Abstract

When a confocal fluorescence microscope with a high-numerical-aperture oil-immersion objective is focused deep into an aqueous medium, aberrations result that degrade image quality. We have designed and fabricated a simple two-level binary phase mask that partially corrects these aberrations, improving axial resolution. We present the design and some confirming results.

© 1995 Optical Society of America

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References

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  1. S. F. Gibson, F. Lanni, J. Opt. Soc. Am. A 8, 1601 (1991).
    [CrossRef]
  2. S. F. F. Gibson, “Modeling the three-dimensional imaging properties of the fluorescence light microscope,” Ph.D. dissertation (Carnegie-Mellon University, Pittsburg, Pa., 1990).
  3. H. T. M. van der Voort, G. J. Brakenhoff, J. Microsc. 158, 43 (1990).
    [CrossRef]
  4. K. Carlsson, J. Microsc. 163, 167 (1991).
    [CrossRef]
  5. C. J. R. Sheppard, M. Gu, Appl. Opt. 30, 3563 (1991).
    [CrossRef] [PubMed]
  6. C. J. R. Sheppard, M. Gu, Opt. Commun. 88, 180 (1992).
    [CrossRef]
  7. S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
    [CrossRef]
  8. M. Brenner, Am. Lab. 26, 14 (1994).
  9. E. W. Hansen, J. P. Zelten, B. A. Wiseman, Proc. Soc. Photo-Opt. Instrum. Eng. 909, 304 (1988).

1994

M. Brenner, Am. Lab. 26, 14 (1994).

1993

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
[CrossRef]

1992

C. J. R. Sheppard, M. Gu, Opt. Commun. 88, 180 (1992).
[CrossRef]

1991

1990

H. T. M. van der Voort, G. J. Brakenhoff, J. Microsc. 158, 43 (1990).
[CrossRef]

1988

E. W. Hansen, J. P. Zelten, B. A. Wiseman, Proc. Soc. Photo-Opt. Instrum. Eng. 909, 304 (1988).

Brakenhoff, G. J.

H. T. M. van der Voort, G. J. Brakenhoff, J. Microsc. 158, 43 (1990).
[CrossRef]

Brenner, M.

M. Brenner, Am. Lab. 26, 14 (1994).

Carlsson, K.

K. Carlsson, J. Microsc. 163, 167 (1991).
[CrossRef]

Cremer, C.

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
[CrossRef]

Gibson, S. F.

Gibson, S. F. F.

S. F. F. Gibson, “Modeling the three-dimensional imaging properties of the fluorescence light microscope,” Ph.D. dissertation (Carnegie-Mellon University, Pittsburg, Pa., 1990).

Gu, M.

C. J. R. Sheppard, M. Gu, Opt. Commun. 88, 180 (1992).
[CrossRef]

C. J. R. Sheppard, M. Gu, Appl. Opt. 30, 3563 (1991).
[CrossRef] [PubMed]

Hansen, E. W.

E. W. Hansen, J. P. Zelten, B. A. Wiseman, Proc. Soc. Photo-Opt. Instrum. Eng. 909, 304 (1988).

Hell, S.

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
[CrossRef]

Lanni, F.

Reiner, G.

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
[CrossRef]

Sheppard, C. J. R.

C. J. R. Sheppard, M. Gu, Opt. Commun. 88, 180 (1992).
[CrossRef]

C. J. R. Sheppard, M. Gu, Appl. Opt. 30, 3563 (1991).
[CrossRef] [PubMed]

Stelzer, E. H. K.

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
[CrossRef]

van der Voort, H. T. M.

H. T. M. van der Voort, G. J. Brakenhoff, J. Microsc. 158, 43 (1990).
[CrossRef]

Wiseman, B. A.

E. W. Hansen, J. P. Zelten, B. A. Wiseman, Proc. Soc. Photo-Opt. Instrum. Eng. 909, 304 (1988).

Zelten, J. P.

E. W. Hansen, J. P. Zelten, B. A. Wiseman, Proc. Soc. Photo-Opt. Instrum. Eng. 909, 304 (1988).

Am. Lab.

M. Brenner, Am. Lab. 26, 14 (1994).

Appl. Opt.

J. Microsc.

H. T. M. van der Voort, G. J. Brakenhoff, J. Microsc. 158, 43 (1990).
[CrossRef]

K. Carlsson, J. Microsc. 163, 167 (1991).
[CrossRef]

S. Hell, G. Reiner, C. Cremer, E. H. K. Stelzer, J. Microsc. 169, 391 (1993).
[CrossRef]

J. Opt. Soc. Am. A

Opt. Commun.

C. J. R. Sheppard, M. Gu, Opt. Commun. 88, 180 (1992).
[CrossRef]

Proc. Soc. Photo-Opt. Instrum. Eng.

E. W. Hansen, J. P. Zelten, B. A. Wiseman, Proc. Soc. Photo-Opt. Instrum. Eng. 909, 304 (1988).

Other

S. F. F. Gibson, “Modeling the three-dimensional imaging properties of the fluorescence light microscope,” Ph.D. dissertation (Carnegie-Mellon University, Pittsburg, Pa., 1990).

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Figures (5)

Fig. 1
Fig. 1

When a high-NA oil-immersion lens images a specimen in an aqueous medium, path differences result from index mismatch. Solid ray, noil > ns (exaggerated for purposes of illustration); dashed ray, noilns.

Fig. 2
Fig. 2

Phase profiles of continuous and two-level fourth-order correcting elements designed for a 1.4-NA oil-immersion objective focusing 40 μm into an aqueous medium.

Fig. 3
Fig. 3

Simulated axial point response for a confocal fluorescence microscope at various depths below the coverslip in an aqueous medium, with (solid curves) and without (dashed curves) a binary corrector designed for 40-μm depth. Δz is measured at the stage (i.e., toil). Axial FWHM is improved by 40–50% over the range 20–60 μm.

Fig. 4
Fig. 4

Averaged lateral intensity of ensembles of thirteen 140-nm beads at best focus, 40-μm depth, with (solid curves) and without (dashed curves) the binary corrector. Samples are spaced at 50 nm. Error bars represent 95% confidence intervals; there is no significant difference between the profiles.

Fig. 5
Fig. 5

Averaged axial intensity of ensembles of 140-nm beads at 40-μm depth in water. Optical sections were taken every 200 nm at the stage (160 nm in water); Δz is measured at the stage (i.e., toil). Solid curve, average of 20 beads imaged with the binary corrector; dashed curve, average of 14 beads imaged without the corrector. Error bars represent 95% confidence intervals. The FWHM is improved by approximately 50%, from ~1.5 to ~0.7 μm.

Equations (3)

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OPD ( ρ ) = n oil ( t oil - t oil * ) [ 1 - ( NA ρ n oil ) 2 ] 1 / 2 + n s t s [ 1 - ( NA ρ n s ) 2 ] 1 / 2 ,
h ( r , z ) = p 1 ( r , z ) 2 [ p 2 ( r , z ) 2 * * P 3 ( r ) ] ,
p 2 ( r , z ) = 0 1 exp [ - i Φ ( ρ ) ] J 0 ( k ρ r f ) ρ d ρ ,

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