Abstract

In 4Pi fluorescence microscopy the point-spread function is composed of a strong central lobe accompanied by interference sidelobes that produce artifacts in the image. We propose to combine two-color two-photon fluorescence microscopy and 4Pi fluorescence microscopy to overcome this sidelobe problem. Simulation results show that a single sharp fluorescence spot can be produced by use of two excitation wavelengths of 400 and 800 nm and detected at 350-nm emission wavelength.

© 2004 Optical Society of America

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References

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  1. M. Minsky, Scanning 10, 128 (1988).
    [CrossRef]
  2. S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
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    [CrossRef]
  4. W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
    [CrossRef]
  5. S. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992).
    [CrossRef]
  6. M. Dyba and S. W. Hell, Phys. Rev. Lett. 88, 163901 (2002).
    [CrossRef]
  7. M. O. Cambaliza and C. Saloma, Opt. Commun. 184, 25 (2000).
    [CrossRef]
  8. J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, J. Phys. Chem. 100, 19406 (1996).
  9. J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, Photochem. Photobiol. 64, 632 (1996).
    [CrossRef]
  10. M. Nagorni and S. W. Hell, J. Opt. Soc. Am. A 18, 36 (2001).
    [CrossRef]
  11. M. Schrader, K. Bahlmann, G. Giese, and S. W. Hell, Biophys. J. 75, 1659 (1998).
    [CrossRef] [PubMed]
  12. O. Haeberlé, Opt. Commun. 216, 55 (2003).

2003 (1)

O. Haeberlé, Opt. Commun. 216, 55 (2003).

2002 (1)

M. Dyba and S. W. Hell, Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef]

2001 (1)

2000 (1)

M. O. Cambaliza and C. Saloma, Opt. Commun. 184, 25 (2000).
[CrossRef]

1998 (1)

M. Schrader, K. Bahlmann, G. Giese, and S. W. Hell, Biophys. J. 75, 1659 (1998).
[CrossRef] [PubMed]

1996 (2)

J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, J. Phys. Chem. 100, 19406 (1996).

J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, Photochem. Photobiol. 64, 632 (1996).
[CrossRef]

1994 (1)

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

1992 (2)

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

S. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992).
[CrossRef]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef]

1988 (1)

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

Bahlmann, K.

M. Schrader, K. Bahlmann, G. Giese, and S. W. Hell, Biophys. J. 75, 1659 (1998).
[CrossRef] [PubMed]

Cambaliza, M. O.

M. O. Cambaliza and C. Saloma, Opt. Commun. 184, 25 (2000).
[CrossRef]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef]

Dyba, M.

M. Dyba and S. W. Hell, Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef]

Giese, G.

M. Schrader, K. Bahlmann, G. Giese, and S. W. Hell, Biophys. J. 75, 1659 (1998).
[CrossRef] [PubMed]

Grycznski, Z.

J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, Photochem. Photobiol. 64, 632 (1996).
[CrossRef]

J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, J. Phys. Chem. 100, 19406 (1996).

Gryczynski, I.

J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, J. Phys. Chem. 100, 19406 (1996).

J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, Photochem. Photobiol. 64, 632 (1996).
[CrossRef]

Haeberlé, O.

O. Haeberlé, Opt. Commun. 216, 55 (2003).

Hell, S.

Hell, S. W.

M. Dyba and S. W. Hell, Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef]

M. Nagorni and S. W. Hell, J. Opt. Soc. Am. A 18, 36 (2001).
[CrossRef]

M. Schrader, K. Bahlmann, G. Giese, and S. W. Hell, Biophys. J. 75, 1659 (1998).
[CrossRef] [PubMed]

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

Lacowicz, J. R.

J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, Photochem. Photobiol. 64, 632 (1996).
[CrossRef]

J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, J. Phys. Chem. 100, 19406 (1996).

Lindek, S.

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

Malak, H.

J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, Photochem. Photobiol. 64, 632 (1996).
[CrossRef]

J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, J. Phys. Chem. 100, 19406 (1996).

Minsky, M.

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

Nagorni, M.

Saloma, C.

M. O. Cambaliza and C. Saloma, Opt. Commun. 184, 25 (2000).
[CrossRef]

Schrader, M.

M. Schrader, K. Bahlmann, G. Giese, and S. W. Hell, Biophys. J. 75, 1659 (1998).
[CrossRef] [PubMed]

Stelzer, E. H. K.

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. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992).
[CrossRef]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef]

Biophys. J. (1)

M. Schrader, K. Bahlmann, G. Giese, and S. W. Hell, Biophys. J. 75, 1659 (1998).
[CrossRef] [PubMed]

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

J. Phys. Chem. (1)

J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, J. Phys. Chem. 100, 19406 (1996).

Opt. Commun. (4)

O. Haeberlé, Opt. Commun. 216, 55 (2003).

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]

M. O. Cambaliza and C. Saloma, Opt. Commun. 184, 25 (2000).
[CrossRef]

Photochem. Photobiol. (1)

J. R. Lacowicz, I. Gryczynski, H. Malak, and Z. Grycznski, Photochem. Photobiol. 64, 632 (1996).
[CrossRef]

Phys. Rev. Lett. (1)

M. Dyba and S. W. Hell, Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef]

Scanning (1)

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

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

Schematic of the experimental setup of 2C2P 4Pi microscopy.

Fig. 2
Fig. 2

Intensities of the light focused by the opposing lenses for the 800-nm wavelength (top row) and the 400-nm wavelength (middle row). The PSF of 2C2P 4Pi microscopy is shown at the bottom. The top and bottom rows are shifted 1 µm upward and 1 µm downward along the radial direction, respectively.

Fig. 3
Fig. 3

(a) Axial profiles of the intensities of light at the foci of the 4Pi microscope for 800 nm and 400 nm. (b) Axial profiles of the PSF of the 2C2P 4Pi microscope and the axial PSF of the one-photon confocal fluorescence microscope using excitation light at 266 nm and detection light at 350 nm. The same objective lenses are used in the two simulations.

Fig. 4
Fig. 4

(a) Axial profiles of the PSFs of the 2C2P 4Pi-A confocal and 2C2P 4Pi-C confocal microscopes with 400- and 800-nm excitation wavelengths and a 350-nm detection wavelength. (b) Identical radial profiles of the PSFs of both 2C2P 4Pi-A and 2C2P 4Pi-C confocal microscopes and the radial profile of the PSF of the 2C2P 4Pi microscope without confocal detection.

Equations (5)

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

hr,z=E1r,zE2r,z2,
E1,2r,z2=ReI1,20r,z2+2 ImI1,21r,z2+ReI1,22r,z2.
I1,20=0αcos1/2 θsin θ1+cos θJ0nk1,2rsin θ×expink1,2z cos θdθ,
I1,21=0αcos1/2 θsin2 θJ1nk1,2rsin θ×expink1,2z cos θdθ,
I1,22=0αcos1/2 θsin θ1-cos θJ2nk1,2rsin θ×expink1,2z cos θdθ,

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