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Applied Optics

Applied Optics


  • Vol. 39, Iss. 10 — Apr. 1, 2000
  • pp: 1652–1658

Polarization effects in 4Pi confocal microscopy studied with water-immersion lenses

Karsten Bahlmann and Stefan W. Hell  »View Author Affiliations

Applied Optics, Vol. 39, Issue 10, pp. 1652-1658 (2000)

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We studied the effect of electric field orientation on the point-spread function (PSF) of a 4Pi microscope. We show that in a standard 4Pi arrangement the orientation of the field can be used for changing between constructive- and destructive-mode 4Pi microscopy. The effect is counteracted by introduction of a phase shift of π into one of the half-arms. This compensation is compulsory during illumination with unpolarized or circularly polarized light. By performing our experiments with 1.2-N.A. water-immersion lenses, we demonstrate that water immersion is suitable for 4Pi confocal microscopy. At a two-photon excitation wavelength of 1064 nm, the water 4Pi confocal PSF features an axial lobe of 40% above and below the focal plane, which, by linear filtering, can be unambiguously removed. The measured axial full width at half-maximum of the PSF is 240 nm. This is 4.3 times narrower than its single-lens confocal counterpart. The 4Pi confocal microscope sets a new resolution benchmark in three-dimensional imaging of watery samples.

© 2000 Optical Society of America

OCIS Codes
(110.0180) Imaging systems : Microscopy
(110.6880) Imaging systems : Three-dimensional image acquisition
(170.1790) Medical optics and biotechnology : Confocal microscopy
(170.6900) Medical optics and biotechnology : Three-dimensional microscopy
(190.4180) Nonlinear optics : Multiphoton processes

Original Manuscript: June 15, 1999
Revised Manuscript: August 30, 1999
Published: April 1, 2000

Karsten Bahlmann and Stefan W. Hell, "Polarization effects in 4Pi confocal microscopy studied with water-immersion lenses," Appl. Opt. 39, 1652-1658 (2000)

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  1. S. W. Hell, “Double-confocal microscope,” European patent0491289 (18December1990).
  2. S. Hell, E. H. K. Stelzer, “Properties of a 4Pi confocal fluorescence microscope,” J. Opt. Soc. Am. A 9, 2159–2166 (1992). [CrossRef]
  3. M. Schrader, S. W. Hell, “4Pi confocal images with axial superresolution,” J. Microsc. 183, 189–193 (1996). [CrossRef]
  4. S. W. Hell, M. Schrader, H. T. M. van der Voort, “Far-field fluorescence microscopy with three-dimensional resolution in the 100 nm range,” J. Microsc. 185, 1–5 (1997). [CrossRef]
  5. M. Schrader, S. W. Hell, H. T. M. van der Voort, “Three-dimensional superresolution with a 4Pi confocal microscope using image restoration,” J. Appl. Phys. 84, 4033–4042 (1998). [CrossRef]
  6. M. Schrader, K. Bahlmann, G. Giese, S. W. Hell, “4Pi confocal imaging in fixed biological specimens,” Biophys. J. 75, 1659–1668 (1998); A. Egner, M. Schrader, S. W. Hell, “Refractive index mismatch induced intensity and phase variations in fluorescence confocal, multiphoton and 4Pi microscopy,” Opt. Commun. 153, 211–217 (1998). [CrossRef] [PubMed]
  7. M. Nagorni, S. W. Hell, “4Pi confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution,” J. Struct. Biol. 123, 236–247 (1998). [CrossRef]
  8. M. Gu, C. J. R. Sheppard, “Three-dimensional transfer functions in 4Pi confocal microscopes,” J. Opt. Soc. Am. A 11, 1619–1627 (1994). [CrossRef]
  9. M. Schrader, M. Kozubek, S. W. Hell, T. Wilson, “Optical transfer functions of 4Pi confocal microscopes: theory and experiment,” Opt. Lett. 22, 436–438 (1997). [CrossRef] [PubMed]
  10. F. Lanni, Applications of Fluorescence in the Biological Sciences, 1st ed. (Liss, New York, 1986).
  11. R. Freimann, S. Pentz, H. Hörler, “Development of a standing-wave fluorescence microscope with high nodal plane flatness,” J. Microsc. 187, 193–200 (1997). [CrossRef] [PubMed]
  12. S. W. Hell, E. H. K. Stelzer, “Fundamental improvement of resolution with a 4Pi confocal fluorescence microscope using two-photon excitation,” Opt. Commun. 93, 277–282 (1992). [CrossRef]
  13. S. Lindek, N. Salmon, C. Cremer, E. H. K. Stelzer, “Theta microscopy allows phase regulation in 4Pi(A)-confocal two-photon fluorescence microscopy,” Optik 98, 15–20 (1994).
  14. S. W. Hell, M. Nagorni, “4Pi confocal microscopy with alternate interference,” Opt. Lett. 23, 1567–1569 (1998). [CrossRef]
  15. M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998). [CrossRef] [PubMed]
  16. P. E. Hänninen, S. W. Hell, J. Salo, C. Cremer, “Two-photon excitation 4Pi confocal microscope: enhanced axial resolution microscope for biological research,” Appl. Phys. Lett. 66, 1698–1700 (1995). [CrossRef]
  17. M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon Press, Oxford, U.K., 1993), p. 612.

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