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

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  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 (1)

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

1993 (1)

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

1992 (1)

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

1991 (3)

1990 (1)

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

1988 (1)

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. (1)

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

Appl. Opt. (1)

J. Microsc. (3)

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 (1)

Opt. Commun. (1)

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

Proc. Soc. Photo-Opt. Instrum. Eng. (1)

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

Other (1)

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

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 (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)

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

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 ,

Metrics