Abstract

We report a new method for microscopic imaging of an object embedded in a turbid medium. The new method is based on the angle-gating mechanism achieved by the use of polarized annular objectives in the illumination and collection paths of a microscopic imaging system. A detailed experimental study is presented of the effects of the size of annular obstructions on image quality when turbid media, including polystyrene microspheres and milk suspensions, are imaged. Images of 22-μm polystyrene microspheres embedded in the turbid media show that misinterpretation can occur when circular objectives are used, because of the detection of mainly multiply scattered photons (i.e., diffusing photons). However, when annular objectives are employed, diffusing photons from a turbid medium can be efficiently suppressed; thus image contrast appears correctly, and image resolution is increased.

© 1998 Optical Society of America

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1997 (8)

B. Tromberg, A. Yodh, E. Sevick, D. Pine, “Diffusing photons in turbid media: introduction to the feature,” Appl. Opt. 36, 9 (1997).
[Crossref] [PubMed]

A. Yodh, B. Tromberg, E. Sevick-Muraca, D. Pine, “Introduction to the special issue on diffusing photons in turbid media,” J. Opt. Soc. Am. A 14, 136 (1997).

S. G. Demos, R. R. Alfano, “Optical polarization imaging,” Appl. Opt. 36, 150–155 (1997).
[Crossref] [PubMed]

S. P. Morgan, M. P. Khong, M. G. Somekh, “Effects of polarization state and scatter concentration on optical imaging through scattering media,” Appl. Opt. 36, 1560–1565 (1997).
[Crossref] [PubMed]

M. Gu, X. Gan, “Monte Carlo simulation for confocal microscopy through turbid media,” Cell Vision 4, 109–110 (1997).

X. Gan, S. Schilders, M. Gu, “Combination of annular aperture and polarization gating methods for efficient microscopic imaging through a turbid medium: theoretical analysis,” Microsc. Microanal. 3, 495–503 (1997).

A. H. Hielscher, J. R. Mourant, I. J. Bigio, “Influence of particle size and concentration on the diffuse backscattering of polarized light from tissue phantoms and biological cell suspensions,” Appl. Opt. 36, 125–135 (1997).
[Crossref] [PubMed]

H. Wabnitz, H. Rinneberg, “Imaging in turbid media by photon density waves: spatial resolution and scaling relations,” Appl. Opt. 36, 64–74 (1997).
[Crossref]

1996 (4)

1995 (2)

1994 (3)

1993 (2)

H. P. Chiang, W. S. Cheng, J. Wang, “Imaging through random scattering media using cw broadband interferometry,” Opt. Lett. 18, 546–548 (1993).
[Crossref] [PubMed]

M. Gu, C. J. R. Sheppard, H. Zhou, “Optimization of axial resolution in confocal imaging using annular pupils,” Optik 93, 87–90 (1993).

1992 (1)

1991 (1)

K. M. Yoo, Q. Xing, R. R. Alfano, “Imaging objects hidden in highly scattering media using femtosecond second-harmonic-generation cross-correlation time gating,” Opt. Lett. 16, 1019–1021 (1991).
[Crossref] [PubMed]

1990 (1)

W. F. Cheong, S. A. Prahl, A. J. Walsh, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[Crossref]

Alfano, R. R.

S. G. Demos, R. R. Alfano, “Optical polarization imaging,” Appl. Opt. 36, 150–155 (1997).
[Crossref] [PubMed]

S. G. Demos, R. R. Alfano, “Temporal gating in highly scattering media by the degree of optical polarization,” Opt. Lett. 21, 161–163 (1996).
[Crossref] [PubMed]

K. M. Yoo, Q. Xing, R. R. Alfano, “Imaging objects hidden in highly scattering media using femtosecond second-harmonic-generation cross-correlation time gating,” Opt. Lett. 16, 1019–1021 (1991).
[Crossref] [PubMed]

Baselman, J. M.

Ben-Lataief, K.

Bigio, I. J.

Bohern, C. F.

C. F. Bohern, D. R. Huffman, Absorption and Scattering of Light by Small Particles (Wiley, New York, 1983).

Bonner, R. F.

Cheng, W. S.

H. P. Chiang, W. S. Cheng, J. Wang, “Imaging through random scattering media using cw broadband interferometry,” Opt. Lett. 18, 546–548 (1993).
[Crossref] [PubMed]

Cheong, W. F.

W. F. Cheong, S. A. Prahl, A. J. Walsh, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[Crossref]

Chiang, H. P.

H. P. Chiang, W. S. Cheng, J. Wang, “Imaging through random scattering media using cw broadband interferometry,” Opt. Lett. 18, 546–548 (1993).
[Crossref] [PubMed]

Cho, Y.

Demos, S. G.

Fujimoto, J. G.

Gan, X.

M. Gu, X. Gan, “Monte Carlo simulation for confocal microscopy through turbid media,” Cell Vision 4, 109–110 (1997).

X. Gan, S. Schilders, M. Gu, “Combination of annular aperture and polarization gating methods for efficient microscopic imaging through a turbid medium: theoretical analysis,” Microsc. Microanal. 3, 495–503 (1997).

X. Gan, M. Gu, “Temporal and angular distribution of photon migration through highly scattering media under ultrashort pulse illumination,” Optik (to be published).

Gandjbakhche, A. H.

Gu, M.

M. Gu, X. Gan, “Monte Carlo simulation for confocal microscopy through turbid media,” Cell Vision 4, 109–110 (1997).

X. Gan, S. Schilders, M. Gu, “Combination of annular aperture and polarization gating methods for efficient microscopic imaging through a turbid medium: theoretical analysis,” Microsc. Microanal. 3, 495–503 (1997).

M. Gu, T. Tannous, C. J. R. Sheppard, “Effect of an annular pupil on confocal imaging through highly scattering media,” Opt. Lett. 21, 312–314 (1996).
[Crossref] [PubMed]

M. Gu, C. J. R. Sheppard, H. Zhou, “Optimization of axial resolution in confocal imaging using annular pupils,” Optik 93, 87–90 (1993).

M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, Singapore, 1996).

X. Gan, M. Gu, “Temporal and angular distribution of photon migration through highly scattering media under ultrashort pulse illumination,” Optik (to be published).

Hashimoto, K.

Hee, M. R.

Hielscher, A. H.

Hooft, G. W.

Horinaka, H.

Huffman, D. R.

C. F. Bohern, D. R. Huffman, Absorption and Scattering of Light by Small Particles (Wiley, New York, 1983).

Izatt, J. A.

Kempe, M.

M. Kempe, W. Rudolph, W. Welsch, “Comparative study of confocal and heterodyne microscopy for imaging through scattering media,” J. Opt. Soc. Am. A 13, 46–52 (1996).
[Crossref]

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

Khong, M. P.

Kniittel, A.

Morgan, S. P.

Mourant, J. R.

Osawa, M.

Owen, G. M.

Papaioannou, G.

Pine, D.

B. Tromberg, A. Yodh, E. Sevick, D. Pine, “Diffusing photons in turbid media: introduction to the feature,” Appl. Opt. 36, 9 (1997).
[Crossref] [PubMed]

A. Yodh, B. Tromberg, E. Sevick-Muraca, D. Pine, “Introduction to the special issue on diffusing photons in turbid media,” J. Opt. Soc. Am. A 14, 136 (1997).

Prahl, S. A.

W. F. Cheong, S. A. Prahl, A. J. Walsh, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[Crossref]

Rinneberg, H.

Rudolph, W.

M. Kempe, W. Rudolph, W. Welsch, “Comparative study of confocal and heterodyne microscopy for imaging through scattering media,” J. Opt. Soc. Am. A 13, 46–52 (1996).
[Crossref]

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

Schilders, S.

X. Gan, S. Schilders, M. Gu, “Combination of annular aperture and polarization gating methods for efficient microscopic imaging through a turbid medium: theoretical analysis,” Microsc. Microanal. 3, 495–503 (1997).

Schmitt, J. M.

Sevick, E.

B. Tromberg, A. Yodh, E. Sevick, D. Pine, “Diffusing photons in turbid media: introduction to the feature,” Appl. Opt. 36, 9 (1997).
[Crossref] [PubMed]

Sevick-Muraca, E.

A. Yodh, B. Tromberg, E. Sevick-Muraca, D. Pine, “Introduction to the special issue on diffusing photons in turbid media,” J. Opt. Soc. Am. A 14, 136 (1997).

Sheppard, C. J. R.

M. Gu, T. Tannous, C. J. R. Sheppard, “Effect of an annular pupil on confocal imaging through highly scattering media,” Opt. Lett. 21, 312–314 (1996).
[Crossref] [PubMed]

M. Gu, C. J. R. Sheppard, H. Zhou, “Optimization of axial resolution in confocal imaging using annular pupils,” Optik 93, 87–90 (1993).

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

Somekh, M. G.

Swanson, E. A.

Tannous, T.

Tromberg, B.

A. Yodh, B. Tromberg, E. Sevick-Muraca, D. Pine, “Introduction to the special issue on diffusing photons in turbid media,” J. Opt. Soc. Am. A 14, 136 (1997).

B. Tromberg, A. Yodh, E. Sevick, D. Pine, “Diffusing photons in turbid media: introduction to the feature,” Appl. Opt. 36, 9 (1997).
[Crossref] [PubMed]

Van Germert, M.

Wabnitz, H.

Wada, K.

Walsh, A. J.

W. F. Cheong, S. A. Prahl, A. J. Walsh, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[Crossref]

Wang, J.

H. P. Chiang, W. S. Cheng, J. Wang, “Imaging through random scattering media using cw broadband interferometry,” Opt. Lett. 18, 546–548 (1993).
[Crossref] [PubMed]

Welsch, W.

Wilson, T.

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

Xing, Q.

K. M. Yoo, Q. Xing, R. R. Alfano, “Imaging objects hidden in highly scattering media using femtosecond second-harmonic-generation cross-correlation time gating,” Opt. Lett. 16, 1019–1021 (1991).
[Crossref] [PubMed]

Yadlowsky, M.

Yodh, A.

B. Tromberg, A. Yodh, E. Sevick, D. Pine, “Diffusing photons in turbid media: introduction to the feature,” Appl. Opt. 36, 9 (1997).
[Crossref] [PubMed]

A. Yodh, B. Tromberg, E. Sevick-Muraca, D. Pine, “Introduction to the special issue on diffusing photons in turbid media,” J. Opt. Soc. Am. A 14, 136 (1997).

Yoo, K. M.

K. M. Yoo, Q. Xing, R. R. Alfano, “Imaging objects hidden in highly scattering media using femtosecond second-harmonic-generation cross-correlation time gating,” Opt. Lett. 16, 1019–1021 (1991).
[Crossref] [PubMed]

Zhou, H.

M. Gu, C. J. R. Sheppard, H. Zhou, “Optimization of axial resolution in confocal imaging using annular pupils,” Optik 93, 87–90 (1993).

Appl. Opt. (1)

B. Tromberg, A. Yodh, E. Sevick, D. Pine, “Diffusing photons in turbid media: introduction to the feature,” Appl. Opt. 36, 9 (1997).
[Crossref] [PubMed]

Appl. Opt. (6)

Cell Vision (1)

M. Gu, X. Gan, “Monte Carlo simulation for confocal microscopy through turbid media,” Cell Vision 4, 109–110 (1997).

IEEE J. Quantum Electron. (1)

W. F. Cheong, S. A. Prahl, A. J. Walsh, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[Crossref]

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

A. Yodh, B. Tromberg, E. Sevick-Muraca, D. Pine, “Introduction to the special issue on diffusing photons in turbid media,” J. Opt. Soc. Am. A 14, 136 (1997).

J. Opt. Soc. Am. A (3)

Microsc. Microanal. (1)

X. Gan, S. Schilders, M. Gu, “Combination of annular aperture and polarization gating methods for efficient microscopic imaging through a turbid medium: theoretical analysis,” Microsc. Microanal. 3, 495–503 (1997).

Opt. Lett. (3)

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

K. M. Yoo, Q. Xing, R. R. Alfano, “Imaging objects hidden in highly scattering media using femtosecond second-harmonic-generation cross-correlation time gating,” Opt. Lett. 16, 1019–1021 (1991).
[Crossref] [PubMed]

H. P. Chiang, W. S. Cheng, J. Wang, “Imaging through random scattering media using cw broadband interferometry,” Opt. Lett. 18, 546–548 (1993).
[Crossref] [PubMed]

Opt. Lett. (4)

Optik (1)

M. Gu, C. J. R. Sheppard, H. Zhou, “Optimization of axial resolution in confocal imaging using annular pupils,” Optik 93, 87–90 (1993).

Other (4)

C. F. Bohern, D. R. Huffman, Absorption and Scattering of Light by Small Particles (Wiley, New York, 1983).

X. Gan, M. Gu, “Temporal and angular distribution of photon migration through highly scattering media under ultrashort pulse illumination,” Optik (to be published).

M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, Singapore, 1996).

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

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Figures (11)

Fig. 1
Fig. 1

Schematic diagram of the experimental microscopic scanning imaging system with angle- and polarization-gating mechanisms: D, diaphragm; P, pinhole; 3D, three-dimensional; other abbreviations defined in text.

Fig. 2
Fig. 2

Simulated photon distribution along the radius of the collection objective when a circular objective (solid curve) or an annular objective (dashed curve; ε = 0.9) is employed for illumination. The NA for the illumination and collection objectives is 0.6. The turbid medium is sample1 (including polystyrene microspheres; diameter, 0.48 μm; anisotropy value, 0.81), which has an optical thickness, n, of 5.2.

Fig. 3
Fig. 3

Dependence of the degree of polarization, γ, on the radius of the central obstruction of the illumination objective, εin. The numerical aperture for objectives O2 and O3 is 0.6.

Fig. 4
Fig. 4

Dependence of γ on the radius of the central obstruction of the collection objective, εout, when εin is given. The numerical aperture for objectives O2 and O3 is 0.6.

Fig. 5
Fig. 5

Dependence of γ on the radius of the central obstruction of a matching pair of annular objectives, ε. The numerical aperture for objectives O2 and O3 is 0.6.

Fig. 6
Fig. 6

Dependence of γ on the NA of a matching pair of illumination and collection objectives.

Fig. 7
Fig. 7

Images of a 22-μm polystyrene bead cluster embedded with no scattering medium. (a), (b) Circular objectives (ε = 0) with parallel polarizers; (c), (d) annular objectives (ε ≈ 0.93) with parallel polarizers. In (a) and (c) the focus is on the dried cluster layer; in (b) and (d) the focus is shifted away from the cluster layer by approximately 5 μm.

Fig. 8
Fig. 8

Images of a 22-μm polystyrene bead cluster embedded in sample1. (a)–(c) Circular objectives (ε = 0) with parallel polarization; (d)–(f) annular objectives (ε ≈ 0.93) with parallel polarization. In (a) and (d) the focus is on the dried cluster layer, in (b) and (e) the focus is shifted away from the cluster layer by approximately 20 μm, and in (c) and (f) the focus is shifted away from the dried cluster layer by approximately 40 μm.

Fig. 9
Fig. 9

Images of a 22-μm polystyrene beads embedded in sample2. (a) Circular objectives (ε = 0) with parallel polarization, (b) annular objectives (ε = 0.93) with parallel polarization, (c) annular objectives (ε ≈ 0.93) with perpendicular polarization.

Fig. 10
Fig. 10

Images of a 22-μm polystyrene bead embedded in sample3. (a) Circular objectives (ε = 0) with parallel polarization, (b) annular objectives (ε ≈ 0.93) with parallel polarization.

Fig. 11
Fig. 11

Simulated dependence of the degree of polarization, γ, on (a) the NA of the circular objectives (dashed curve) and (b) the radius of the central annular obstruction for a matching pair of annular objectives with a NA of 0.6 (solid curve). Sample1 is considered.

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