Abstract

In a confocal theta fluorescence microscope, two objective lenses with circular apertures are used, one to illuminate the sample and the other to detect the emitted light at an angle to the illumination axis. We show that annular illumination and detection apertures lead to a reduction in the extent of the point-spread function. A spatial resolution improved by more than 50% can be achieved with a central obstruction blocking the inner 80% of the diameter. For the limit of a very narrow annular aperture and a numerical aperture of 0.75, the volume at half-maximum of the point-spread function is reduced from 15 to 5 aL. A mixed setup with an annular illumination aperture and a circular detection aperture is also considered.

© 1996 Optical Society of America

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References

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  1. R. W. Wijnaendts van Resandt, H. J. B. Marsman, R. Kaplan, J. Davoust, E. H. K. Stelzer, R. Stricker, “Optical fluorescence microscopy in three dimensions: microtomoscopy,” J. Microsc. 138, 29–34 (1985).
    [CrossRef]
  2. D. M. Shotton, “Confocal scanning optical microscopy and its applications for biological specimens,” J. Cell Sci. 94, 175–206 (1989).
  3. C. J. R. Sheppard, A. Choudhury, “Image formation in the scanning microscope,” Opt. Acta 24, 1051–1073 (1977).
    [CrossRef]
  4. G. J. Brakenhoff, H. T. M. van der Voort, E. A. van Spronsen, N. Nanninga, “3-dimensional imaging of biological structures by high resolution confocal scanning laser microscopy,” Scanning Microsc. 2, 33–40 (1988).
    [PubMed]
  5. T. Wilson, “Optical sectioning in confocal fluorescent microscopes,” J. Microsc. 154, 143–156 (1989).
    [CrossRef]
  6. C. J. R. Sheppard, “Axial resolution of confocal fluorescence microscopy,” J. Microsc. 154, 237–241 (1989).
    [CrossRef]
  7. C. J. R. Sheppard, Y. Gong, “Improvement in axial resolution by interference confocal microscopy,” Optik 87, 129–132 (1991).
  8. C. J. R. Sheppard, M. Gu, “Improvement of axial resolution in confocal microscopy using an annular pupil,” Opt. Commun. 84, 7–13 (1991).
    [CrossRef]
  9. S. Hell, E. H. K. Stelzer, “Properties of a 4Pi confocal fluorescence microscope,” J. Opt. Soc. Am. A 9, 2159–2166 (1992).
    [CrossRef]
  10. S. 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]
  11. M. Gu, C. J. R. Sheppard, H. Zhou, “Optimization of axial resolution in confocal imaging using annular pupils,” Optik 93, 87–90 (1993).
  12. M. Gu, T. Tannous, C. J. R. Sheppard, “Improved axial resolution in confocal fluorescence microscopy using annular pupils,” Opt. Commun. 110, 533–539 (1994).
    [CrossRef]
  13. S. Lindek, E. H. K. Stelzer, “Confocal theta microscopy and 4Pi-confocal theta microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing, C. J. Cogswell, K. Carlsson, eds., Proc. Soc. Photo-Opt. Instrum. Eng.2184, 188–194 (1994).
  14. E. H. K. Stelzer, S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111, 536–547 (1994).
    [CrossRef]
  15. S. Lindek, R. Pick, E. H. K. Stelzer, “Confocal theta microscope with three objective lenses,” Rev. Sci. Instrum. 65, 3367–3372 (1994).
    [CrossRef]
  16. A. T. Young, “Photometric error analysis. X: Encircled energy 1total illuminance2 calculations for annular apertures,” Appl. Opt. 9, 1874–1878 (1970).
    [PubMed]
  17. E. H. Linfoot, E. Wolf, “Diffraction images in systems with annular apertures,” Proc. Phys. Soc. London Sect. B 66, 145–149 (1953).
    [CrossRef]
  18. C. J. R. Sheppard, M. Gu, “Optical sectioning in confocal microscopes with annular pupil,” Optik 86, 169–172 (1991).
  19. X. S. Gan, C. J. R. Sheppard, “Imaging in a confocal microscope with one circular and one annular lens,” Opt. Commun. 103, 254–264 (1993).
    [CrossRef]
  20. K. Sieracki, E. W. Hansen, “A leaky annular pupil for improved axial resolution in confocal fluorescence microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing, C. J. Cogswell, K. Carlsson, eds., Proc. Soc. Photo-Opt. Instrum. Eng.2184, 120–126 (1994).
  21. M. Born, E. Wolf, Principles of Optics (Pergamon, Oxford, UK, 1980), p. 437.
  22. S. Wolfram, Mathematica—A System for Doing Mathematics by Computer (Addison-Wesley, Redwood City, Calif., 1991).

1994 (3)

M. Gu, T. Tannous, C. J. R. Sheppard, “Improved axial resolution in confocal fluorescence microscopy using annular pupils,” Opt. Commun. 110, 533–539 (1994).
[CrossRef]

E. H. K. Stelzer, S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111, 536–547 (1994).
[CrossRef]

S. Lindek, R. Pick, E. H. K. Stelzer, “Confocal theta microscope with three objective lenses,” Rev. Sci. Instrum. 65, 3367–3372 (1994).
[CrossRef]

1993 (2)

M. Gu, C. J. R. Sheppard, H. Zhou, “Optimization of axial resolution in confocal imaging using annular pupils,” Optik 93, 87–90 (1993).

X. S. Gan, C. J. R. Sheppard, “Imaging in a confocal microscope with one circular and one annular lens,” Opt. Commun. 103, 254–264 (1993).
[CrossRef]

1992 (2)

S. 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]

1991 (3)

C. J. R. Sheppard, M. Gu, “Optical sectioning in confocal microscopes with annular pupil,” Optik 86, 169–172 (1991).

C. J. R. Sheppard, Y. Gong, “Improvement in axial resolution by interference confocal microscopy,” Optik 87, 129–132 (1991).

C. J. R. Sheppard, M. Gu, “Improvement of axial resolution in confocal microscopy using an annular pupil,” Opt. Commun. 84, 7–13 (1991).
[CrossRef]

1989 (3)

T. Wilson, “Optical sectioning in confocal fluorescent microscopes,” J. Microsc. 154, 143–156 (1989).
[CrossRef]

C. J. R. Sheppard, “Axial resolution of confocal fluorescence microscopy,” J. Microsc. 154, 237–241 (1989).
[CrossRef]

D. M. Shotton, “Confocal scanning optical microscopy and its applications for biological specimens,” J. Cell Sci. 94, 175–206 (1989).

1988 (1)

G. J. Brakenhoff, H. T. M. van der Voort, E. A. van Spronsen, N. Nanninga, “3-dimensional imaging of biological structures by high resolution confocal scanning laser microscopy,” Scanning Microsc. 2, 33–40 (1988).
[PubMed]

1985 (1)

R. W. Wijnaendts van Resandt, H. J. B. Marsman, R. Kaplan, J. Davoust, E. H. K. Stelzer, R. Stricker, “Optical fluorescence microscopy in three dimensions: microtomoscopy,” J. Microsc. 138, 29–34 (1985).
[CrossRef]

1977 (1)

C. J. R. Sheppard, A. Choudhury, “Image formation in the scanning microscope,” Opt. Acta 24, 1051–1073 (1977).
[CrossRef]

1970 (1)

1953 (1)

E. H. Linfoot, E. Wolf, “Diffraction images in systems with annular apertures,” Proc. Phys. Soc. London Sect. B 66, 145–149 (1953).
[CrossRef]

Born, M.

M. Born, E. Wolf, Principles of Optics (Pergamon, Oxford, UK, 1980), p. 437.

Brakenhoff, G. J.

G. J. Brakenhoff, H. T. M. van der Voort, E. A. van Spronsen, N. Nanninga, “3-dimensional imaging of biological structures by high resolution confocal scanning laser microscopy,” Scanning Microsc. 2, 33–40 (1988).
[PubMed]

Choudhury, A.

C. J. R. Sheppard, A. Choudhury, “Image formation in the scanning microscope,” Opt. Acta 24, 1051–1073 (1977).
[CrossRef]

Davoust, J.

R. W. Wijnaendts van Resandt, H. J. B. Marsman, R. Kaplan, J. Davoust, E. H. K. Stelzer, R. Stricker, “Optical fluorescence microscopy in three dimensions: microtomoscopy,” J. Microsc. 138, 29–34 (1985).
[CrossRef]

Gan, X. S.

X. S. Gan, C. J. R. Sheppard, “Imaging in a confocal microscope with one circular and one annular lens,” Opt. Commun. 103, 254–264 (1993).
[CrossRef]

Gong, Y.

C. J. R. Sheppard, Y. Gong, “Improvement in axial resolution by interference confocal microscopy,” Optik 87, 129–132 (1991).

Gu, M.

M. Gu, T. Tannous, C. J. R. Sheppard, “Improved axial resolution in confocal fluorescence microscopy using annular pupils,” Opt. Commun. 110, 533–539 (1994).
[CrossRef]

M. Gu, C. J. R. Sheppard, H. Zhou, “Optimization of axial resolution in confocal imaging using annular pupils,” Optik 93, 87–90 (1993).

C. J. R. Sheppard, M. Gu, “Optical sectioning in confocal microscopes with annular pupil,” Optik 86, 169–172 (1991).

C. J. R. Sheppard, M. Gu, “Improvement of axial resolution in confocal microscopy using an annular pupil,” Opt. Commun. 84, 7–13 (1991).
[CrossRef]

Hansen, E. W.

K. Sieracki, E. W. Hansen, “A leaky annular pupil for improved axial resolution in confocal fluorescence microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing, C. J. Cogswell, K. Carlsson, eds., Proc. Soc. Photo-Opt. Instrum. Eng.2184, 120–126 (1994).

Hell, S.

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

S. 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]

Kaplan, R.

R. W. Wijnaendts van Resandt, H. J. B. Marsman, R. Kaplan, J. Davoust, E. H. K. Stelzer, R. Stricker, “Optical fluorescence microscopy in three dimensions: microtomoscopy,” J. Microsc. 138, 29–34 (1985).
[CrossRef]

Lindek, S.

E. H. K. Stelzer, S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111, 536–547 (1994).
[CrossRef]

S. Lindek, R. Pick, E. H. K. Stelzer, “Confocal theta microscope with three objective lenses,” Rev. Sci. Instrum. 65, 3367–3372 (1994).
[CrossRef]

S. Lindek, E. H. K. Stelzer, “Confocal theta microscopy and 4Pi-confocal theta microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing, C. J. Cogswell, K. Carlsson, eds., Proc. Soc. Photo-Opt. Instrum. Eng.2184, 188–194 (1994).

Linfoot, E. H.

E. H. Linfoot, E. Wolf, “Diffraction images in systems with annular apertures,” Proc. Phys. Soc. London Sect. B 66, 145–149 (1953).
[CrossRef]

Marsman, H. J. B.

R. W. Wijnaendts van Resandt, H. J. B. Marsman, R. Kaplan, J. Davoust, E. H. K. Stelzer, R. Stricker, “Optical fluorescence microscopy in three dimensions: microtomoscopy,” J. Microsc. 138, 29–34 (1985).
[CrossRef]

Nanninga, N.

G. J. Brakenhoff, H. T. M. van der Voort, E. A. van Spronsen, N. Nanninga, “3-dimensional imaging of biological structures by high resolution confocal scanning laser microscopy,” Scanning Microsc. 2, 33–40 (1988).
[PubMed]

Pick, R.

S. Lindek, R. Pick, E. H. K. Stelzer, “Confocal theta microscope with three objective lenses,” Rev. Sci. Instrum. 65, 3367–3372 (1994).
[CrossRef]

Sheppard, C. J. R.

M. Gu, T. Tannous, C. J. R. Sheppard, “Improved axial resolution in confocal fluorescence microscopy using annular pupils,” Opt. Commun. 110, 533–539 (1994).
[CrossRef]

X. S. Gan, C. J. R. Sheppard, “Imaging in a confocal microscope with one circular and one annular lens,” Opt. Commun. 103, 254–264 (1993).
[CrossRef]

M. Gu, C. J. R. Sheppard, H. Zhou, “Optimization of axial resolution in confocal imaging using annular pupils,” Optik 93, 87–90 (1993).

C. J. R. Sheppard, M. Gu, “Optical sectioning in confocal microscopes with annular pupil,” Optik 86, 169–172 (1991).

C. J. R. Sheppard, Y. Gong, “Improvement in axial resolution by interference confocal microscopy,” Optik 87, 129–132 (1991).

C. J. R. Sheppard, M. Gu, “Improvement of axial resolution in confocal microscopy using an annular pupil,” Opt. Commun. 84, 7–13 (1991).
[CrossRef]

C. J. R. Sheppard, “Axial resolution of confocal fluorescence microscopy,” J. Microsc. 154, 237–241 (1989).
[CrossRef]

C. J. R. Sheppard, A. Choudhury, “Image formation in the scanning microscope,” Opt. Acta 24, 1051–1073 (1977).
[CrossRef]

Shotton, D. M.

D. M. Shotton, “Confocal scanning optical microscopy and its applications for biological specimens,” J. Cell Sci. 94, 175–206 (1989).

Sieracki, K.

K. Sieracki, E. W. Hansen, “A leaky annular pupil for improved axial resolution in confocal fluorescence microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing, C. J. Cogswell, K. Carlsson, eds., Proc. Soc. Photo-Opt. Instrum. Eng.2184, 120–126 (1994).

Stelzer, E. H. K.

S. Lindek, R. Pick, E. H. K. Stelzer, “Confocal theta microscope with three objective lenses,” Rev. Sci. Instrum. 65, 3367–3372 (1994).
[CrossRef]

E. H. K. Stelzer, S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111, 536–547 (1994).
[CrossRef]

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

S. 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]

R. W. Wijnaendts van Resandt, H. J. B. Marsman, R. Kaplan, J. Davoust, E. H. K. Stelzer, R. Stricker, “Optical fluorescence microscopy in three dimensions: microtomoscopy,” J. Microsc. 138, 29–34 (1985).
[CrossRef]

S. Lindek, E. H. K. Stelzer, “Confocal theta microscopy and 4Pi-confocal theta microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing, C. J. Cogswell, K. Carlsson, eds., Proc. Soc. Photo-Opt. Instrum. Eng.2184, 188–194 (1994).

Stricker, R.

R. W. Wijnaendts van Resandt, H. J. B. Marsman, R. Kaplan, J. Davoust, E. H. K. Stelzer, R. Stricker, “Optical fluorescence microscopy in three dimensions: microtomoscopy,” J. Microsc. 138, 29–34 (1985).
[CrossRef]

Tannous, T.

M. Gu, T. Tannous, C. J. R. Sheppard, “Improved axial resolution in confocal fluorescence microscopy using annular pupils,” Opt. Commun. 110, 533–539 (1994).
[CrossRef]

van der Voort, H. T. M.

G. J. Brakenhoff, H. T. M. van der Voort, E. A. van Spronsen, N. Nanninga, “3-dimensional imaging of biological structures by high resolution confocal scanning laser microscopy,” Scanning Microsc. 2, 33–40 (1988).
[PubMed]

van Spronsen, E. A.

G. J. Brakenhoff, H. T. M. van der Voort, E. A. van Spronsen, N. Nanninga, “3-dimensional imaging of biological structures by high resolution confocal scanning laser microscopy,” Scanning Microsc. 2, 33–40 (1988).
[PubMed]

Wijnaendts van Resandt, R. W.

R. W. Wijnaendts van Resandt, H. J. B. Marsman, R. Kaplan, J. Davoust, E. H. K. Stelzer, R. Stricker, “Optical fluorescence microscopy in three dimensions: microtomoscopy,” J. Microsc. 138, 29–34 (1985).
[CrossRef]

Wilson, T.

T. Wilson, “Optical sectioning in confocal fluorescent microscopes,” J. Microsc. 154, 143–156 (1989).
[CrossRef]

Wolf, E.

E. H. Linfoot, E. Wolf, “Diffraction images in systems with annular apertures,” Proc. Phys. Soc. London Sect. B 66, 145–149 (1953).
[CrossRef]

M. Born, E. Wolf, Principles of Optics (Pergamon, Oxford, UK, 1980), p. 437.

Wolfram, S.

S. Wolfram, Mathematica—A System for Doing Mathematics by Computer (Addison-Wesley, Redwood City, Calif., 1991).

Young, A. T.

Zhou, H.

M. Gu, C. J. R. Sheppard, H. Zhou, “Optimization of axial resolution in confocal imaging using annular pupils,” Optik 93, 87–90 (1993).

Appl. Opt. (1)

J. Cell Sci. (1)

D. M. Shotton, “Confocal scanning optical microscopy and its applications for biological specimens,” J. Cell Sci. 94, 175–206 (1989).

J. Microsc. (3)

T. Wilson, “Optical sectioning in confocal fluorescent microscopes,” J. Microsc. 154, 143–156 (1989).
[CrossRef]

C. J. R. Sheppard, “Axial resolution of confocal fluorescence microscopy,” J. Microsc. 154, 237–241 (1989).
[CrossRef]

R. W. Wijnaendts van Resandt, H. J. B. Marsman, R. Kaplan, J. Davoust, E. H. K. Stelzer, R. Stricker, “Optical fluorescence microscopy in three dimensions: microtomoscopy,” J. Microsc. 138, 29–34 (1985).
[CrossRef]

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

Opt. Acta (1)

C. J. R. Sheppard, A. Choudhury, “Image formation in the scanning microscope,” Opt. Acta 24, 1051–1073 (1977).
[CrossRef]

Opt. Commun. (5)

C. J. R. Sheppard, M. Gu, “Improvement of axial resolution in confocal microscopy using an annular pupil,” Opt. Commun. 84, 7–13 (1991).
[CrossRef]

S. 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]

M. Gu, T. Tannous, C. J. R. Sheppard, “Improved axial resolution in confocal fluorescence microscopy using annular pupils,” Opt. Commun. 110, 533–539 (1994).
[CrossRef]

E. H. K. Stelzer, S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111, 536–547 (1994).
[CrossRef]

X. S. Gan, C. J. R. Sheppard, “Imaging in a confocal microscope with one circular and one annular lens,” Opt. Commun. 103, 254–264 (1993).
[CrossRef]

Optik (3)

M. Gu, C. J. R. Sheppard, H. Zhou, “Optimization of axial resolution in confocal imaging using annular pupils,” Optik 93, 87–90 (1993).

C. J. R. Sheppard, Y. Gong, “Improvement in axial resolution by interference confocal microscopy,” Optik 87, 129–132 (1991).

C. J. R. Sheppard, M. Gu, “Optical sectioning in confocal microscopes with annular pupil,” Optik 86, 169–172 (1991).

Proc. Phys. Soc. London Sect. B (1)

E. H. Linfoot, E. Wolf, “Diffraction images in systems with annular apertures,” Proc. Phys. Soc. London Sect. B 66, 145–149 (1953).
[CrossRef]

Rev. Sci. Instrum. (1)

S. Lindek, R. Pick, E. H. K. Stelzer, “Confocal theta microscope with three objective lenses,” Rev. Sci. Instrum. 65, 3367–3372 (1994).
[CrossRef]

Scanning Microsc. (1)

G. J. Brakenhoff, H. T. M. van der Voort, E. A. van Spronsen, N. Nanninga, “3-dimensional imaging of biological structures by high resolution confocal scanning laser microscopy,” Scanning Microsc. 2, 33–40 (1988).
[PubMed]

Other (4)

S. Lindek, E. H. K. Stelzer, “Confocal theta microscopy and 4Pi-confocal theta microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing, C. J. Cogswell, K. Carlsson, eds., Proc. Soc. Photo-Opt. Instrum. Eng.2184, 188–194 (1994).

K. Sieracki, E. W. Hansen, “A leaky annular pupil for improved axial resolution in confocal fluorescence microscopy,” in Three-Dimensional Microscopy: Image Acquisition and Processing, C. J. Cogswell, K. Carlsson, eds., Proc. Soc. Photo-Opt. Instrum. Eng.2184, 120–126 (1994).

M. Born, E. Wolf, Principles of Optics (Pergamon, Oxford, UK, 1980), p. 437.

S. Wolfram, Mathematica—A System for Doing Mathematics by Computer (Addison-Wesley, Redwood City, Calif., 1991).

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Figures (3)

Fig. 1
Fig. 1

a, PSFs along the z axis in confocal (left) and confocal theta (right) microscopes with circular (∊ = 0, continuous curve) and identical annular apertures (∊ = 0.5 and ∊ = 0.99, dashed curves) for illumination and detection. b, PSFs along the x axis for the same setups.

Fig. 2
Fig. 2

a, PSFs along the z axis in confocal (left) and confocal theta (right) microscopes with circular (∊ = 0, continuous curve) and one circular (detection) and one annular (illumination) aperture (∊ = 0.5 and ∊ = 0.99, dashed curves). b, PSFs along the x axis for the same setups. The three confocal theta PSFs overlap because the annular illumination has almost no effect on their axial extent.

Fig. 3
Fig. 3

VHM of the confocal PSF (upper graph) and the confocal theta PSF (lower graph) as a function of the obstruction ratio ∊ in a microscope with, a, annular illumination and detection apertures and, b, a circular detection and an annular illumination aperture.

Tables (1)

Tables Icon

Table 1 FWHMa along all Three Axes and the VHM of the PSFs in Confocal and Confocal Theta Microscopes with Circular (Rows 1–2), Annular (Rows 3–6), and Circular and Annular Apertures (Rows 7–10) a

Equations (4)

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| h ( u , 0 ) | 2 = 1 ( 1 2 ) 2 { sin [ ( 1 2 ) u / 4 ] u / 4 } 2 ,
| h ( 0 , υ ) | 2 = 1 ( 1 2 ) 2 [ 2 J 1 ( υ ) υ 2 2 J 1 ( υ ) υ ] 2 ,
u = n k 0 z sin 2 α , υ = n k 0 ( x 2 + y 2 ) 1 / 2 sin α ,
VHM = 4 π 3 FWHM x 2 FWHM y 2 FWHM z 2 .

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