Abstract

We investigate limits of the confocal microscope when applied to imaging through scattering layers and compare its performance with that of a correlation (heterodyne) microscope. Confocal laser scanning microscopy is shown to make possible imaging through scattering media owing to the spatial filtering of the signal back-reflected from the sample. Its performance is limited by the noise of the detection system and/or insufficient rejection of scattered light, depending on the sample under investigation. Correlation microscopy with narrow or broad bandwidth light can extend these limits by selective, optical amplification of the image information.

© 1996 Optical Society of America

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References

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    [CrossRef]
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1994

1990

P. M. Saulnier, M. P. Zinkin, G. H. Watson, “Scatterer correlation effects on photon transport in dense random media,” Phys. Rev. B 42, 2621–2623 (1990).
[CrossRef]

G. S. Kino, S. C. Chim, “Mirau correlation microscope,” Appl. Opt. 29, 3775–3783 (1990).
[CrossRef] [PubMed]

1988

T. Wilson, A. R. Carlini, “Three-dimensional imaging in confocal imaging systems with finite sized detectors,” J. Microsc. 149, 51–66 (1988).
[CrossRef]

1983

1978

C. J. R. Sheppard, T. Wilson, “Image formation in scanning microscopes with partially coherent source and detector,” Opt. Acta 25, 315–325 (1978).
[CrossRef]

1973

Alfano, R. R.

Anderson, G. E.

Born, M.

M. Born, E. Wolf, Principles of Optics (Pergamon, London, 1989).

Carlini, A. R.

T. Wilson, A. R. Carlini, “Three-dimensional imaging in confocal imaging systems with finite sized detectors,” J. Microsc. 149, 51–66 (1988).
[CrossRef]

Chen, Y.

Cheung, R. L.-T.

Chim, S. C.

Cohen, F.

M. Davidson, K. Kaufman, I. Mazor, F. Cohen, “An application of interference microscopy to integrated circuit inspection and metrology,” in Integrated Circuit Metrology, Inspection, and Process Control, K. M. Monahan, ed., Proc. Soc. Photo.775, 233–241 (1987).
[CrossRef]

Davidson, M.

M. Davidson, K. Kaufman, I. Mazor, F. Cohen, “An application of interference microscopy to integrated circuit inspection and metrology,” in Integrated Circuit Metrology, Inspection, and Process Control, K. M. Monahan, ed., Proc. Soc. Photo.775, 233–241 (1987).
[CrossRef]

Fujimoto, J. G.

Goodman, J. W.

J. W. Goodman, Statistical Optics (Wiley, New York, 1985).

Hee, M. R.

Ishimaru, A.

Izatt, J. A.

Kaufman, K.

M. Davidson, K. Kaufman, I. Mazor, F. Cohen, “An application of interference microscopy to integrated circuit inspection and metrology,” in Integrated Circuit Metrology, Inspection, and Process Control, K. M. Monahan, ed., Proc. Soc. Photo.775, 233–241 (1987).
[CrossRef]

Kempe, M.

M. Kempe, W. Rudolph, “Scanning microscopy through thick layers based on linear correlation,” Opt. Lett. 19, 1919–1921 (1994).
[CrossRef] [PubMed]

M. Kempe, W. Rudolph, “Microscopy with ultrashort light pulses,” Nonl. Opt. 7, 129–151 (1994).

M. Kempe, A. Thon, W. Rudolph, “Resolution limits of microscopy through scattering layers,” Opt. Commun. 110, 492–496 (1994).
[CrossRef]

Kino, G. S.

Knuettel, A.

J. M. Schmitt, A. Knuettel, M. Yadlowsky, “Confocal microscopy in turbid media,” J. Opt. Soc. Am. A 11, 2226–2235 (1994).
[CrossRef]

J. M. Schmitt, A. Knuettel, M. Yadlowsky, “Interferometric versus confocal techniques for imaging microstructures in turbid biological media,” in Advances in Laser and Light Spectroscopy to Diagnose Cancer and Other Diseases, R. R. Alfano, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2135, 251–262 (1994).
[CrossRef]

Kuga, Y.

Liu, F.

Mazor, I.

M. Davidson, K. Kaufman, I. Mazor, F. Cohen, “An application of interference microscopy to integrated circuit inspection and metrology,” in Integrated Circuit Metrology, Inspection, and Process Control, K. M. Monahan, ed., Proc. Soc. Photo.775, 233–241 (1987).
[CrossRef]

Moon, J. A.

Owen, G. M.

Reintjes, J.

Rudolph, W.

M. Kempe, W. Rudolph, “Microscopy with ultrashort light pulses,” Nonl. Opt. 7, 129–151 (1994).

M. Kempe, W. Rudolph, “Scanning microscopy through thick layers based on linear correlation,” Opt. Lett. 19, 1919–1921 (1994).
[CrossRef] [PubMed]

M. Kempe, A. Thon, W. Rudolph, “Resolution limits of microscopy through scattering layers,” Opt. Commun. 110, 492–496 (1994).
[CrossRef]

Saulnier, P. M.

P. M. Saulnier, M. P. Zinkin, G. H. Watson, “Scatterer correlation effects on photon transport in dense random media,” Phys. Rev. B 42, 2621–2623 (1990).
[CrossRef]

Sawatari, T.

Schmitt, J. M.

J. M. Schmitt, A. Knuettel, M. Yadlowsky, “Confocal microscopy in turbid media,” J. Opt. Soc. Am. A 11, 2226–2235 (1994).
[CrossRef]

J. M. Schmitt, A. Knuettel, M. Yadlowsky, “Interferometric versus confocal techniques for imaging microstructures in turbid biological media,” in Advances in Laser and Light Spectroscopy to Diagnose Cancer and Other Diseases, R. R. Alfano, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2135, 251–262 (1994).
[CrossRef]

Sheppard, C. J. R.

C. J. R. Sheppard, T. Wilson, “Image formation in scanning microscopes with partially coherent source and detector,” Opt. Acta 25, 315–325 (1978).
[CrossRef]

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

Shimizu, K.

Swanson, E. A.

Thon, A.

M. Kempe, A. Thon, W. Rudolph, “Resolution limits of microscopy through scattering layers,” Opt. Commun. 110, 492–496 (1994).
[CrossRef]

Watson, G. H.

P. M. Saulnier, M. P. Zinkin, G. H. Watson, “Scatterer correlation effects on photon transport in dense random media,” Phys. Rev. B 42, 2621–2623 (1990).
[CrossRef]

Wilson, T.

T. Wilson, A. R. Carlini, “Three-dimensional imaging in confocal imaging systems with finite sized detectors,” J. Microsc. 149, 51–66 (1988).
[CrossRef]

C. J. R. Sheppard, T. Wilson, “Image formation in scanning microscopes with partially coherent source and detector,” Opt. Acta 25, 315–325 (1978).
[CrossRef]

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

Wolf, E.

M. Born, E. Wolf, Principles of Optics (Pergamon, London, 1989).

Yadlowsky, M.

J. M. Schmitt, A. Knuettel, M. Yadlowsky, “Confocal microscopy in turbid media,” J. Opt. Soc. Am. A 11, 2226–2235 (1994).
[CrossRef]

J. M. Schmitt, A. Knuettel, M. Yadlowsky, “Interferometric versus confocal techniques for imaging microstructures in turbid biological media,” in Advances in Laser and Light Spectroscopy to Diagnose Cancer and Other Diseases, R. R. Alfano, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2135, 251–262 (1994).
[CrossRef]

Zinkin, M. P.

P. M. Saulnier, M. P. Zinkin, G. H. Watson, “Scatterer correlation effects on photon transport in dense random media,” Phys. Rev. B 42, 2621–2623 (1990).
[CrossRef]

Appl. Opt.

J. Microsc.

T. Wilson, A. R. Carlini, “Three-dimensional imaging in confocal imaging systems with finite sized detectors,” J. Microsc. 149, 51–66 (1988).
[CrossRef]

J. Opt. Soc. Am.

J. Opt. Soc. Am. A

Nonl. Opt.

M. Kempe, W. Rudolph, “Microscopy with ultrashort light pulses,” Nonl. Opt. 7, 129–151 (1994).

Opt. Acta

C. J. R. Sheppard, T. Wilson, “Image formation in scanning microscopes with partially coherent source and detector,” Opt. Acta 25, 315–325 (1978).
[CrossRef]

Opt. Commun.

M. Kempe, A. Thon, W. Rudolph, “Resolution limits of microscopy through scattering layers,” Opt. Commun. 110, 492–496 (1994).
[CrossRef]

Opt. Lett.

Phys. Rev. B

P. M. Saulnier, M. P. Zinkin, G. H. Watson, “Scatterer correlation effects on photon transport in dense random media,” Phys. Rev. B 42, 2621–2623 (1990).
[CrossRef]

Other

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

J. M. Schmitt, A. Knuettel, M. Yadlowsky, “Interferometric versus confocal techniques for imaging microstructures in turbid biological media,” in Advances in Laser and Light Spectroscopy to Diagnose Cancer and Other Diseases, R. R. Alfano, ed., Proc. Soc. Photo-Opt. Instrum. Eng.2135, 251–262 (1994).
[CrossRef]

M. Davidson, K. Kaufman, I. Mazor, F. Cohen, “An application of interference microscopy to integrated circuit inspection and metrology,” in Integrated Circuit Metrology, Inspection, and Process Control, K. M. Monahan, ed., Proc. Soc. Photo.775, 233–241 (1987).
[CrossRef]

M. Born, E. Wolf, Principles of Optics (Pergamon, London, 1989).

T. Wilson, ed., Confocal Microscopy (Academic, London, 1991).

J. W. Goodman, Statistical Optics (Wiley, New York, 1985).

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Figures (9)

Fig. 1
Fig. 1

Experimental setup of a laser scanning microscope with heterodyne detection.

Fig. 2
Fig. 2

Normalized signal of the confocal (● and ◆) and correlation microscope (■ and ▼) as a function of the concentration of 135-nm latex spheres and two different layer thicknesses. For comparison, the results of the confocal microscope with a large pinhole are also shown (★). Solid curves, expected signal attenuation according to Mie theory; dashed curves, attenuation according to the corrected Mie theory.

Fig. 3
Fig. 3

Depth scan through a cover glass into the scattering medium for the confocal microscope (■ and solid curves) and the correlation microscope (+ and dotted curves). The inset shows the logarithmic plot of the signal near the glass–scatterer interface. Δz is the distance the object has been moved through the focus as measured externally.

Fig. 4
Fig. 4

Contrast obtained with different imaging modes. (a) For a 3.8-mm layer (confocal, ●; correlation, ■) and (b) for a 1.3-mm layer (confocal, ◆; large pinhole, ★) on top of the object. Dotted curves, calculated contrast for the confocal microscope.

Fig. 5
Fig. 5

Lateral resolution versus optical depth through the 3.8-mm layer, determined by scanning the focus spot across a straight edge and measuring the lateral displacement between the 10% and 90% signal levels. The solid line represents the diffraction limit.17

Fig. 6
Fig. 6

Images of a grating structure through a 3.8-mm-thick scattering layer. Upper row, confocal image; lower row, corresponding correlation images. The concentration of latex spheres (in percent solid content) and the MFP values according to the corrected Mie theory are (a) 0/0, (b) 1/3.1, (c) 1.5/4.6, (d) 2/6.0, and (e) 2.5/7.4.

Fig. 7
Fig. 7

Images of a grating structure through a 1.3-mm-thick layer obtained with a true confocal setup (upper row) and with a setup with large pinhole (vp ≈ 50) (lower row). The concentration of latex spheres (in percent solid content) and the MFP values according to the corrected Mie theory are (a) 0/0, (b) 2.5/2.5, (c) 5.0/4.6, and (d) 7.5/6.4.

Fig. 8
Fig. 8

Line scans across the grating images shown in Fig. 6.

Fig. 9
Fig. 9

Calculated light backscattered from a layer of thickness dz and detected in the confocal microscope versus depth in the sample. The signal is normalized to the coherent signal from the (in focus) object layer. The focus is in a fixed depth z = L. The parameters are μL = 5, f = 12.5 mm, a = 5.5 mm, R = 0.01, λ = 830 nm, n = 1.33.

Equations (5)

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C = S max - S min S max + S min ,
C [ 1 + 2 S scatt R S 0 exp ( - 2 μ L ) ] - 1 ,
1 S 0 d d z S scatt = q μ 2 exp ( - 2 μ z ) 0 d v v h 2 [ v , u ( z ) ] 2 + q μ R 2 exp ( - 2 μ L ) 0 d v v h 2 [ v , - u ( z ) ] 2 + q μ R 2 exp ( - 2 μ L ) 0 d v v h 2 [ v , u ( z ) ] 2 + q μ R 2 2 exp [ - 2 μ ( 2 L - z ) ] × 0 d v v h 2 [ v , - u ( z ) ] 2 ,
S interf = S 0 q R i 0 d v v h [ v , u ( z = 0 ) ] 2 .
S coh = S 0 q R e - 2 μ L | 0 d v v h 2 ( v , 0 ) | 2 .

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