Abstract

Three-dimensional imaging characteristics of a laser-scan fluorescence microscope with various sizes of detector are quantitatively analyzed through the studies and calculations of the three-dimensional optical transfer function of the microscope. It is found that a detector of an Airy-disk size formed by the objective lens in the detector plane provides both reasonable energy-collection efficiency and three-dimensionally resolved imaging capability. It is also found that a slit detector provides longitudinal resolution of the sample structure similarly to a confocal microscope, while the lateral resolution is approximately the same as that of a conventional microscope.

© 1991 Optical Society of America

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References

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  1. T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), Chaps. 3.7 and 5.
  2. D. K. Hamilton, T. Wilson, C. J. R. Sheppard, “Experimental observations of the depth-discrimination properties of scanning microscopes,” Opt. Lett. 6, 625–626 (1981).
    [CrossRef] [PubMed]
  3. T. R. Corle, C. H. Chou, G. S. Kino, “Depth response of confocal microscope,” Opt. Lett. 11, 770–772 (1986).
    [CrossRef] [PubMed]
  4. T. Wilson, A. R. Carlini, “Size of the detector in confocal imaging systems,” Opt. Lett. 12, 227–229 (1987).
    [CrossRef] [PubMed]
  5. I. J. Cox, C. R. Sheppard, T. Wilson, “Super resolution by confocal fluorescent microscopy,” Optik 60, 391–396 (1982).
  6. T. Wilson, “Optical sectioning in confocal fluorescent microscopes,” J. Microsc. (Oxford) 154, 143–156 (1989).
    [CrossRef]
  7. S. Kawata, O. Nakamura, S. Minami, “Three-dimensional imaging by laser scanning microscope,” presented at the Eighteenth Joint Conference on Image Technology, Tokyo, 1987; O. Nakamura, S. Kawata, “Three-dimensional transfer-function analysis of the tomographic capability of a confocal fluorescence microscope,” J. Opt. Soc. Am. A 7, 522–526 (1990).
    [CrossRef] [PubMed]
  8. S. Kimura, C. Munakata, “Calculation of a three-dimensional optical transfer function for a confocal scanning fluorescent microscope,” J. Opt. Soc. Am. A 6, 1015–1019 (1989).
    [CrossRef]
  9. H. T. M. van der Voort, G. J. Brakenhoff, G. C. A. M. Janssen, “Determination of 3-dimensional properties of a confocal scanning laser microscope,” Optik 78, 48–53 (1988).
  10. N. Streibl, “Depth transfer by an imaging system,” Opt. Acta 31, 1233–1241 (1984).
    [CrossRef]
  11. C. J. R. Sheppard, A. Choudhury, “Image formation in the scanning microscope,” Opt. Acta 24, 1051–1073 (1977).
    [CrossRef]
  12. M. Y. Chiu, H. H. Barrett, R. G. Simpson, C. Chou, J. W. Arendt, G. R. Gindi, “Three-dimensional radiographic imaging with a restricted view angle,”J. Opt. Soc. Am. 69, 1323–1332 (1979).
    [CrossRef]
  13. C. J. Koester, “Scanning mirror microscope with optical sectioning characteristics: applications in ophthalmology,” Appl. Opt. 19, 1749–1757 (1980).
    [CrossRef] [PubMed]
  14. D. Awamura, T. Ode, M. Yonezawa, “Color laser microscope,” in Imaging Sensors and Displays, C. F. Freeman, ed., Proc. Soc. Photo-Opt. Instrum. Eng.765, 53–60 (1987).
    [CrossRef]

1989 (2)

1988 (1)

H. T. M. van der Voort, G. J. Brakenhoff, G. C. A. M. Janssen, “Determination of 3-dimensional properties of a confocal scanning laser microscope,” Optik 78, 48–53 (1988).

1987 (1)

1986 (1)

1984 (1)

N. Streibl, “Depth transfer by an imaging system,” Opt. Acta 31, 1233–1241 (1984).
[CrossRef]

1982 (1)

I. J. Cox, C. R. Sheppard, T. Wilson, “Super resolution by confocal fluorescent microscopy,” Optik 60, 391–396 (1982).

1981 (1)

1980 (1)

1979 (1)

1977 (1)

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

Arendt, J. W.

Awamura, D.

D. Awamura, T. Ode, M. Yonezawa, “Color laser microscope,” in Imaging Sensors and Displays, C. F. Freeman, ed., Proc. Soc. Photo-Opt. Instrum. Eng.765, 53–60 (1987).
[CrossRef]

Barrett, H. H.

Brakenhoff, G. J.

H. T. M. van der Voort, G. J. Brakenhoff, G. C. A. M. Janssen, “Determination of 3-dimensional properties of a confocal scanning laser microscope,” Optik 78, 48–53 (1988).

Carlini, A. R.

Chiu, M. Y.

Chou, C.

Chou, C. H.

Choudhury, A.

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

Corle, T. R.

Cox, I. J.

I. J. Cox, C. R. Sheppard, T. Wilson, “Super resolution by confocal fluorescent microscopy,” Optik 60, 391–396 (1982).

Gindi, G. R.

Hamilton, D. K.

Janssen, G. C. A. M.

H. T. M. van der Voort, G. J. Brakenhoff, G. C. A. M. Janssen, “Determination of 3-dimensional properties of a confocal scanning laser microscope,” Optik 78, 48–53 (1988).

Kawata, S.

S. Kawata, O. Nakamura, S. Minami, “Three-dimensional imaging by laser scanning microscope,” presented at the Eighteenth Joint Conference on Image Technology, Tokyo, 1987; O. Nakamura, S. Kawata, “Three-dimensional transfer-function analysis of the tomographic capability of a confocal fluorescence microscope,” J. Opt. Soc. Am. A 7, 522–526 (1990).
[CrossRef] [PubMed]

Kimura, S.

Kino, G. S.

Koester, C. J.

Minami, S.

S. Kawata, O. Nakamura, S. Minami, “Three-dimensional imaging by laser scanning microscope,” presented at the Eighteenth Joint Conference on Image Technology, Tokyo, 1987; O. Nakamura, S. Kawata, “Three-dimensional transfer-function analysis of the tomographic capability of a confocal fluorescence microscope,” J. Opt. Soc. Am. A 7, 522–526 (1990).
[CrossRef] [PubMed]

Munakata, C.

Nakamura, O.

S. Kawata, O. Nakamura, S. Minami, “Three-dimensional imaging by laser scanning microscope,” presented at the Eighteenth Joint Conference on Image Technology, Tokyo, 1987; O. Nakamura, S. Kawata, “Three-dimensional transfer-function analysis of the tomographic capability of a confocal fluorescence microscope,” J. Opt. Soc. Am. A 7, 522–526 (1990).
[CrossRef] [PubMed]

Ode, T.

D. Awamura, T. Ode, M. Yonezawa, “Color laser microscope,” in Imaging Sensors and Displays, C. F. Freeman, ed., Proc. Soc. Photo-Opt. Instrum. Eng.765, 53–60 (1987).
[CrossRef]

Sheppard, C.

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), Chaps. 3.7 and 5.

Sheppard, C. J. R.

Sheppard, C. R.

I. J. Cox, C. R. Sheppard, T. Wilson, “Super resolution by confocal fluorescent microscopy,” Optik 60, 391–396 (1982).

Simpson, R. G.

Streibl, N.

N. Streibl, “Depth transfer by an imaging system,” Opt. Acta 31, 1233–1241 (1984).
[CrossRef]

van der Voort, H. T. M.

H. T. M. van der Voort, G. J. Brakenhoff, G. C. A. M. Janssen, “Determination of 3-dimensional properties of a confocal scanning laser microscope,” Optik 78, 48–53 (1988).

Wilson, T.

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

T. Wilson, A. R. Carlini, “Size of the detector in confocal imaging systems,” Opt. Lett. 12, 227–229 (1987).
[CrossRef] [PubMed]

I. J. Cox, C. R. Sheppard, T. Wilson, “Super resolution by confocal fluorescent microscopy,” Optik 60, 391–396 (1982).

D. K. Hamilton, T. Wilson, C. J. R. Sheppard, “Experimental observations of the depth-discrimination properties of scanning microscopes,” Opt. Lett. 6, 625–626 (1981).
[CrossRef] [PubMed]

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), Chaps. 3.7 and 5.

Yonezawa, M.

D. Awamura, T. Ode, M. Yonezawa, “Color laser microscope,” in Imaging Sensors and Displays, C. F. Freeman, ed., Proc. Soc. Photo-Opt. Instrum. Eng.765, 53–60 (1987).
[CrossRef]

Appl. Opt. (1)

J. Microsc. (Oxford) (1)

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

J. Opt. Soc. Am. (1)

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

Opt. Acta (2)

N. Streibl, “Depth transfer by an imaging system,” Opt. Acta 31, 1233–1241 (1984).
[CrossRef]

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

Opt. Lett. (3)

Optik (2)

I. J. Cox, C. R. Sheppard, T. Wilson, “Super resolution by confocal fluorescent microscopy,” Optik 60, 391–396 (1982).

H. T. M. van der Voort, G. J. Brakenhoff, G. C. A. M. Janssen, “Determination of 3-dimensional properties of a confocal scanning laser microscope,” Optik 78, 48–53 (1988).

Other (3)

S. Kawata, O. Nakamura, S. Minami, “Three-dimensional imaging by laser scanning microscope,” presented at the Eighteenth Joint Conference on Image Technology, Tokyo, 1987; O. Nakamura, S. Kawata, “Three-dimensional transfer-function analysis of the tomographic capability of a confocal fluorescence microscope,” J. Opt. Soc. Am. A 7, 522–526 (1990).
[CrossRef] [PubMed]

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), Chaps. 3.7 and 5.

D. Awamura, T. Ode, M. Yonezawa, “Color laser microscope,” in Imaging Sensors and Displays, C. F. Freeman, ed., Proc. Soc. Photo-Opt. Instrum. Eng.765, 53–60 (1987).
[CrossRef]

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Figures (5)

Fig. 1
Fig. 1

Optical diagram for laser-scan fluorescence microscope.

Fig. 2
Fig. 2

3-D OTF’s of laser-scan fluorescence microscopes with different detector sizes: (a) point detector (confocal system), (b) Airy-disk size, (c) 2 × Airy-disk size, and (d) large-area detector (conventional system).

Fig. 3
Fig. 3

Same as Fig. 1 but on a log scale: (a) point detector (confocal system), (b) Airy-disk size, (c) 2 × Airy-disk size, and (d) large-area detector (conventional system).

Fig. 4
Fig. 4

3-D OTF’s of laser-scan fluorescence microscopes with a slit detector and circular detectors: (a) 3-D OTF of confocal microscope with a point detector (type 2), (b) 2-D (ξζ) cut of a 3-D OTF of a laser-scan microscope with a slit detector opened along the y axis (type 1.5), (c) same as (b) but the cut is ηζ (type 1.5), (d) 3-D OTF of nonconfocal microscope with a large-area detector (type 1).

Fig. 5
Fig. 5

Same as Fig. 4 but on a log scale: (a) type 2, (b) type 1.5 (ξζ cut), (c) type 1.5 (ηζ cut), (d) type 1.

Equations (9)

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g ( x , y , z ) = h 1 ( x , y , z ) h 2 ( x , y , z ) ,
G ( ξ , η , ζ ) = H 1 ( ξ , η , ζ ) * * * H 2 ( ξ , η , ζ ) ,
G conv ( ξ , η , ζ ) = H 2 ( ξ , η , ζ ) .
H ( ξ , η , ζ ) = P ( ξ + ξ 2 , η + η 2 ) P * ( ξ - ξ 2 , η - η 2 ) × exp { - 2 π i z [ ζ + λ ( ξ ξ + η η ) ] } d ξ d η d z ,
P ( ξ , η ) = { 1 ( ξ 2 + η 2 ) 1 / 2 < N λ 0 otherwise .
H ( ξ , η , ζ ) = P ( ξ + ξ 2 , η + η 2 ) P * ( ξ - ξ 2 , η - η 2 ) × z L sinc { z L [ ζ + λ ( ξ ξ + η η ) ] } d ξ d η
g ( x , y , z ) = h 1 ( x , y , z ) [ h 2 ( x , y , z ) * * f 2 ( x , y ) ] ,
f 2 ( x , y ) = { 1 ( x 2 + y 2 ) 1 / 2 a 0 otherwise ,
G ( ρ , ζ ) = H 1 ( ρ , ζ ) * * * [ H 2 ( ρ , ζ ) J 1 ( 2 π a ρ ) / ( π a ρ ) ] ,

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