Abstract

A method for increasing lateral as well as axial resolution in fluorescence microscopy is presented. A passband with a high cutoff frequency throughout reciprocal space can be achieved by illumination of the object with spatially harmonic excitation patterns generated by the interference of two collimated laser beams. Theoretical calculations show an almost isotropic point-spread function with a FWHM near 100  nm.

© 2001 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. M. Born and E. Wolf, Principles of Optics (Cambridge U. Press, Cambridge, 1980).
  2. T. Wilson, in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), p. 167.
    [CrossRef]
  3. S. W. Hell, M. Schrader, and H. T. M. van der Voort, J. Microsc. 187, 1 (1997).
    [CrossRef] [PubMed]
  4. J. T. Frohn, H. F. Knapp, and A. Stemmer, Proc. Natl. Acad. Sci. USA 97, 7232 (2000).
    [CrossRef]
  5. M. G. L. Gustafsson, J. Microsc. 198, 82 (2000).
    [CrossRef] [PubMed]
  6. B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
    [CrossRef] [PubMed]
  7. M. A. A. Neil, R. Juškaitis, and T. Wilson, Opt. Lett. 153, 1 (1998).
  8. V. Krishnamurthi, B. Bailey, and F. Lanni, Proc. SPIE 2655, 18 (1996).
    [CrossRef]
  9. Absolute fringe position Δ1 of the first image can easily be calculated from the measured fluorescence images because the original passband and the shifted copies overlap. This fact simplifies a practical 3D HELM setup because the beam deflection units are not required to maintain a fixed phase relationship during operation.
  10. A. Erhardt, G. Zinser, D. Komitowski, and J. Bille, Appl. Opt. 24, 194 (1998).
    [CrossRef]
  11. The choice of a cosine bell apodization results in image points with negative intensities. One normalizes the traces in Figs.  2–4 by setting the most negative value to zero.
  12. The spatial frequency of excitation u equals the difference of the propagation vectors p1 and p2 of the interfering waves. Therefore a rotation of both p1 and p2 about an axis parallel to p1–p2 leaves the pattern unaffected. By an appropriate rotation of p1 and p2, any desired value of u can be realized by interference of two plane waves whose propagation vectors reside in lower and upper half-spaces, respectively, relative to the object plane.

2000 (2)

J. T. Frohn, H. F. Knapp, and A. Stemmer, Proc. Natl. Acad. Sci. USA 97, 7232 (2000).
[CrossRef]

M. G. L. Gustafsson, J. Microsc. 198, 82 (2000).
[CrossRef] [PubMed]

1998 (2)

M. A. A. Neil, R. Juškaitis, and T. Wilson, Opt. Lett. 153, 1 (1998).

A. Erhardt, G. Zinser, D. Komitowski, and J. Bille, Appl. Opt. 24, 194 (1998).
[CrossRef]

1997 (1)

S. W. Hell, M. Schrader, and H. T. M. van der Voort, J. Microsc. 187, 1 (1997).
[CrossRef] [PubMed]

1996 (1)

V. Krishnamurthi, B. Bailey, and F. Lanni, Proc. SPIE 2655, 18 (1996).
[CrossRef]

1993 (1)

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
[CrossRef] [PubMed]

Bailey, B.

V. Krishnamurthi, B. Bailey, and F. Lanni, Proc. SPIE 2655, 18 (1996).
[CrossRef]

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
[CrossRef] [PubMed]

Bille, J.

Born, M.

M. Born and E. Wolf, Principles of Optics (Cambridge U. Press, Cambridge, 1980).

Erhardt, A.

Farkas, D. L.

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
[CrossRef] [PubMed]

Frohn, J. T.

J. T. Frohn, H. F. Knapp, and A. Stemmer, Proc. Natl. Acad. Sci. USA 97, 7232 (2000).
[CrossRef]

Gustafsson, M. G. L.

M. G. L. Gustafsson, J. Microsc. 198, 82 (2000).
[CrossRef] [PubMed]

Hell, S. W.

S. W. Hell, M. Schrader, and H. T. M. van der Voort, J. Microsc. 187, 1 (1997).
[CrossRef] [PubMed]

Juškaitis, R.

M. A. A. Neil, R. Juškaitis, and T. Wilson, Opt. Lett. 153, 1 (1998).

Knapp, H. F.

J. T. Frohn, H. F. Knapp, and A. Stemmer, Proc. Natl. Acad. Sci. USA 97, 7232 (2000).
[CrossRef]

Komitowski, D.

Krishnamurthi, V.

V. Krishnamurthi, B. Bailey, and F. Lanni, Proc. SPIE 2655, 18 (1996).
[CrossRef]

Lanni, F.

V. Krishnamurthi, B. Bailey, and F. Lanni, Proc. SPIE 2655, 18 (1996).
[CrossRef]

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
[CrossRef] [PubMed]

Neil, M. A. A.

M. A. A. Neil, R. Juškaitis, and T. Wilson, Opt. Lett. 153, 1 (1998).

Schrader, M.

S. W. Hell, M. Schrader, and H. T. M. van der Voort, J. Microsc. 187, 1 (1997).
[CrossRef] [PubMed]

Stemmer, A.

J. T. Frohn, H. F. Knapp, and A. Stemmer, Proc. Natl. Acad. Sci. USA 97, 7232 (2000).
[CrossRef]

Taylor, D. L.

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
[CrossRef] [PubMed]

van der Voort, H. T. M.

S. W. Hell, M. Schrader, and H. T. M. van der Voort, J. Microsc. 187, 1 (1997).
[CrossRef] [PubMed]

Wilson, T.

M. A. A. Neil, R. Juškaitis, and T. Wilson, Opt. Lett. 153, 1 (1998).

T. Wilson, in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), p. 167.
[CrossRef]

Wolf, E.

M. Born and E. Wolf, Principles of Optics (Cambridge U. Press, Cambridge, 1980).

Zinser, G.

Appl. Opt. (1)

J. Microsc. (2)

S. W. Hell, M. Schrader, and H. T. M. van der Voort, J. Microsc. 187, 1 (1997).
[CrossRef] [PubMed]

M. G. L. Gustafsson, J. Microsc. 198, 82 (2000).
[CrossRef] [PubMed]

Nature (1)

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
[CrossRef] [PubMed]

Opt. Lett. (1)

M. A. A. Neil, R. Juškaitis, and T. Wilson, Opt. Lett. 153, 1 (1998).

Proc. Natl. Acad. Sci. USA (1)

J. T. Frohn, H. F. Knapp, and A. Stemmer, Proc. Natl. Acad. Sci. USA 97, 7232 (2000).
[CrossRef]

Proc. SPIE (1)

V. Krishnamurthi, B. Bailey, and F. Lanni, Proc. SPIE 2655, 18 (1996).
[CrossRef]

Other (5)

Absolute fringe position Δ1 of the first image can easily be calculated from the measured fluorescence images because the original passband and the shifted copies overlap. This fact simplifies a practical 3D HELM setup because the beam deflection units are not required to maintain a fixed phase relationship during operation.

The choice of a cosine bell apodization results in image points with negative intensities. One normalizes the traces in Figs.  2–4 by setting the most negative value to zero.

The spatial frequency of excitation u equals the difference of the propagation vectors p1 and p2 of the interfering waves. Therefore a rotation of both p1 and p2 about an axis parallel to p1–p2 leaves the pattern unaffected. By an appropriate rotation of p1 and p2, any desired value of u can be realized by interference of two plane waves whose propagation vectors reside in lower and upper half-spaces, respectively, relative to the object plane.

M. Born and E. Wolf, Principles of Optics (Cambridge U. Press, Cambridge, 1980).

T. Wilson, in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), p. 167.
[CrossRef]

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

Schematic representation of the passband regions obtained by standard FM and by 3D HELM by use of the combinations of shift vectors given in Table  1. Shown are projections upon the kxkz plane (top row) and upon the kxky-plane (bottom row).

Fig. 2
Fig. 2

Lateral point-spread function for standard FM and for 3D HELM for cases a–c from Table  1. Because the point-spread function for case a is not symmetric with respect to the x and y coordinates, the values along both axes are given.

Fig. 3
Fig. 3

Axial point-spread function for standard FM and for 3D-HELM for the cases a–c of Table  1.

Fig. 4
Fig. 4

Axial response to a thin, circular disk (diameter, 2 μm) oriented parallel to the object plane for standard FM and for 3D HELM for cases a–c of Table  1.

Fig. 5
Fig. 5

3D HELM setup. Two lenses (L1 and L2) form focal spots in the back apertures of two objectives. A rotatable mirror sets the common off-axis distance of these spots, and two metal-coated rotatable Abbe–Koenig prisms are used to turn the spots about the optical axis. The positions of these spots determine u, whereas the piezo-actuated mirror sets Δ.

Tables (1)

Tables Icon

Table 1 Simulations Performed for Some Combinations of Shift Vectorsa

Equations (5)

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

Ir=1+Mcosur+Δ,
ϕr=Irψr,
θ˜k=Tkϕ˜k,
θ˜lk=Tk2ψ˜k+MexpiΔlψ˜k+u+Mexp-iΔlψ˜k-u,
M=λ216π2n2u2,

Metrics