Abstract

In the image of a confocal microscope, only those parts of an object appear bright that are located in the focal plane of the objective. Because of an axial chromatic aberration deliberately introduced into the microscope objective, the location of the focal plane depends on the wavelength used. By using a white-light source and examining an object with a depth variation less than the axial range of the chromatic focus, we find that all parts of the object appear sharp and bright in the image, but according to its height they appear in different colors. A camera with black-and-white film sequentially combines, with three selected chromatic filters, intensity and tone of color of each object point. For each tone of color one can assign a height by using a calibration curve. This assignment could be made unequivocal by the selection of filters with adequate chromatic transmission.

© 1994 Optical Society of America

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References

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  1. T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984).
  2. C. J. Brakenhoff, P. Blom, P. Barends, “Confocal scanning light microscopy with high aperture immersion lenses,” J. Microsc. 117, 219–232 (1979).
    [Crossref]
  3. K. Carlsson, N. Aslund, “Confocal imaging for 3-D digital microscopy,” Appl. Opt. 26, 3232–3238 (1987).
    [Crossref] [PubMed]
  4. D. A. Agard, J. W. Sedat, “Three-dimensional architecture of apolytene nucleus,” Nature (London) 302, 676–681 (1983).
    [Crossref]
  5. A. Erhardt, G. Zinser, D. Komitowski, J. Bille, “Reconstructing 3-D light microscopic images by digital image processing,” Appl. Opt. 24, 194–200 (1985).
    [Crossref] [PubMed]
  6. G. Q. Xiao, T. R. Corle, G. S. Kino, “Real time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
    [Crossref]
  7. G. S. Kino, G. Q. Xiao, “Real time scanning optical microscopes,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), pp. 361–387.
  8. C. J. R. Sheppard, H. J. Matthews, “The extended-focus, auto-focus and surface-profiling techniques of confocal microscopy,” J. Mod. Opt. 35, 145–154 (1988).
    [Crossref]
  9. N. S. Levine, “Three dimensional image visualisation using the real-time confocal scanning optical microscope,” in Integrated Circuit Metrology, Inspection, and Process Control TV, W. H. Arnold, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1261, 91–101 (1990).

1988 (2)

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

C. J. R. Sheppard, H. J. Matthews, “The extended-focus, auto-focus and surface-profiling techniques of confocal microscopy,” J. Mod. Opt. 35, 145–154 (1988).
[Crossref]

1987 (1)

1985 (1)

1983 (1)

D. A. Agard, J. W. Sedat, “Three-dimensional architecture of apolytene nucleus,” Nature (London) 302, 676–681 (1983).
[Crossref]

1979 (1)

C. J. Brakenhoff, P. Blom, P. Barends, “Confocal scanning light microscopy with high aperture immersion lenses,” J. Microsc. 117, 219–232 (1979).
[Crossref]

Agard, D. A.

D. A. Agard, J. W. Sedat, “Three-dimensional architecture of apolytene nucleus,” Nature (London) 302, 676–681 (1983).
[Crossref]

Aslund, N.

Barends, P.

C. J. Brakenhoff, P. Blom, P. Barends, “Confocal scanning light microscopy with high aperture immersion lenses,” J. Microsc. 117, 219–232 (1979).
[Crossref]

Bille, J.

Blom, P.

C. J. Brakenhoff, P. Blom, P. Barends, “Confocal scanning light microscopy with high aperture immersion lenses,” J. Microsc. 117, 219–232 (1979).
[Crossref]

Brakenhoff, C. J.

C. J. Brakenhoff, P. Blom, P. Barends, “Confocal scanning light microscopy with high aperture immersion lenses,” J. Microsc. 117, 219–232 (1979).
[Crossref]

Carlsson, K.

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]

Erhardt, A.

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]

G. S. Kino, G. Q. Xiao, “Real time scanning optical microscopes,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), pp. 361–387.

Komitowski, D.

Levine, N. S.

N. S. Levine, “Three dimensional image visualisation using the real-time confocal scanning optical microscope,” in Integrated Circuit Metrology, Inspection, and Process Control TV, W. H. Arnold, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1261, 91–101 (1990).

Matthews, H. J.

C. J. R. Sheppard, H. J. Matthews, “The extended-focus, auto-focus and surface-profiling techniques of confocal microscopy,” J. Mod. Opt. 35, 145–154 (1988).
[Crossref]

Sedat, J. W.

D. A. Agard, J. W. Sedat, “Three-dimensional architecture of apolytene nucleus,” Nature (London) 302, 676–681 (1983).
[Crossref]

Sheppard, C. J. R.

C. J. R. Sheppard, H. J. Matthews, “The extended-focus, auto-focus and surface-profiling techniques of confocal microscopy,” J. Mod. Opt. 35, 145–154 (1988).
[Crossref]

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

Wilson, T.

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

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]

G. S. Kino, G. Q. Xiao, “Real time scanning optical microscopes,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), pp. 361–387.

Zinser, G.

Appl. Opt. (2)

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. Microsc. (1)

C. J. Brakenhoff, P. Blom, P. Barends, “Confocal scanning light microscopy with high aperture immersion lenses,” J. Microsc. 117, 219–232 (1979).
[Crossref]

J. Mod. Opt. (1)

C. J. R. Sheppard, H. J. Matthews, “The extended-focus, auto-focus and surface-profiling techniques of confocal microscopy,” J. Mod. Opt. 35, 145–154 (1988).
[Crossref]

Nature (London) (1)

D. A. Agard, J. W. Sedat, “Three-dimensional architecture of apolytene nucleus,” Nature (London) 302, 676–681 (1983).
[Crossref]

Other (3)

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

N. S. Levine, “Three dimensional image visualisation using the real-time confocal scanning optical microscope,” in Integrated Circuit Metrology, Inspection, and Process Control TV, W. H. Arnold, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1261, 91–101 (1990).

G. S. Kino, G. Q. Xiao, “Real time scanning optical microscopes,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), pp. 361–387.

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Figures (10)

Fig. 1
Fig. 1

Depth discrimination with confocal reproduction.

Fig. 2
Fig. 2

Experimental setup of the scanning confocal microscope with the Nipkow disk.

Fig. 3
Fig. 3

Topography of a microchip calculated out of 256 height sections (z scanning, center of gravity calculation).

Fig. 4
Fig. 4

Spectral sensitivity of the three kinds of cone of the human retina.9

Fig. 5
Fig. 5

Spectral transmission of the selected blue filter.

Fig. 6
Fig. 6

Spectral transmission of the selected yellow filter.

Fig. 7
Fig. 7

Spectral transmission of the selected red filter.

Fig. 8
Fig. 8

Measured dependence of the color components S i on the axial shift z by using the three selected filters.

Fig. 9
Fig. 9

Curve of tones of color by using the three selected filters.

Fig. 10
Fig. 10

Topography of two microchips measured with the chromatic method.

Tables (1)

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Table 1 Confocal Microscope Data Obtained by Using the 160×/0.95 Objective

Equations (7)

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I ( z ) = { sin [ k z ( 1 cos α ) ] k z ( 1 cos α ) } 2 ,
FWHM = 0 . 44 λ 1 cos α .
S 1 = k I ( λ ) E 1 ( λ ) d λ , S 2 = k I ( λ ) E 2 ( λ ) d λ , S 3 = k I ( λ ) E 3 ( λ ) d λ .
I = S 1 + S 2 + S 3 , ( X f , Y f ) = ( S 1 S 1 + S 2 + S 3 , S 2 S 1 + S 2 + S 3 ) .
X f = S 1 ( 1 ± σ r ) S 1 ( 1 ± σ r ) + S 2 ( 1 ± σ r ) + S 3 ( 1 ± σ r ) .
X f , max = S 1 ( 1 + σ r ) S 1 ( 1 + σ r ) + S 2 ( 1 σ r ) + S 3 ( 1 σ r ) , X f , min = S 1 ( 1 σ r ) S 1 ( 1 σ r ) + S 2 ( 1 + σ r ) + S 3 ( 1 + σ r ) .
Δ X f = X f , max X f , min = 4 σ r 2 ( 1 + σ r 2 ) + ( 1 σ r 2 ) [ ( S 1 / S 2 + S 3 ) + ( S 2 + S 3 / S 1 ) ] σ r ,

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