Abstract

The resolution of microscopes is limited by the sizes of their point-spread functions. The invention of confocal, theta, and 4Pi microscopes has permitted the classic Abbe limit to be exceeded. We propose the use of a combination of 4Pi and theta microscopy to decrease resolution by using four illumination objectives and two detection objectives. Using middle numerical aperture, long-working-distance objectives yielded a resolution near 100  nm in the three dimensions, which opens the possibility of exploring large volumes with a high resolution.

© 2001 Optical Society of America

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References

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  1. T. Wilson and C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984).
  2. D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, in Fluorescence Microscopy in Three Dimensions, D. L. Taylor and Y. Wang, eds., Vol. 30 of Methods in Cell Biology (Academic, San Diego, Calif., 1989), p. 353.
  3. S. W. Hell, E. Lehtonen, and E. H. K. Stelzer, in New Dimensions of Visualization in Biomedical Microscopies, A. Kritte, ed. (Verlag Chemie, Weinheim, Germany, 1992), p. 145.
  4. M. Minsky, Scanning 10, 128 (1988).
    [CrossRef]
  5. S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
    [CrossRef]
  6. E. H. K. Stelzer and S. Lindek, Opt. Commun. 111, 536 (1994).
    [CrossRef]
  7. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
    [CrossRef]
  8. B. Richards and E. Wolf, Proc. R. Soc. London Ser. A 253, 349 (1959).
    [CrossRef]
  9. C. J. R. Sheppard and P. Török, Bioimaging 5, 205 (1989).
    [CrossRef]
  10. P. D. Higdon, P. Török, and T. Wilson, J. Microsc. 193, 127 (1999).
    [CrossRef]
  11. S. W. Hell, in Nonlinear and Two-Photon Induced Fluorescence, J. Lakowicz, ed., Vol. 5 of Topics in Fluorescence Microscopy (Plenum, New York, 1997), p. 361.
  12. M. Nagorni and S. W. Hell, J. Opt. Soc. Am. A 18, 49 (2001).
    [CrossRef]
  13. R. Clappier, Olympus France SA, 75 Rue d’Areueil 94533 Rungis, France (personal communication, 2001).
  14. T. Tanikawa and T. Arai, IEEE Trans. Robot. Autom. 15, 152 (1999).
    [CrossRef]
  15. J. Swoger, S. Lindek, T. Stefany, F.-M. Haar, and E. H. K. Stelzer, Rev. Sci. Instrum. 69, 2956 (1998).
    [CrossRef]

2001

2000

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[CrossRef]

1999

T. Tanikawa and T. Arai, IEEE Trans. Robot. Autom. 15, 152 (1999).
[CrossRef]

P. D. Higdon, P. Török, and T. Wilson, J. Microsc. 193, 127 (1999).
[CrossRef]

1998

J. Swoger, S. Lindek, T. Stefany, F.-M. Haar, and E. H. K. Stelzer, Rev. Sci. Instrum. 69, 2956 (1998).
[CrossRef]

1994

E. H. K. Stelzer and S. Lindek, Opt. Commun. 111, 536 (1994).
[CrossRef]

1992

S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

1989

C. J. R. Sheppard and P. Török, Bioimaging 5, 205 (1989).
[CrossRef]

1988

M. Minsky, Scanning 10, 128 (1988).
[CrossRef]

1959

B. Richards and E. Wolf, Proc. R. Soc. London Ser. A 253, 349 (1959).
[CrossRef]

Agard, D. A.

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, in Fluorescence Microscopy in Three Dimensions, D. L. Taylor and Y. Wang, eds., Vol. 30 of Methods in Cell Biology (Academic, San Diego, Calif., 1989), p. 353.

Arai, T.

T. Tanikawa and T. Arai, IEEE Trans. Robot. Autom. 15, 152 (1999).
[CrossRef]

Clappier, R.

R. Clappier, Olympus France SA, 75 Rue d’Areueil 94533 Rungis, France (personal communication, 2001).

Dyba, M.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[CrossRef]

Egner, A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[CrossRef]

Haar, F.-M.

J. Swoger, S. Lindek, T. Stefany, F.-M. Haar, and E. H. K. Stelzer, Rev. Sci. Instrum. 69, 2956 (1998).
[CrossRef]

Hell, S. W.

M. Nagorni and S. W. Hell, J. Opt. Soc. Am. A 18, 49 (2001).
[CrossRef]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[CrossRef]

S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

S. W. Hell, E. Lehtonen, and E. H. K. Stelzer, in New Dimensions of Visualization in Biomedical Microscopies, A. Kritte, ed. (Verlag Chemie, Weinheim, Germany, 1992), p. 145.

S. W. Hell, in Nonlinear and Two-Photon Induced Fluorescence, J. Lakowicz, ed., Vol. 5 of Topics in Fluorescence Microscopy (Plenum, New York, 1997), p. 361.

Higdon, P. D.

P. D. Higdon, P. Török, and T. Wilson, J. Microsc. 193, 127 (1999).
[CrossRef]

Hiraoka, Y.

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, in Fluorescence Microscopy in Three Dimensions, D. L. Taylor and Y. Wang, eds., Vol. 30 of Methods in Cell Biology (Academic, San Diego, Calif., 1989), p. 353.

Jakobs, S.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[CrossRef]

Klar, T. A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[CrossRef]

Lehtonen, E.

S. W. Hell, E. Lehtonen, and E. H. K. Stelzer, in New Dimensions of Visualization in Biomedical Microscopies, A. Kritte, ed. (Verlag Chemie, Weinheim, Germany, 1992), p. 145.

Lindek, S.

J. Swoger, S. Lindek, T. Stefany, F.-M. Haar, and E. H. K. Stelzer, Rev. Sci. Instrum. 69, 2956 (1998).
[CrossRef]

E. H. K. Stelzer and S. Lindek, Opt. Commun. 111, 536 (1994).
[CrossRef]

Minsky, M.

M. Minsky, Scanning 10, 128 (1988).
[CrossRef]

Nagorni, M.

Richards, B.

B. Richards and E. Wolf, Proc. R. Soc. London Ser. A 253, 349 (1959).
[CrossRef]

Sedat, J. W.

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, in Fluorescence Microscopy in Three Dimensions, D. L. Taylor and Y. Wang, eds., Vol. 30 of Methods in Cell Biology (Academic, San Diego, Calif., 1989), p. 353.

Shaw, P.

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, in Fluorescence Microscopy in Three Dimensions, D. L. Taylor and Y. Wang, eds., Vol. 30 of Methods in Cell Biology (Academic, San Diego, Calif., 1989), p. 353.

Sheppard, C. J. R.

C. J. R. Sheppard and P. Török, Bioimaging 5, 205 (1989).
[CrossRef]

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

Stefany, T.

J. Swoger, S. Lindek, T. Stefany, F.-M. Haar, and E. H. K. Stelzer, Rev. Sci. Instrum. 69, 2956 (1998).
[CrossRef]

Stelzer, E. H. K.

J. Swoger, S. Lindek, T. Stefany, F.-M. Haar, and E. H. K. Stelzer, Rev. Sci. Instrum. 69, 2956 (1998).
[CrossRef]

E. H. K. Stelzer and S. Lindek, Opt. Commun. 111, 536 (1994).
[CrossRef]

S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

S. W. Hell, E. Lehtonen, and E. H. K. Stelzer, in New Dimensions of Visualization in Biomedical Microscopies, A. Kritte, ed. (Verlag Chemie, Weinheim, Germany, 1992), p. 145.

Swoger, J.

J. Swoger, S. Lindek, T. Stefany, F.-M. Haar, and E. H. K. Stelzer, Rev. Sci. Instrum. 69, 2956 (1998).
[CrossRef]

Tanikawa, T.

T. Tanikawa and T. Arai, IEEE Trans. Robot. Autom. 15, 152 (1999).
[CrossRef]

Török, P.

P. D. Higdon, P. Török, and T. Wilson, J. Microsc. 193, 127 (1999).
[CrossRef]

C. J. R. Sheppard and P. Török, Bioimaging 5, 205 (1989).
[CrossRef]

Wilson, T.

P. D. Higdon, P. Török, and T. Wilson, J. Microsc. 193, 127 (1999).
[CrossRef]

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

Wolf, E.

B. Richards and E. Wolf, Proc. R. Soc. London Ser. A 253, 349 (1959).
[CrossRef]

Bioimaging

C. J. R. Sheppard and P. Török, Bioimaging 5, 205 (1989).
[CrossRef]

IEEE Trans. Robot. Autom.

T. Tanikawa and T. Arai, IEEE Trans. Robot. Autom. 15, 152 (1999).
[CrossRef]

J. Microsc.

P. D. Higdon, P. Török, and T. Wilson, J. Microsc. 193, 127 (1999).
[CrossRef]

J. Opt. Soc. Am. A

Opt. Commun.

S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

E. H. K. Stelzer and S. Lindek, Opt. Commun. 111, 536 (1994).
[CrossRef]

Proc. Natl. Acad. Sci. USA

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[CrossRef]

Proc. R. Soc. London Ser. A

B. Richards and E. Wolf, Proc. R. Soc. London Ser. A 253, 349 (1959).
[CrossRef]

Rev. Sci. Instrum.

J. Swoger, S. Lindek, T. Stefany, F.-M. Haar, and E. H. K. Stelzer, Rev. Sci. Instrum. 69, 2956 (1998).
[CrossRef]

Scanning

M. Minsky, Scanning 10, 128 (1988).
[CrossRef]

Other

R. Clappier, Olympus France SA, 75 Rue d’Areueil 94533 Rungis, France (personal communication, 2001).

S. W. Hell, in Nonlinear and Two-Photon Induced Fluorescence, J. Lakowicz, ed., Vol. 5 of Topics in Fluorescence Microscopy (Plenum, New York, 1997), p. 361.

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

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, in Fluorescence Microscopy in Three Dimensions, D. L. Taylor and Y. Wang, eds., Vol. 30 of Methods in Cell Biology (Academic, San Diego, Calif., 1989), p. 353.

S. W. Hell, E. Lehtonen, and E. H. K. Stelzer, in New Dimensions of Visualization in Biomedical Microscopies, A. Kritte, ed. (Verlag Chemie, Weinheim, Germany, 1992), p. 145.

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Figures (4)

Fig. 1
Fig. 1

Light linearly polarized along the x axis and focused by a lens in a single medium. Cylindrical coordinates are used.

Fig. 2
Fig. 2

Multiple-objective configuration considered in this Letter. Illumination comes from coherent addition of the focal fields of objectives 1–4. Objectives 5 and 6 are used in the 4Pi detection mode.

Fig. 3
Fig. 3

Computed intensity PSF of a MOM: (a) illumination PSF in the x,y,z=0 plane, (b) illumination PSF in the x,y=0,z plane, (c) detection PSF in the x,y,z=0 plane, (d) detection PSF in the x,y=0,z plane (central part only), (e) final intensity PSF in the x,y,z=0 plane, (f) final intensity PSF in the x,y=0,z plane, (g) lateral intensity distribution, (h) z-axis intensity distribution. The lateral resolution is 108  nm, and the longitudinal resolution is 89  nm. Illumination wavelength, 400  nm; detection wavelength, 450  nm (Cascade Blue from Molecular Probes); N.A.  of water-immersion objectives, 0.8.

Fig. 4
Fig. 4

Computed intensity PSFs for (a) single-objective, (b) 4Pi type C, (c) theta, and (d) MOM microscopes with N.A.=0.8 water-immersion objective (same conditions as for Fig.  3). 4(d) Intensity PSF of a MOM with N.A.=0.4 objective. The resultant interference pattern is more complex and exhibits several secondary maxima.

Equations (8)

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

Exx,y,z=-iI0+I2cos2ϕ,Eyx,y,z=-iI2sin2ϕ,Ezx,y,z=-2I1cosϕ,
I0=0aaθsinθ1+cosθJ0krsinθexpikzcosθdθ,I1=0aaθsin2θJ1krsinθexpikzcosθdθ,I2=0aaθsinθ1-cosθJ2krsinθexpikzcosθdθ.
PSFillx,y,z=Ex,y,z2=I02+4I12cos2ϕ+I22+2 ReI0I2*cos2ϕ.
PSFdetx,y,z=I02+2I12+I22.
PSFill4 objx,y,z=Exx,y,z+Exx,y,z+Eyx,y,z+Eyx,y,z2.
PSFill4 objx,y,z=ReI02+2 ReI12+ReI22+ReI02+2 ReI12+ReI22+2 ReI0ReI0.
PSFdet2 objx,y,z=ReI02+2 ReI12+ReI22,
In=Inx,y,z,In=Iny,z,-x,In=Iny,z,x   n=1,2,3.

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