Abstract

The fluorescent confocal scanning optical microscope is capable of 3-D observation, for which depth discrimination is vital. To characterize the depth discrimination capability of this microscope, intensity variations depending on fluorescence wavelength are simulated for fluorescent films being moved in the depth direction. Also, the resolution between two films is calculated for a range of fluorescence wavelengths. Results of experiments using photoresist films are compared to simulations. As a practical application, through-holes opened in a thick photoresist film are observed by slice images in the depth direction.

© 1990 Optical Society of America

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References

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  1. T. Wilson, C. Sheppard, Theory and Practice of Scanning Microscopy (Academic, London, 1984).
  2. C. J. R. Sheppard, T. Wilson, “Depth of Field in the Scanning Microscope,” Opt. Lett. 3, 115–117 (1978).
    [CrossRef] [PubMed]
  3. C. J. R. Sheppard, “The Spatial Frequency Cut-Off in Three-Dimensional Imaging,” Optik 72, 131–133 (1986).
  4. C. J. R. Sheppard, “The Spatial Frequency Cut-Off in Three-Dimensional Imaging II,” Optik 74, 128–129 (1986).
  5. D. K. Hamilton, T. Wilson, “Surface Profile Measurement Using the Confocal Microscope,” J. Appl. Phys. 53, 5320–5322 (1982).
    [CrossRef]
  6. D. K. Hamilton, T. Wilson, “Three-Dimensional Surface Measurement Using the Confocal Scanning Microscope,” Appl. Phys. B 27, 211–213 (1982).
    [CrossRef]
  7. I. J. Cox, C. J. R. Sheppard, T. Wilson, “Super-Resolution by Confocal Fluorescent Microscopy,” Optik 60, 391–396 (1982).
  8. I. J. Cox, “Scanning Optical Fluorescence Microscopy,” J. Microsc. 133, 149–154 (1984).
    [CrossRef] [PubMed]
  9. T. Wilson, “Optical Sectioning in Confocal Fluorescent Microscopes,” J. Microsc. 154, 143–156 (1989).
    [CrossRef]
  10. K. Carlsson, N. Åslund, “Confocal Imaging for 3-D Digital Microscopy,” Appl. Opt. 26, 3232–3238 (1987).
    [CrossRef] [PubMed]
  11. R. W. Wijnaends 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]
  12. W. B. Amos, J. G. White, M. Fordham, “Use of Confocal Imaging in the Study of Biological Structures,” Appl. Opt. 26, 3239–3243 (1987).
    [CrossRef] [PubMed]
  13. 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 Micros. 2, 33–40 (1988).
  14. H. T. M. van der Voort, G. J. Brakenhoff, G. C. A. M. Janssen, “Determination of the 3-Dimensional Optical Properties of a Confocal Scanning Laser Microscope,” Optik 78, 48–53 (1988).
  15. H. H. Hopkins, “The Frequency Response of a Defocused Optical System,” Proc. R. Soc. London Ser. A 231, 91–103 (1955).
    [CrossRef]
  16. P. A. Stokseth, “Properties of a Defocused Optical System,” J. Opt. Soc. Am. 59, 1314–1321 (1969).
    [CrossRef]
  17. T. Wilson, A. R. Carlini, “Depth Discrimination Criteria in Confocal Optical Systems,” Optik 76, 164–166 (1987).
  18. F. A. Jenkins, H. E. White, Fundamentals of Optics (McGraw-Hill, Tokyo, 1981), p. 38.

1989 (1)

T. Wilson, “Optical Sectioning in Confocal Fluorescent Microscopes,” J. Microsc. 154, 143–156 (1989).
[CrossRef]

1988 (2)

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 Micros. 2, 33–40 (1988).

H. T. M. van der Voort, G. J. Brakenhoff, G. C. A. M. Janssen, “Determination of the 3-Dimensional Optical Properties of a Confocal Scanning Laser Microscope,” Optik 78, 48–53 (1988).

1987 (3)

1986 (2)

C. J. R. Sheppard, “The Spatial Frequency Cut-Off in Three-Dimensional Imaging,” Optik 72, 131–133 (1986).

C. J. R. Sheppard, “The Spatial Frequency Cut-Off in Three-Dimensional Imaging II,” Optik 74, 128–129 (1986).

1985 (1)

R. W. Wijnaends 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]

1984 (1)

I. J. Cox, “Scanning Optical Fluorescence Microscopy,” J. Microsc. 133, 149–154 (1984).
[CrossRef] [PubMed]

1982 (3)

D. K. Hamilton, T. Wilson, “Surface Profile Measurement Using the Confocal Microscope,” J. Appl. Phys. 53, 5320–5322 (1982).
[CrossRef]

D. K. Hamilton, T. Wilson, “Three-Dimensional Surface Measurement Using the Confocal Scanning Microscope,” Appl. Phys. B 27, 211–213 (1982).
[CrossRef]

I. J. Cox, C. J. R. Sheppard, T. Wilson, “Super-Resolution by Confocal Fluorescent Microscopy,” Optik 60, 391–396 (1982).

1978 (1)

1969 (1)

1955 (1)

H. H. Hopkins, “The Frequency Response of a Defocused Optical System,” Proc. R. Soc. London Ser. A 231, 91–103 (1955).
[CrossRef]

Amos, W. B.

Åslund, N.

Brakenhoff, G. J.

H. T. M. van der Voort, G. J. Brakenhoff, G. C. A. M. Janssen, “Determination of the 3-Dimensional Optical Properties of a Confocal Scanning Laser Microscope,” Optik 78, 48–53 (1988).

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 Micros. 2, 33–40 (1988).

Carlini, A. R.

T. Wilson, A. R. Carlini, “Depth Discrimination Criteria in Confocal Optical Systems,” Optik 76, 164–166 (1987).

Carlsson, K.

Cox, I. J.

I. J. Cox, “Scanning Optical Fluorescence Microscopy,” J. Microsc. 133, 149–154 (1984).
[CrossRef] [PubMed]

I. J. Cox, C. J. R. Sheppard, T. Wilson, “Super-Resolution by Confocal Fluorescent Microscopy,” Optik 60, 391–396 (1982).

Davoust, J.

R. W. Wijnaends 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]

Fordham, M.

Hamilton, D. K.

D. K. Hamilton, T. Wilson, “Surface Profile Measurement Using the Confocal Microscope,” J. Appl. Phys. 53, 5320–5322 (1982).
[CrossRef]

D. K. Hamilton, T. Wilson, “Three-Dimensional Surface Measurement Using the Confocal Scanning Microscope,” Appl. Phys. B 27, 211–213 (1982).
[CrossRef]

Hopkins, H. H.

H. H. Hopkins, “The Frequency Response of a Defocused Optical System,” Proc. R. Soc. London Ser. A 231, 91–103 (1955).
[CrossRef]

Janssen, G. C. A. M.

H. T. M. van der Voort, G. J. Brakenhoff, G. C. A. M. Janssen, “Determination of the 3-Dimensional Optical Properties of a Confocal Scanning Laser Microscope,” Optik 78, 48–53 (1988).

Jenkins, F. A.

F. A. Jenkins, H. E. White, Fundamentals of Optics (McGraw-Hill, Tokyo, 1981), p. 38.

Kaplan, R.

R. W. Wijnaends 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]

Marsman, H. J. B.

R. W. Wijnaends 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 Micros. 2, 33–40 (1988).

Sheppard, C.

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

Sheppard, C. J. R.

C. J. R. Sheppard, “The Spatial Frequency Cut-Off in Three-Dimensional Imaging II,” Optik 74, 128–129 (1986).

C. J. R. Sheppard, “The Spatial Frequency Cut-Off in Three-Dimensional Imaging,” Optik 72, 131–133 (1986).

I. J. Cox, C. J. R. Sheppard, T. Wilson, “Super-Resolution by Confocal Fluorescent Microscopy,” Optik 60, 391–396 (1982).

C. J. R. Sheppard, T. Wilson, “Depth of Field in the Scanning Microscope,” Opt. Lett. 3, 115–117 (1978).
[CrossRef] [PubMed]

Stelzer, E. H. K.

R. W. Wijnaends 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]

Stokseth, P. A.

Stricker, R.

R. W. Wijnaends 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]

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 Micros. 2, 33–40 (1988).

H. T. M. van der Voort, G. J. Brakenhoff, G. C. A. M. Janssen, “Determination of the 3-Dimensional Optical Properties of a Confocal Scanning Laser Microscope,” Optik 78, 48–53 (1988).

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 Micros. 2, 33–40 (1988).

White, H. E.

F. A. Jenkins, H. E. White, Fundamentals of Optics (McGraw-Hill, Tokyo, 1981), p. 38.

White, J. G.

Wijnaends van Resandt, R. W.

R. W. Wijnaends 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]

T. Wilson, A. R. Carlini, “Depth Discrimination Criteria in Confocal Optical Systems,” Optik 76, 164–166 (1987).

I. J. Cox, C. J. R. Sheppard, T. Wilson, “Super-Resolution by Confocal Fluorescent Microscopy,” Optik 60, 391–396 (1982).

D. K. Hamilton, T. Wilson, “Three-Dimensional Surface Measurement Using the Confocal Scanning Microscope,” Appl. Phys. B 27, 211–213 (1982).
[CrossRef]

D. K. Hamilton, T. Wilson, “Surface Profile Measurement Using the Confocal Microscope,” J. Appl. Phys. 53, 5320–5322 (1982).
[CrossRef]

C. J. R. Sheppard, T. Wilson, “Depth of Field in the Scanning Microscope,” Opt. Lett. 3, 115–117 (1978).
[CrossRef] [PubMed]

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

Appl. Opt. (2)

Appl. Phys. B (1)

D. K. Hamilton, T. Wilson, “Three-Dimensional Surface Measurement Using the Confocal Scanning Microscope,” Appl. Phys. B 27, 211–213 (1982).
[CrossRef]

J. Appl. Phys. (1)

D. K. Hamilton, T. Wilson, “Surface Profile Measurement Using the Confocal Microscope,” J. Appl. Phys. 53, 5320–5322 (1982).
[CrossRef]

J. Microsc. (3)

I. J. Cox, “Scanning Optical Fluorescence Microscopy,” J. Microsc. 133, 149–154 (1984).
[CrossRef] [PubMed]

T. Wilson, “Optical Sectioning in Confocal Fluorescent Microscopes,” J. Microsc. 154, 143–156 (1989).
[CrossRef]

R. W. Wijnaends 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. (1)

Opt. Lett. (1)

Optik (5)

H. T. M. van der Voort, G. J. Brakenhoff, G. C. A. M. Janssen, “Determination of the 3-Dimensional Optical Properties of a Confocal Scanning Laser Microscope,” Optik 78, 48–53 (1988).

T. Wilson, A. R. Carlini, “Depth Discrimination Criteria in Confocal Optical Systems,” Optik 76, 164–166 (1987).

I. J. Cox, C. J. R. Sheppard, T. Wilson, “Super-Resolution by Confocal Fluorescent Microscopy,” Optik 60, 391–396 (1982).

C. J. R. Sheppard, “The Spatial Frequency Cut-Off in Three-Dimensional Imaging,” Optik 72, 131–133 (1986).

C. J. R. Sheppard, “The Spatial Frequency Cut-Off in Three-Dimensional Imaging II,” Optik 74, 128–129 (1986).

Proc. R. Soc. London Ser. A (1)

H. H. Hopkins, “The Frequency Response of a Defocused Optical System,” Proc. R. Soc. London Ser. A 231, 91–103 (1955).
[CrossRef]

Scanning Micros. (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 Micros. 2, 33–40 (1988).

Other (2)

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

F. A. Jenkins, H. E. White, Fundamentals of Optics (McGraw-Hill, Tokyo, 1981), p. 38.

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Figures (8)

Fig. 1
Fig. 1

Basic optical arrangement of fluorescent CSOM.

Fig. 2
Fig. 2

Calculated intensity variations of fluorescence when an infinitely thin fluorescent film is moved in a depth direction. The parameter β denotes the ratio of fluorescence wavelength to excitation light. The dotted line denotes the variation in reflected light from a flat surface.

Fig. 3
Fig. 3

Two-plane resolution dependence on the fluorescence wavelength. The resolution is represented in optical units.

Fig. 4
Fig. 4

Schematic diagram of the experimental setup of the fluorescent CSOM.

Fig. 5
Fig. 5

Fluorescence intensity variations calculated and experimentally observed. The focusing lens was moved in a depth direction relative to AZ1350J photoresist films with thicknesses of (a) 300 and (b) 950 nm.

Fig. 6
Fig. 6

Experimental and simulated results of intensity variation of fluorescence emitted from the thicker OFPR5000 photoresist of 3.4 μm.

Fig. 7
Fig. 7

Observation of an OFPR5000 photoresist step: (a) cross section of the resist; (b) x-z slice image.

Fig. 8
Fig. 8

Through-hole x-z slice images and cross sections. The through-holes in the OFPR5000 photoresist film were 0.8 μm in diameter. Their depths were (a) 3.4 and (b) 2.1 μm.

Equations (19)

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I ( x s , y s , z s ) = - h 1 ( x , y , z ) 2 h 2 ( x , y , z ) 2 × o ( x - x s , y - y s , z - z s ) d x d y d z ,
o ( x - x s , y - y s , z - z s ) = C 1 δ ( z - z s ) ,
H ( f x , f y ; z s ) = - h 1 ( x , y , z s ) h 2 ( x , y , z s ) 2 × exp [ - 2 π i ( f x + f y ) ] d x d y , = { F [ h 1 ( x , y , z s ) ] F [ h 1 * ( x , y , z s ) ] } { F [ h 2 ( x , y , z s ) ] F [ h 2 * ( x , y , z s ) ] } ,
I ( x s , y s z s ) = C 1 - h 1 ( x , y , z s ) 2 h 2 ( x , y , z s ) 2 d x d y , = C 1 H ( 0 , 0 ; z s ) .
v x = 2 π λ x sin α , v y = 2 π λ y sin α , u = 2 π λ z sin 2 α .
h n 1 ( v x , v y , u ) = 1 2 π - f ( x L , y L u ) × exp [ - i ( v x x L + v y y L ) ] d x L d y L ,
F [ h n 1 ( v x , u y , u ) ] = 1 ( 2 π ) 2 - f ( x L , y L , u ) × exp { - i [ ( x L + s ) v x + ( y L + t ) v y ] } d x L d y L d v x d v y , = f ( s , t , u ) ,
h n 2 ( v x ¯ , v y ¯ , u ¯ ) = h n 1 ( v x β , v y β , u β ) ,
T n 1 ( s , t ; u ) = f ( s , t ; u ) f * ( s , t ; u ) .
T n 2 ( s , t ; u ) = T n 1 ( β s , β t ; u β ) ,
T n 1 ( s , 0 ; u ) = { g 1 ( s ) { J 1 [ u g 2 ( s ) ] u g 2 ( s ) } , ( 0 < s < 2 ) , 0 , ( 2 s ) ,
g 1 ( s ) = 2 ( 1 - 0.69 s + 0.0076 s 2 + 0.043 s 3 ) , g 2 ( s ) = s - 0.5 s 2 ,
T n 1 ( s , t ; u ) = T n 1 ( s 2 + t 2 , 0 ; u ) .
H n ( s , t ; u ) = T n 1 ( s , t ; u ) T n 1 ( β s , β t ; u β ) = - T n 1 ( s - s , t - t ; u ) T n 1 ( β s , β t ; u β ) d s d t = - T n 1 [ ( s - s ) 2 + ( t - t ) 2 , 0 ; u ] × T n 1 ( β s 2 + t 2 , 0 ; μ β ) d s d t .
I n ( v x , v y , u ) = H n ( 0 , 0 ; u + d 2 ) + H n ( 0 , 0 ; u + d 2 ) ,
o ( x - x s , y - y s , z - z s ) = C 2 Π [ n ( z - z s ) l ] ,
Π ( x ) = { 1 , ( x < 1 2 ) , 0 , ( x > 1 2 ) .
I ( x s , y s , z s ) = C 2 - l 2 n 1 2 n H ( 0 , 0 ; z - z s ) d z .
T n 1 ( s , 0 ; u ) = 4 π u s 0 1 - ( 1 2 s ) 2 sin [ u s ( 1 - y 2 - 1 2 s ) ] d y .

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