scheme. For clarity the lenses for focusing the laser beams into the pinhole plane are not shown.

Fig. 3
Fig. 3

Population probability n2(ν) of L2 after Gaussian STED-beam pulses of peak intenisties of 3.4, 34, 170, and 1300 MW/cm2 for curves a, b, c, and d, respectively, have left the focal region. (The computational error of the numerical data is less than 0.1%. These curves and curve a of Fig. 4 have been calculated with a density of 150–200 points per curve.)

Fig. 4
Fig. 4

PSF’s for the STED fluorescence microscope with Δν = 3.9 (curve a) and the confocal (curve b) and conventional scanning microscopes in the focal plane.

Fig. 5
Fig. 5

Intensity maximum versus the FWHM of the effective PSF of the STED fluorescence microscope.

Equations (1)

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d n 0 d t = h exc σ 01 ( n 1 - n 0 ) + 1 τ vibr n 3 , d n 1 d t = h exc σ 01 ( n 0 - n 1 ) - 1 τ vibr n 1 , d n 2 d t = 1 τ vibr n 1 + h STED σ 23 ( n 3 - n 2 ) - ( 1 τ fluor + Q ) n 2 , d n 3 d t = h STED σ 23 ( n 2 - n 3 ) + ( 1 τ fluor + Q ) n 2 - 1 τ vibr n 3 ,

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