Abstract

We present a novel scanning fluorescence microscopy technique, nonconfocal theta microscopy (NCTM), that provides almost isotropic resolution. In NCTM, multiphoton absorption from two orthogonal illumination directions is used to induce fluorescence emission. Therefore the point-spread function of the microscope is described by the product of illumination point-spread functions with reduced spatial overlap, which provides the resolution improvement and the more isotropic observation volume. We discuss the technical details of this new method.

© 1999 Optical Society of America

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References

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  1. M. Minsky, “Microscopy apparatus,” U.S. patent3,012,467 (December19, 1961).
  2. C. W. McCutchen, J. Opt. Soc. Am. 57, 1190 (1967).
    [CrossRef] [PubMed]
  3. C. J. R. Sheppard, J. Microsc. 154, 237 (1989).
    [CrossRef]
  4. E. H. K. Stelzer, S. Lindek, and R. Pick, “Konfokales Mikroskop” German patentDE 43 26 473 (December6, 1996).
  5. E. H. K. Stelzer and S. Lindek, Opt. Commun. 111, 536 (1994).
    [CrossRef]
  6. E. H. K. Stelzer, S. Lindek, S. Albrecht, R. Pick, G. Ritter, N. Salmon, and R. Stricker, J. Microsc. 179, 1 (1995).
    [CrossRef]
  7. S. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992).
    [CrossRef]
  8. S. Lindek, R. Pick, and E. H. K. Stelzer, Rev. Sci. Instrum. 65, 3367 (1994).
    [CrossRef]
  9. E. H. K. Stelzer and S. Lindek, “Konfokales Mikroskop mit einem Doppelobjektiv-System,” German patentDE 196 29 725 (December19, 1997).
  10. S. Lindek, T. Sefany, and E. H. K. Stelzer, J. Microsc. 188, 280 (1997).
    [CrossRef]
  11. W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
    [CrossRef] [PubMed]
  12. J. R. Lakowicz, I. Gryczynski, H. Malak, and Z. Gryczynski, J. Phys. Chem. 100, 19406 (1996).
  13. J. R. Lakowicz, I. Gryczynski, H. Malak, and Z. Gryczynski, Photochem. Photobiol. 64, 632 (1996).
    [CrossRef] [PubMed]
  14. N. M. Kroll, Phys. Rev. 127, 1207 (1962).
    [CrossRef]
  15. J. A. Giordmaine and R. C. Miller, Phys. Rev. Lett. 14, 973 (1965).
    [CrossRef]
  16. S. Lindek, C. Cremer, and E. H. K. Stelzer, Appl. Opt. 35, 126 (1996).
    [CrossRef] [PubMed]
  17. J. J. Hopfield, J. M. Worlock, and K. Park, Phys. Rev. Lett. 11, 414 (1963).
    [CrossRef]
  18. D. Fröhlich and H. Mahr, Phys. Rev. Lett. 16, 895 (1966).
    [CrossRef]
  19. S. Lindek, J. Swoger, and E. H. K. Stelzer, J. Mod. Opt. 46, 843 (1999).
    [CrossRef]

1999 (1)

S. Lindek, J. Swoger, and E. H. K. Stelzer, J. Mod. Opt. 46, 843 (1999).
[CrossRef]

1997 (1)

S. Lindek, T. Sefany, and E. H. K. Stelzer, J. Microsc. 188, 280 (1997).
[CrossRef]

1996 (3)

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

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

S. Lindek, C. Cremer, and E. H. K. Stelzer, Appl. Opt. 35, 126 (1996).
[CrossRef] [PubMed]

1995 (1)

E. H. K. Stelzer, S. Lindek, S. Albrecht, R. Pick, G. Ritter, N. Salmon, and R. Stricker, J. Microsc. 179, 1 (1995).
[CrossRef]

1994 (2)

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

S. Lindek, R. Pick, and E. H. K. Stelzer, Rev. Sci. Instrum. 65, 3367 (1994).
[CrossRef]

1992 (1)

1990 (1)

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

1989 (1)

C. J. R. Sheppard, J. Microsc. 154, 237 (1989).
[CrossRef]

1967 (1)

1966 (1)

D. Fröhlich and H. Mahr, Phys. Rev. Lett. 16, 895 (1966).
[CrossRef]

1965 (1)

J. A. Giordmaine and R. C. Miller, Phys. Rev. Lett. 14, 973 (1965).
[CrossRef]

1963 (1)

J. J. Hopfield, J. M. Worlock, and K. Park, Phys. Rev. Lett. 11, 414 (1963).
[CrossRef]

1962 (1)

N. M. Kroll, Phys. Rev. 127, 1207 (1962).
[CrossRef]

Albrecht, S.

E. H. K. Stelzer, S. Lindek, S. Albrecht, R. Pick, G. Ritter, N. Salmon, and R. Stricker, J. Microsc. 179, 1 (1995).
[CrossRef]

Cremer, C.

Denk, W.

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

Fröhlich, D.

D. Fröhlich and H. Mahr, Phys. Rev. Lett. 16, 895 (1966).
[CrossRef]

Giordmaine, J. A.

J. A. Giordmaine and R. C. Miller, Phys. Rev. Lett. 14, 973 (1965).
[CrossRef]

Gryczynski, I.

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

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

Gryczynski, Z.

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

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

Hell, S.

Hopfield, J. J.

J. J. Hopfield, J. M. Worlock, and K. Park, Phys. Rev. Lett. 11, 414 (1963).
[CrossRef]

Kroll, N. M.

N. M. Kroll, Phys. Rev. 127, 1207 (1962).
[CrossRef]

Lakowicz, J. R.

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

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

Lindek, S.

S. Lindek, J. Swoger, and E. H. K. Stelzer, J. Mod. Opt. 46, 843 (1999).
[CrossRef]

S. Lindek, T. Sefany, and E. H. K. Stelzer, J. Microsc. 188, 280 (1997).
[CrossRef]

S. Lindek, C. Cremer, and E. H. K. Stelzer, Appl. Opt. 35, 126 (1996).
[CrossRef] [PubMed]

E. H. K. Stelzer, S. Lindek, S. Albrecht, R. Pick, G. Ritter, N. Salmon, and R. Stricker, J. Microsc. 179, 1 (1995).
[CrossRef]

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

S. Lindek, R. Pick, and E. H. K. Stelzer, Rev. Sci. Instrum. 65, 3367 (1994).
[CrossRef]

E. H. K. Stelzer, S. Lindek, and R. Pick, “Konfokales Mikroskop” German patentDE 43 26 473 (December6, 1996).

E. H. K. Stelzer and S. Lindek, “Konfokales Mikroskop mit einem Doppelobjektiv-System,” German patentDE 196 29 725 (December19, 1997).

Mahr, H.

D. Fröhlich and H. Mahr, Phys. Rev. Lett. 16, 895 (1966).
[CrossRef]

Malak, H.

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

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

McCutchen, C. W.

Miller, R. C.

J. A. Giordmaine and R. C. Miller, Phys. Rev. Lett. 14, 973 (1965).
[CrossRef]

Minsky, M.

M. Minsky, “Microscopy apparatus,” U.S. patent3,012,467 (December19, 1961).

Park, K.

J. J. Hopfield, J. M. Worlock, and K. Park, Phys. Rev. Lett. 11, 414 (1963).
[CrossRef]

Pick, R.

E. H. K. Stelzer, S. Lindek, S. Albrecht, R. Pick, G. Ritter, N. Salmon, and R. Stricker, J. Microsc. 179, 1 (1995).
[CrossRef]

S. Lindek, R. Pick, and E. H. K. Stelzer, Rev. Sci. Instrum. 65, 3367 (1994).
[CrossRef]

E. H. K. Stelzer, S. Lindek, and R. Pick, “Konfokales Mikroskop” German patentDE 43 26 473 (December6, 1996).

Ritter, G.

E. H. K. Stelzer, S. Lindek, S. Albrecht, R. Pick, G. Ritter, N. Salmon, and R. Stricker, J. Microsc. 179, 1 (1995).
[CrossRef]

Salmon, N.

E. H. K. Stelzer, S. Lindek, S. Albrecht, R. Pick, G. Ritter, N. Salmon, and R. Stricker, J. Microsc. 179, 1 (1995).
[CrossRef]

Sefany, T.

S. Lindek, T. Sefany, and E. H. K. Stelzer, J. Microsc. 188, 280 (1997).
[CrossRef]

Sheppard, C. J. R.

C. J. R. Sheppard, J. Microsc. 154, 237 (1989).
[CrossRef]

Stelzer, E. H. K.

S. Lindek, J. Swoger, and E. H. K. Stelzer, J. Mod. Opt. 46, 843 (1999).
[CrossRef]

S. Lindek, T. Sefany, and E. H. K. Stelzer, J. Microsc. 188, 280 (1997).
[CrossRef]

S. Lindek, C. Cremer, and E. H. K. Stelzer, Appl. Opt. 35, 126 (1996).
[CrossRef] [PubMed]

E. H. K. Stelzer, S. Lindek, S. Albrecht, R. Pick, G. Ritter, N. Salmon, and R. Stricker, J. Microsc. 179, 1 (1995).
[CrossRef]

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

S. Lindek, R. Pick, and E. H. K. Stelzer, Rev. Sci. Instrum. 65, 3367 (1994).
[CrossRef]

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

E. H. K. Stelzer and S. Lindek, “Konfokales Mikroskop mit einem Doppelobjektiv-System,” German patentDE 196 29 725 (December19, 1997).

E. H. K. Stelzer, S. Lindek, and R. Pick, “Konfokales Mikroskop” German patentDE 43 26 473 (December6, 1996).

Stricker, R.

E. H. K. Stelzer, S. Lindek, S. Albrecht, R. Pick, G. Ritter, N. Salmon, and R. Stricker, J. Microsc. 179, 1 (1995).
[CrossRef]

Strickler, J. H.

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

Swoger, J.

S. Lindek, J. Swoger, and E. H. K. Stelzer, J. Mod. Opt. 46, 843 (1999).
[CrossRef]

Webb, W. W.

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

Worlock, J. M.

J. J. Hopfield, J. M. Worlock, and K. Park, Phys. Rev. Lett. 11, 414 (1963).
[CrossRef]

Appl. Opt. (1)

J. Microsc. (3)

S. Lindek, T. Sefany, and E. H. K. Stelzer, J. Microsc. 188, 280 (1997).
[CrossRef]

C. J. R. Sheppard, J. Microsc. 154, 237 (1989).
[CrossRef]

E. H. K. Stelzer, S. Lindek, S. Albrecht, R. Pick, G. Ritter, N. Salmon, and R. Stricker, J. Microsc. 179, 1 (1995).
[CrossRef]

J. Mod. Opt. (1)

S. Lindek, J. Swoger, and E. H. K. Stelzer, J. Mod. Opt. 46, 843 (1999).
[CrossRef]

J. Opt. Soc. Am. (1)

J. Opt. Soc. Am. A (1)

J. Phys. Chem. (1)

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

Opt. Commun. (1)

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

Photochem. Photobiol. (1)

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

Phys. Rev. (1)

N. M. Kroll, Phys. Rev. 127, 1207 (1962).
[CrossRef]

Phys. Rev. Lett. (3)

J. A. Giordmaine and R. C. Miller, Phys. Rev. Lett. 14, 973 (1965).
[CrossRef]

J. J. Hopfield, J. M. Worlock, and K. Park, Phys. Rev. Lett. 11, 414 (1963).
[CrossRef]

D. Fröhlich and H. Mahr, Phys. Rev. Lett. 16, 895 (1966).
[CrossRef]

Rev. Sci. Instrum. (1)

S. Lindek, R. Pick, and E. H. K. Stelzer, Rev. Sci. Instrum. 65, 3367 (1994).
[CrossRef]

Science (1)

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

Other (3)

E. H. K. Stelzer and S. Lindek, “Konfokales Mikroskop mit einem Doppelobjektiv-System,” German patentDE 196 29 725 (December19, 1997).

M. Minsky, “Microscopy apparatus,” U.S. patent3,012,467 (December19, 1961).

E. H. K. Stelzer, S. Lindek, and R. Pick, “Konfokales Mikroskop” German patentDE 43 26 473 (December6, 1996).

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Figures (2)

Fig. 1
Fig. 1

Two NCTM setups. A, A basic setup with two lasers (L1, L2) that illuminate the sample through two orthogonal objective lenses (OL1, OL2). The fluorescence light is detected through OL1. B, A setup with two lasers that illuminate the sample through a single objective lens (SLTM). Solid lines, illumination paths; dotted lines, detection paths; D’s, detectors; DM’s, dichroic mirrors; F’s, foci; MU, mirror unit; TL, tube lens.

Fig. 2
Fig. 2

Dependence of NCTM resolution on wavelengths λ1 and λ2; the FWHM’s and the VHM. The abscissa shows the larger wavelength λ2 in units of λ0. The FWHM’s are also given in units of λ0. The VHM is the product of the three normalized FWHM’s.

Tables (1)

Tables Icon

Table 1 Resolution for Standard Microscopy and NCTM with a NA of 0.75

Metrics