Abstract

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

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References

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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.

1998 (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]

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]

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]

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]

S. W. Hell, M. Nagorni, “4Pi confocal microscopy with alternate interference,” Opt. Lett. 23, 1567–1569 (1998).
[CrossRef]

1997 (3)

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]

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]

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]

1996 (1)

M. Schrader, S. W. Hell, “4Pi confocal images with axial superresolution,” J. Microsc. 183, 189–193 (1996).
[CrossRef]

1995 (1)

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]

1994 (2)

M. Gu, C. J. R. Sheppard, “Three-dimensional transfer functions in 4Pi confocal microscopes,” J. Opt. Soc. Am. A 11, 1619–1627 (1994).
[CrossRef]

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).

1992 (2)

S. Hell, E. H. K. Stelzer, “Properties of a 4Pi confocal fluorescence microscope,” J. Opt. Soc. Am. A 9, 2159–2166 (1992).
[CrossRef]

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]

Bahlmann, K.

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]

Born, M.

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon Press, Oxford, U.K., 1993), p. 612.

Cremer, C.

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]

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).

Freimann, R.

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]

Giese, G.

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]

Gu, M.

Hänninen, P. E.

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]

Hell, S.

Hell, S. W.

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]

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]

S. W. Hell, M. Nagorni, “4Pi confocal microscopy with alternate interference,” Opt. Lett. 23, 1567–1569 (1998).
[CrossRef]

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]

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]

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]

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]

M. Schrader, S. W. Hell, “4Pi confocal images with axial superresolution,” J. Microsc. 183, 189–193 (1996).
[CrossRef]

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]

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]

S. W. Hell, “Double-confocal microscope,” European patent0491289 (18December1990).

Hofmann, U. G.

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]

Hörler, H.

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]

Kozubek, M.

Lanni, F.

F. Lanni, Applications of Fluorescence in the Biological Sciences, 1st ed. (Liss, New York, 1986).

Lindek, S.

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).

Nagorni, M.

S. W. Hell, M. Nagorni, “4Pi confocal microscopy with alternate interference,” Opt. Lett. 23, 1567–1569 (1998).
[CrossRef]

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]

Pentz, S.

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]

Salmon, N.

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).

Salo, J.

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]

Schrader, M.

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]

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]

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]

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]

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]

M. Schrader, S. W. Hell, “4Pi confocal images with axial superresolution,” J. Microsc. 183, 189–193 (1996).
[CrossRef]

Sheppard, C. J. R.

Stelzer, E. H. K.

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).

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]

S. Hell, E. H. K. Stelzer, “Properties of a 4Pi confocal fluorescence microscope,” J. Opt. Soc. Am. A 9, 2159–2166 (1992).
[CrossRef]

van der Voort, H. T. M.

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]

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]

Wilson, T.

Wolf, E.

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon Press, Oxford, U.K., 1993), p. 612.

Appl. Phys. Lett. (1)

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]

Biophys. J. (1)

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]

J. Appl. Phys. (1)

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]

J. Microsc. (4)

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]

M. Schrader, S. W. Hell, “4Pi confocal images with axial superresolution,” J. Microsc. 183, 189–193 (1996).
[CrossRef]

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]

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]

J. Opt. Soc. Am. A (2)

J. Struct. Biol. (1)

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]

Opt. Commun. (1)

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]

Opt. Lett. (2)

Optik (1)

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).

Other (3)

S. W. Hell, “Double-confocal microscope,” European patent0491289 (18December1990).

F. Lanni, Applications of Fluorescence in the Biological Sciences, 1st ed. (Liss, New York, 1986).

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon Press, Oxford, U.K., 1993), p. 612.

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Figures (6)

Fig. 1
Fig. 1

(a) Axial response, I(z), to an ultrathin fluorescent plane of the water-immersion two-photon microscope operating at 1064-nm excitation featuring a N.A. of 1.2; (b) FWHM of I(z) for different collar adjustments a–i. The adjustment for the standard 170-µm cover glass thickness is marked by a thin line.

Fig. 2
Fig. 2

(a) 4Pi confocal microscope and relative orientation of the electric field. The rotatable λ/2-plate changes the polarization of the incoming field E; θ is the angle between the axis of the plate with respect to the plane of the setup. BS denotes a neutral beam splitter, and k is the wave vector. (b) Measured 4Pi confocal PSF’s: xz sections containing the optic axis (left-hand side) and corresponding profiles along the optic axis (right-hand side). The confocal PSF is shown for comparison (below). (c) Orientation of the polarization before the beam splitter (left-hand side) and the corresponding orientation of the polarization of the two beams at the focal point for a multiple of 2π phase delay between the paths. Note that the change of the orientation of the fields (change of 2θ from 0° to 90°) changes the 4Pi PSF from constructive to destructive mode.

Fig. 3
Fig. 3

(a) As in Fig. 2, however, with a λ/2 plate with the fast axis perpendicular to the plane of the setup. In contrast to Fig. 2, the structure of the constructive-mode 4Pi confocal PSF remains unchanged upon rotation of the first λ/2-plate.

Fig. 4
Fig. 4

(a) As in Figs. 2 and 3, however, with left- and right-hand circularly polarized light, respectively. The rotatable λ/4 plate changes the polarization of the field E from linear to circular. Note the degraded interference for circular polarization.

Fig. 5
Fig. 5

(a) As in Fig. 3; note the unchanged structure of the constructive-mode 4Pi confocal PSF for varying orientation of the λ/4 plate.

Fig. 6
Fig. 6

(a) 4Pi confocal PSF of 1.2-N.A. water-immersion lens at 1064-nm excitation and 540–650-nm fluorescence detection, (b) the inverse filter l –1(z), and (c) the point-deconvolved effective PSF featuring a lateral FWHM of 360 ± 25 nm and an axial FWHM of 240 ± 20 nm. (d) The single-lens confocal counterpart has an axial FWHM of 1040 ± 50 nm. The comparison of (c) with (d) indicates that the 4Pi confocal microscope features an axial resolution with 4.3-fold improvement.

Equations (4)

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

Hill,4Pir, z=H1,illr, z+H2,illr, -zexpiϕ.
hconf,4Pi=Hill,4Pir, z2nhdetr, z=Hill,4Pir, z2nHillnr, nz2,
Iz=0H1r, z4rdr.
hconf,4Pi=lzzhpeakr, z.

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