Abstract

Laser sources offer a potentially low-cost means of improving the light throughput in tandem-scanning confocal microscopy because of their high beam directionality. We measure and compare the optical sectioning characteristics of the tandem-scanning microscope (TSM) employing (i) the traditional choice of incoherent light from a Xe arc lamp and (ii) a cited alternative—coherent light from a He–Ne laser source. In general the laser source is found to result in axial responses with pronounced sidelobes, the sizes and locations of which are extremely sensitive to the alignment of the pinhole array. The implications of these results for practical TSM systems are discussed.

© 1998 Optical Society of America

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  1. M. Petráň, M. Hadravský, M. D. Egger, R. Galambos, “Tandem-scanning reflected-light microscope,” J. Opt. Soc. Am. 58, 661–664 (1968).
    [CrossRef]
  2. A. Boyde, “The tandem scanning reflected light microscope. Part 2—Pre-Micro ’84 applications at UCL,” Proc. R. Microsc. Soc. 20, 131–139 (1985).
  3. G. Q. Xiao, T. R. Corle, G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
    [CrossRef]
  4. T. Wilson, S. J. Hewlett, “Optical sectioning strength of the direct-view microscope employing finite-sized pin-hole arrays,” J. Microsc. 163, 131–150 (1991).
    [CrossRef]
  5. E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, J. Hegarty, “Direct-view microscopy: optical sectioning strength for finite-sized, multiple-pinhole arrays,” J. Microsc. 184, 95–105 (1996).
    [CrossRef]
  6. B. R. Masters, “Confocal microscopy of ocular tissue,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), Chap. 11, pp. 305–324.
  7. A. Boyde, S. J. Jones, M. L. Taylor, L. A. Wolfe, T. F. Watson, “Fluorescence in the tandem scanning microscope,” J. Microsc. 157, 39–49 (1990).
    [CrossRef] [PubMed]
  8. T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), Chap. 1, pp. 1–11.
  9. R. Hard, R. Zeh, R. D. Allen, “Phase-randomized laser illumination for microscopy,” J. Cell Sci. 23, 335–343 (1977).
    [PubMed]
  10. G. W. Ellis, “A fiber-optic phase-randomizer for microscope illumination by laser,” J. Cell Biol. 83, 303a (1979).
  11. S. J. Hewlett, D. T. Fewer, E. M. McCabe, “Influence of source coherence and aperture distribution on the imaging properties in direct-view microscopy,” J. Opt. Soc. Am. A 14, 1066–1075 (1997).
    [CrossRef]
  12. T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), pp. 16–22.
  13. A circularly symmetric objective lens is assumed throughout, and all premultiplying constants in the equation are neglected.
  14. M. Abramowitz, I. A. Stegun, Handbook of Mathematical Functions (Dover, New York, 1965), Chap. 9, pp. 358–433.
  15. D. T. Fewer, S. J. Hewlett, E. M. McCabe, J. Hegarty, “Direct-view microscopy: experimental investigation of the dependence of the optical sectioning characteristics on pinhole-array configuration,” J. Microsc. 187, 54–61 (1997).
    [CrossRef]
  16. T. R. Corle, C.-H. Chou, G. S. Kino, “Depth response of confocal optical microscopes,” Opt. Lett. 11, 770–772 (1986).
    [CrossRef] [PubMed]
  17. T. Wilson, “Optical aspects of confocal microscopy,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), Chap. 3, pp. 93–141.
  18. H. J. Matthews, D. K. Hamilton, C. J. R. Sheppard, “Aberration measurement by confocal interferometry,” J. Mod. Opt. 36, 233–250 (1989).
    [CrossRef]

1997 (2)

D. T. Fewer, S. J. Hewlett, E. M. McCabe, J. Hegarty, “Direct-view microscopy: experimental investigation of the dependence of the optical sectioning characteristics on pinhole-array configuration,” J. Microsc. 187, 54–61 (1997).
[CrossRef]

S. J. Hewlett, D. T. Fewer, E. M. McCabe, “Influence of source coherence and aperture distribution on the imaging properties in direct-view microscopy,” J. Opt. Soc. Am. A 14, 1066–1075 (1997).
[CrossRef]

1996 (1)

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, J. Hegarty, “Direct-view microscopy: optical sectioning strength for finite-sized, multiple-pinhole arrays,” J. Microsc. 184, 95–105 (1996).
[CrossRef]

1991 (1)

T. Wilson, S. J. Hewlett, “Optical sectioning strength of the direct-view microscope employing finite-sized pin-hole arrays,” J. Microsc. 163, 131–150 (1991).
[CrossRef]

1990 (1)

A. Boyde, S. J. Jones, M. L. Taylor, L. A. Wolfe, T. F. Watson, “Fluorescence in the tandem scanning microscope,” J. Microsc. 157, 39–49 (1990).
[CrossRef] [PubMed]

1989 (1)

H. J. Matthews, D. K. Hamilton, C. J. R. Sheppard, “Aberration measurement by confocal interferometry,” J. Mod. Opt. 36, 233–250 (1989).
[CrossRef]

1988 (1)

G. Q. Xiao, T. R. Corle, G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[CrossRef]

1986 (1)

1985 (1)

A. Boyde, “The tandem scanning reflected light microscope. Part 2—Pre-Micro ’84 applications at UCL,” Proc. R. Microsc. Soc. 20, 131–139 (1985).

1979 (1)

G. W. Ellis, “A fiber-optic phase-randomizer for microscope illumination by laser,” J. Cell Biol. 83, 303a (1979).

1977 (1)

R. Hard, R. Zeh, R. D. Allen, “Phase-randomized laser illumination for microscopy,” J. Cell Sci. 23, 335–343 (1977).
[PubMed]

1968 (1)

Abramowitz, M.

M. Abramowitz, I. A. Stegun, Handbook of Mathematical Functions (Dover, New York, 1965), Chap. 9, pp. 358–433.

Allen, R. D.

R. Hard, R. Zeh, R. D. Allen, “Phase-randomized laser illumination for microscopy,” J. Cell Sci. 23, 335–343 (1977).
[PubMed]

Boyde, A.

A. Boyde, S. J. Jones, M. L. Taylor, L. A. Wolfe, T. F. Watson, “Fluorescence in the tandem scanning microscope,” J. Microsc. 157, 39–49 (1990).
[CrossRef] [PubMed]

A. Boyde, “The tandem scanning reflected light microscope. Part 2—Pre-Micro ’84 applications at UCL,” Proc. R. Microsc. Soc. 20, 131–139 (1985).

Chou, C.-H.

Corle, T. R.

G. Q. Xiao, T. R. Corle, G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[CrossRef]

T. R. Corle, C.-H. Chou, G. S. Kino, “Depth response of confocal optical microscopes,” Opt. Lett. 11, 770–772 (1986).
[CrossRef] [PubMed]

Egger, M. D.

Ellis, G. W.

G. W. Ellis, “A fiber-optic phase-randomizer for microscope illumination by laser,” J. Cell Biol. 83, 303a (1979).

Fewer, D. T.

D. T. Fewer, S. J. Hewlett, E. M. McCabe, J. Hegarty, “Direct-view microscopy: experimental investigation of the dependence of the optical sectioning characteristics on pinhole-array configuration,” J. Microsc. 187, 54–61 (1997).
[CrossRef]

S. J. Hewlett, D. T. Fewer, E. M. McCabe, “Influence of source coherence and aperture distribution on the imaging properties in direct-view microscopy,” J. Opt. Soc. Am. A 14, 1066–1075 (1997).
[CrossRef]

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, J. Hegarty, “Direct-view microscopy: optical sectioning strength for finite-sized, multiple-pinhole arrays,” J. Microsc. 184, 95–105 (1996).
[CrossRef]

Galambos, R.

Hadravský, M.

Hamilton, D. K.

H. J. Matthews, D. K. Hamilton, C. J. R. Sheppard, “Aberration measurement by confocal interferometry,” J. Mod. Opt. 36, 233–250 (1989).
[CrossRef]

Hard, R.

R. Hard, R. Zeh, R. D. Allen, “Phase-randomized laser illumination for microscopy,” J. Cell Sci. 23, 335–343 (1977).
[PubMed]

Hegarty, J.

D. T. Fewer, S. J. Hewlett, E. M. McCabe, J. Hegarty, “Direct-view microscopy: experimental investigation of the dependence of the optical sectioning characteristics on pinhole-array configuration,” J. Microsc. 187, 54–61 (1997).
[CrossRef]

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, J. Hegarty, “Direct-view microscopy: optical sectioning strength for finite-sized, multiple-pinhole arrays,” J. Microsc. 184, 95–105 (1996).
[CrossRef]

Hewlett, S. J.

D. T. Fewer, S. J. Hewlett, E. M. McCabe, J. Hegarty, “Direct-view microscopy: experimental investigation of the dependence of the optical sectioning characteristics on pinhole-array configuration,” J. Microsc. 187, 54–61 (1997).
[CrossRef]

S. J. Hewlett, D. T. Fewer, E. M. McCabe, “Influence of source coherence and aperture distribution on the imaging properties in direct-view microscopy,” J. Opt. Soc. Am. A 14, 1066–1075 (1997).
[CrossRef]

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, J. Hegarty, “Direct-view microscopy: optical sectioning strength for finite-sized, multiple-pinhole arrays,” J. Microsc. 184, 95–105 (1996).
[CrossRef]

T. Wilson, S. J. Hewlett, “Optical sectioning strength of the direct-view microscope employing finite-sized pin-hole arrays,” J. Microsc. 163, 131–150 (1991).
[CrossRef]

Jones, S. J.

A. Boyde, S. J. Jones, M. L. Taylor, L. A. Wolfe, T. F. Watson, “Fluorescence in the tandem scanning microscope,” J. Microsc. 157, 39–49 (1990).
[CrossRef] [PubMed]

Kino, G. S.

G. Q. Xiao, T. R. Corle, G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[CrossRef]

T. R. Corle, C.-H. Chou, G. S. Kino, “Depth response of confocal optical microscopes,” Opt. Lett. 11, 770–772 (1986).
[CrossRef] [PubMed]

Masters, B. R.

B. R. Masters, “Confocal microscopy of ocular tissue,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), Chap. 11, pp. 305–324.

Matthews, H. J.

H. J. Matthews, D. K. Hamilton, C. J. R. Sheppard, “Aberration measurement by confocal interferometry,” J. Mod. Opt. 36, 233–250 (1989).
[CrossRef]

McCabe, E. M.

S. J. Hewlett, D. T. Fewer, E. M. McCabe, “Influence of source coherence and aperture distribution on the imaging properties in direct-view microscopy,” J. Opt. Soc. Am. A 14, 1066–1075 (1997).
[CrossRef]

D. T. Fewer, S. J. Hewlett, E. M. McCabe, J. Hegarty, “Direct-view microscopy: experimental investigation of the dependence of the optical sectioning characteristics on pinhole-array configuration,” J. Microsc. 187, 54–61 (1997).
[CrossRef]

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, J. Hegarty, “Direct-view microscopy: optical sectioning strength for finite-sized, multiple-pinhole arrays,” J. Microsc. 184, 95–105 (1996).
[CrossRef]

Ottewill, A. C.

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, J. Hegarty, “Direct-view microscopy: optical sectioning strength for finite-sized, multiple-pinhole arrays,” J. Microsc. 184, 95–105 (1996).
[CrossRef]

Petrán, M.

Sheppard, C. J. R.

H. J. Matthews, D. K. Hamilton, C. J. R. Sheppard, “Aberration measurement by confocal interferometry,” J. Mod. Opt. 36, 233–250 (1989).
[CrossRef]

T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), Chap. 1, pp. 1–11.

T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), pp. 16–22.

Stegun, I. A.

M. Abramowitz, I. A. Stegun, Handbook of Mathematical Functions (Dover, New York, 1965), Chap. 9, pp. 358–433.

Taylor, M. L.

A. Boyde, S. J. Jones, M. L. Taylor, L. A. Wolfe, T. F. Watson, “Fluorescence in the tandem scanning microscope,” J. Microsc. 157, 39–49 (1990).
[CrossRef] [PubMed]

Watson, T. F.

A. Boyde, S. J. Jones, M. L. Taylor, L. A. Wolfe, T. F. Watson, “Fluorescence in the tandem scanning microscope,” J. Microsc. 157, 39–49 (1990).
[CrossRef] [PubMed]

Wilson, T.

T. Wilson, S. J. Hewlett, “Optical sectioning strength of the direct-view microscope employing finite-sized pin-hole arrays,” J. Microsc. 163, 131–150 (1991).
[CrossRef]

T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), pp. 16–22.

T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), Chap. 1, pp. 1–11.

T. Wilson, “Optical aspects of confocal microscopy,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), Chap. 3, pp. 93–141.

Wolfe, L. A.

A. Boyde, S. J. Jones, M. L. Taylor, L. A. Wolfe, T. F. Watson, “Fluorescence in the tandem scanning microscope,” J. Microsc. 157, 39–49 (1990).
[CrossRef] [PubMed]

Xiao, G. Q.

G. Q. Xiao, T. R. Corle, G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[CrossRef]

Zeh, R.

R. Hard, R. Zeh, R. D. Allen, “Phase-randomized laser illumination for microscopy,” J. Cell Sci. 23, 335–343 (1977).
[PubMed]

Appl. Phys. Lett. (1)

G. Q. Xiao, T. R. Corle, G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[CrossRef]

J. Cell Biol. (1)

G. W. Ellis, “A fiber-optic phase-randomizer for microscope illumination by laser,” J. Cell Biol. 83, 303a (1979).

J. Cell Sci. (1)

R. Hard, R. Zeh, R. D. Allen, “Phase-randomized laser illumination for microscopy,” J. Cell Sci. 23, 335–343 (1977).
[PubMed]

J. Microsc. (4)

A. Boyde, S. J. Jones, M. L. Taylor, L. A. Wolfe, T. F. Watson, “Fluorescence in the tandem scanning microscope,” J. Microsc. 157, 39–49 (1990).
[CrossRef] [PubMed]

T. Wilson, S. J. Hewlett, “Optical sectioning strength of the direct-view microscope employing finite-sized pin-hole arrays,” J. Microsc. 163, 131–150 (1991).
[CrossRef]

E. M. McCabe, D. T. Fewer, A. C. Ottewill, S. J. Hewlett, J. Hegarty, “Direct-view microscopy: optical sectioning strength for finite-sized, multiple-pinhole arrays,” J. Microsc. 184, 95–105 (1996).
[CrossRef]

D. T. Fewer, S. J. Hewlett, E. M. McCabe, J. Hegarty, “Direct-view microscopy: experimental investigation of the dependence of the optical sectioning characteristics on pinhole-array configuration,” J. Microsc. 187, 54–61 (1997).
[CrossRef]

J. Mod. Opt. (1)

H. J. Matthews, D. K. Hamilton, C. J. R. Sheppard, “Aberration measurement by confocal interferometry,” J. Mod. Opt. 36, 233–250 (1989).
[CrossRef]

J. Opt. Soc. Am. (1)

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

Opt. Lett. (1)

Proc. R. Microsc. Soc. (1)

A. Boyde, “The tandem scanning reflected light microscope. Part 2—Pre-Micro ’84 applications at UCL,” Proc. R. Microsc. Soc. 20, 131–139 (1985).

Other (6)

T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), Chap. 1, pp. 1–11.

B. R. Masters, “Confocal microscopy of ocular tissue,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), Chap. 11, pp. 305–324.

T. Wilson, “Optical aspects of confocal microscopy,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), Chap. 3, pp. 93–141.

T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), pp. 16–22.

A circularly symmetric objective lens is assumed throughout, and all premultiplying constants in the equation are neglected.

M. Abramowitz, I. A. Stegun, Handbook of Mathematical Functions (Dover, New York, 1965), Chap. 9, pp. 358–433.

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Figures (5)

Fig. 1
Fig. 1

Theoretical axial responses to a perfect planar reflector in a TSM employing (spatially) incoherent and coherent light sources operating at 632 nm. A 50×, 0.6-numerical-aperture objective lens is used, together with a 10 × 10 square array of circular pinholes with r p = 10 μm and D = 100 μm.

Fig. 2
Fig. 2

(a) Geometrical image of a 3 × 3 square array of pointlike pinholes in the focal plane of the objective lens. The nearest-neighbor spacing between pinhole images is D/ M, and each individual image has been labeled for identification purposes. (b) Light rays showing one possible contribution to cross talk between pinhole images 1, 2, and 3. Constructive interference will occur at pinhole image 1 when the difference in optical path lengths between the two rays is an integer number of wavelengths. The corresponding defocus position is denoted by z 321.

Fig. 3
Fig. 3

Experimental setup used to record the axial responses to a plane-mirror specimen by use of incoherent illumination from a Xe arc lamp and coherent illumination from a He–Ne laser source.

Fig. 4
Fig. 4

Experimental axial responses to a plane-mirror specimen in a TSM employing incoherent illumination from a Xe arc lamp (dashed curve) and coherent illumination from a He–Ne laser source (solid curve).

Fig. 5
Fig. 5

Experimental axial response to a plane-mirror specimen in a TSM employing coherent illumination from a He–Ne laser source and a slightly tilted pinhole array is introduced. The sidelobe level now drops.

Tables (1)

Tables Icon

Table 1 Solutions of Eqs. (6)a

Equations (6)

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u = 8 π n λ   z   sin 2 α 2 ,     t = 2 π n λ r   sin   α .
I plane u = 0 2   F S 1 2 ρ / M Σ ρ g 2 u ,   ρ d ρ .
Σ ρ = ρ i = 1 N j = 1 N   J 0 ρ M | t i - t j | .
P u ,   ρ = exp - ju ρ 2 / 2 0 ρ 1 otherwise .
I plane u = j = 1 N     S 1 t i = 1 N 0 1   P 2 u ,   ρ × F S 1 A ρ M J 0 ρ M | t i - t j - t | ρ d ρ 2 d 2 t ,
z ijk = ± 1 4 m λ a - b 2 D M 4 - 2 a + b × m 2 λ 2 D M 2 + m 4 λ 4 1 / 2 ,

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