Abstract

We present a method for selective detection of size-dependent scattering characteristics of epithelial cells in vivo based on polarized illumination and polarization sensitive detection of scattered light. We illustrate the method using phantoms designed to simulate squamous epithelial tissue and progressing to epithelial tissue in vitro and in vivo. Elastic light scattering spectroscopy with polarized illumination/detection dramatically reduces background signals due to both diffuse stromal scattering and hemoglobin absorption. Resulting spectra can be described as a linear combination of forward and backscattering components determined from Mie theory. Nuclear sizes and refractive indices extracted by fitting experimental spectra to this model agree well with previous measurements. Reflectance spectroscopy with polarized light can provide quantitative morphological information which could potentially be used for non-invasive detection of neoplastic changes.

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References

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  1. American Cancer Society (1993) Cancer Facts and Figures. Publication No. 93-400M, No. 5008-03.
  2. J. R. Mourant, T. Fuselier, J. Boyer, T. M. Johnson, and I. J. Bigio, "Predictions and Measurements of Scattering and Absorption Over Broad Wavelength Ranges in Tissue Phantoms," Appl. Opt. 36, 949-57 (1997).
    [CrossRef] [PubMed]
  3. L. T. Perelman, V. Backman, M. Wallace, G. Zonios, R. Manoharan, A. Nusrat, S. Shields, M. Seiler, C. Lima, T. Hamano, I. Itzkan, J. Van Dam, J. M. Crawford, and M. S. Feld, "Observation of Periodic Fine Structure in Reflectance from Biological Tissue: A New Technique for Measuring Nuclear Size Distribution," Phys. Rev. Lett. 80, 627-630 (1998).
    [CrossRef]
  4. R. R. Anderson, "Polarized Light Examination and Photography of the Skin," Arch. Dermatol. 127, 1000-5 (1991).
    [CrossRef] [PubMed]
  5. http://omlc.ogi.edu/news/feb98/polarization/index.html
  6. V. Backman, R. Gurjar, K. Badizadegan, I. Itzkan, R. Dasari, L. T. Perelman, and M. S. Feld, "Polarized Light Scattering Spectroscopy for Quantitative Measurements of Epithelial Cellular Structures In Situ," IEEE, J. Selected Topics in Quantum Electronics on Lasers in Medicine and Biology 5, (1999).
  7. T.M. Johnson and J.R. Mourant, "Polarized Wavelength-Dependant Measurements of Turbid Media," Opt. Express 4, 200-216 (1999). http://www.opticsexpress.org/oearchive/source/8492.htm
    [CrossRef] [PubMed]
  8. V. Sankaran, K. Sch�nenberger, J. T. Walsh, Jr., and D. J. Maitland, "Polarization Discrimination of Coherently Propagating Light in Turbid Media," Appl. Opt. 38, 4252-4261 (1999).
    [CrossRef]
  9. G. Jarry, E. Steimer, V. Damaschini, M. Epifanie, M. Jurczak, and R. Kaiser, "Coherence and Polarization of Light Propagating Through scattering Media and Biological Tissues," Appl. Opt. 37, 7357-7367 (1998).
    [CrossRef]
  10. D. Bicout, C. Brosseau, A. S. Martinez, J. M. Schmitt, "Depolarization of Multiply Scattered Waves by Spherical Diffusers: Influence of the Size Parameter," Phys. Rev. E 49, 1767-1770 (1994).
    [CrossRef]
  11. R. Barer, "Refractometry and Interferometry of Living Cells," J. Opt. Soc. Am. 47, 545-56 (1957).
    [CrossRef] [PubMed]
  12. C. Smithpeter, A. Dunn, R. Drezek, T. Collier, and R. Richards-Kortum, "Near Real Time Confocal Microscopy of Cultured Amelanotic Cells: Sources of Signal, Contrast Agents and Limits of Contrast," J. Biomed. Opt. 3, 429-36 (1998).
    [CrossRef] [PubMed]
  13. H.D. Young. Statistical Treatment of Experimental Data. (McGraw-Hill, New York, NY, 1962).
  14. H. van de Hulst. Light Scattering by Small Particles. (Dover, New York, NY, 1957).
  15. R. Kudo, S. Sagae, O. Hayakawa, E. Ito, E. Horimoto, and M. Hashimoto, "Morphology of Adenocarcinoma in Situ and Microinvasive Adenocarcinoma of the Uterine Cervix. A Cytologic and Ultrastructural Study," Acta Cytol. 35, 109-16 (1991).
    [PubMed]
  16. N. Wheeler, S. C. Suffin, T. L. Hall, and D. L. Rosenthal "Predictions of Cervical Neoplasia Diagnosis Groups. Discriminant Analysis on Digitized Cell Images," Analyt. Quant. Cytol. Histol. 9, 169-81 (1987).
  17. E. Artacho-Perula, R. Roldan-Villalobos, J. Salas-Molina, and R. Vaamonde-Lemos, "Histomorphometry of Normal and Abnormal Cervical Samples," Analyt. Quant. Cytol. Histol. 15, 290-97 (1993).
  18. L. Burke, D. A. Antonioli, B. S. Ducatman, Colposcopy. Text and Atlas, (Appleton & Lance, CA 1991).
  19. A. Dunn, C. Smithpeter, A. J. Welch, R. Richards-Kortum, "FDTD Simulation of Light Scattering from Single Cells," J. Biomed. Optic. 2, 262-66 (1997).
    [CrossRef]
  20. R. Drezek, A. Dunn, and R. Richards-Kortum, "Light Scattering from Cells: Finite-Difference Time-Domain Simulations and Goniometric Measurements," Appl. Opt. 38, 3651-3661 (1999).
    [CrossRef]
  21. J. R. Mourant, J. P. freyer, A. H. Hielscher, A. A. Eick, D. Shen, and T. M. Johnson, "Mechanism of Light Scattering from Biological Cells Relevant to Noninvasive Optical-Tissue Diagnostics," Appl. Opt. 37, 3586-3593 (1998).
    [CrossRef]
  22. A. Brunsting and P. F. Mullaney, "Differential Light Scattering from Spherical Mammalian Cells," Biophys. J. 14, 439-453 (1974).
    [CrossRef] [PubMed]
  23. J. M. Schmitt and G. Kumar, "Optical Scattering Properties of Soft Tissue: a Discrete Particle Model," Appl. Opt. 37, 2788-2797 (1998).
    [CrossRef]
  24. S. Asano and M. Sato, "Light Scattering by Randomly Oriented Spheroidal Perticles," Appl. Opt. 19, 962- 974 (1980).
    [CrossRef] [PubMed]

Other

American Cancer Society (1993) Cancer Facts and Figures. Publication No. 93-400M, No. 5008-03.

J. R. Mourant, T. Fuselier, J. Boyer, T. M. Johnson, and I. J. Bigio, "Predictions and Measurements of Scattering and Absorption Over Broad Wavelength Ranges in Tissue Phantoms," Appl. Opt. 36, 949-57 (1997).
[CrossRef] [PubMed]

L. T. Perelman, V. Backman, M. Wallace, G. Zonios, R. Manoharan, A. Nusrat, S. Shields, M. Seiler, C. Lima, T. Hamano, I. Itzkan, J. Van Dam, J. M. Crawford, and M. S. Feld, "Observation of Periodic Fine Structure in Reflectance from Biological Tissue: A New Technique for Measuring Nuclear Size Distribution," Phys. Rev. Lett. 80, 627-630 (1998).
[CrossRef]

R. R. Anderson, "Polarized Light Examination and Photography of the Skin," Arch. Dermatol. 127, 1000-5 (1991).
[CrossRef] [PubMed]

http://omlc.ogi.edu/news/feb98/polarization/index.html

V. Backman, R. Gurjar, K. Badizadegan, I. Itzkan, R. Dasari, L. T. Perelman, and M. S. Feld, "Polarized Light Scattering Spectroscopy for Quantitative Measurements of Epithelial Cellular Structures In Situ," IEEE, J. Selected Topics in Quantum Electronics on Lasers in Medicine and Biology 5, (1999).

T.M. Johnson and J.R. Mourant, "Polarized Wavelength-Dependant Measurements of Turbid Media," Opt. Express 4, 200-216 (1999). http://www.opticsexpress.org/oearchive/source/8492.htm
[CrossRef] [PubMed]

V. Sankaran, K. Sch�nenberger, J. T. Walsh, Jr., and D. J. Maitland, "Polarization Discrimination of Coherently Propagating Light in Turbid Media," Appl. Opt. 38, 4252-4261 (1999).
[CrossRef]

G. Jarry, E. Steimer, V. Damaschini, M. Epifanie, M. Jurczak, and R. Kaiser, "Coherence and Polarization of Light Propagating Through scattering Media and Biological Tissues," Appl. Opt. 37, 7357-7367 (1998).
[CrossRef]

D. Bicout, C. Brosseau, A. S. Martinez, J. M. Schmitt, "Depolarization of Multiply Scattered Waves by Spherical Diffusers: Influence of the Size Parameter," Phys. Rev. E 49, 1767-1770 (1994).
[CrossRef]

R. Barer, "Refractometry and Interferometry of Living Cells," J. Opt. Soc. Am. 47, 545-56 (1957).
[CrossRef] [PubMed]

C. Smithpeter, A. Dunn, R. Drezek, T. Collier, and R. Richards-Kortum, "Near Real Time Confocal Microscopy of Cultured Amelanotic Cells: Sources of Signal, Contrast Agents and Limits of Contrast," J. Biomed. Opt. 3, 429-36 (1998).
[CrossRef] [PubMed]

H.D. Young. Statistical Treatment of Experimental Data. (McGraw-Hill, New York, NY, 1962).

H. van de Hulst. Light Scattering by Small Particles. (Dover, New York, NY, 1957).

R. Kudo, S. Sagae, O. Hayakawa, E. Ito, E. Horimoto, and M. Hashimoto, "Morphology of Adenocarcinoma in Situ and Microinvasive Adenocarcinoma of the Uterine Cervix. A Cytologic and Ultrastructural Study," Acta Cytol. 35, 109-16 (1991).
[PubMed]

N. Wheeler, S. C. Suffin, T. L. Hall, and D. L. Rosenthal "Predictions of Cervical Neoplasia Diagnosis Groups. Discriminant Analysis on Digitized Cell Images," Analyt. Quant. Cytol. Histol. 9, 169-81 (1987).

E. Artacho-Perula, R. Roldan-Villalobos, J. Salas-Molina, and R. Vaamonde-Lemos, "Histomorphometry of Normal and Abnormal Cervical Samples," Analyt. Quant. Cytol. Histol. 15, 290-97 (1993).

L. Burke, D. A. Antonioli, B. S. Ducatman, Colposcopy. Text and Atlas, (Appleton & Lance, CA 1991).

A. Dunn, C. Smithpeter, A. J. Welch, R. Richards-Kortum, "FDTD Simulation of Light Scattering from Single Cells," J. Biomed. Optic. 2, 262-66 (1997).
[CrossRef]

R. Drezek, A. Dunn, and R. Richards-Kortum, "Light Scattering from Cells: Finite-Difference Time-Domain Simulations and Goniometric Measurements," Appl. Opt. 38, 3651-3661 (1999).
[CrossRef]

J. R. Mourant, J. P. freyer, A. H. Hielscher, A. A. Eick, D. Shen, and T. M. Johnson, "Mechanism of Light Scattering from Biological Cells Relevant to Noninvasive Optical-Tissue Diagnostics," Appl. Opt. 37, 3586-3593 (1998).
[CrossRef]

A. Brunsting and P. F. Mullaney, "Differential Light Scattering from Spherical Mammalian Cells," Biophys. J. 14, 439-453 (1974).
[CrossRef] [PubMed]

J. M. Schmitt and G. Kumar, "Optical Scattering Properties of Soft Tissue: a Discrete Particle Model," Appl. Opt. 37, 2788-2797 (1998).
[CrossRef]

S. Asano and M. Sato, "Light Scattering by Randomly Oriented Spheroidal Perticles," Appl. Opt. 19, 962- 974 (1980).
[CrossRef] [PubMed]

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Figures (7)

Fig. 1.
Fig. 1.

A diagram of spectrometer used for reflectance measurements with polarized illumination/detection.

Fig. 2.
Fig. 2.

Schematic presentation of polarized light scattering from diffusely scattering substrate alone (top) and from spherical scatterers placed atop the substrate (bottom). Five different types of scattering events (I–V) can take place and are described in the text.

Fig. 3.
Fig. 3.

Reflectance spectra and forward and backward scattering spectra calculated from Mie theory for: 5 µm polystyrene beads in water (A); 5 µm polystyrene beads in glycerol (B); and 10 µm polystyrene beads in glycerol (C). Reflectance spectrum obtained using unpolarized detection is shown in purple; depolarization ratio spectra in black; fits to equation (12) in green; forward components of Mie scattering in red; backscattering Mie components in blue. The theoretical curves are multiplied by coefficients extracted from the least-square algorithm used to achieve the best fits for the experimental data. Arbitrary DC offsets are added to theoretical curves in order to facilitate comparison of all curves on one graph.

Fig. 4.
Fig. 4.

Density dependence of scattering of 5 µm polystyrene beads in water: (A) a semi-regular monolayer of 5 µm polystyrene beads; (B) depolarization ratio spectra of a water suspension (blue) and of a monolayer of the beads (red).

Figure 5.
Figure 5.

Scattering spectra of SiHa cells: (A) phase-contrast photographs of cells in: a pure PBS buffer (left), BSA/PBS solution that matches the refractive index of the cytoplasm (middle), and acetic acid/PBS solution (right); (B) reflectance spectra of SiHa cells in the presence of acetic acid (red), in the PBS buffer (black), and in the presence of high concentration of BSA (blue). Experimentally measured depolarization ratio spectra and Mie theory calculations for scattering of: (C) cells in BSA/PBS (measured depolarization ratio spectrum - black, the best fit to equation (12) - green, forward Mie scattering component of the nucleus - red, backscattering of the nucleus - blue); (D) cells in PBS (measured depolarization ratio spectrum - black, the best fit to equation (12) - green, forward Mie scattering component of the cytoplasm - red, backscattering of the cytoplasm - blue, forward Mie scattering component of the nucleus - purple, backscattering of the nucleus - brown); (E) cells in acetic acid/PBS (measured depolarization ratio spectrum - black, the best fit to equation (12) - green, forward Mie scattering component of the nucleus - red, backscattering of the nucleus - blue). The theoretical curves are multiplied by coefficients extracted from the least-square algorithm used to achieve the best fits for the experimental data. Arbitrary DC offsets are added to theoretical curves in order to facilitate comparison within one plot.

Fig. 6.
Fig. 6.

Measured reflectance spectra and Mie calculations for normal cervical biopsy: (A) reflectance spectra obtained using unpolarized (blue) and polarized (black) illumination/detection; (B) depolarization ratio spectrum (black), calculated fit to equation (12) (green), forward Mie component of nucleus (red), forward Mie component of cytoplasm (orange), and backward Mie component of cytoplasm (blue). The theoretical curves are multiplied by coefficients extracted from the least-square algorithm used to achieve the best fits for the experimental data. Arbitrary DC offsets are added to theoretical curves in order to facilitate comparison within one plot.

Fig. 7.
Fig. 7.

Oral cavity reflectance spectra obtained in vivo and corresponding Mie theory calculations: (A) reflectance spectra obtained using unpolarized (blue) and polarized (black) illumination/detection. (B) Polarized reflectance spectrum (black), calculated fit to equation (12) (green), forward Mie component of scattering of nucleus (red), and backscattering Mie component of nucleus (blue). Arbitrary DC offsets are added to the theoretical curves in order to facilitate comparison within one plot.

Tables (1)

Tables Icon

Table 1. Summary of collection parameters and fitting procedures for samples studied.

Equations (15)

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D ( λ ) = I II ( λ ) I ( λ ) I II ( λ ) + I ( λ )
I II S ( λ ) = I 0 ( λ ) S ( λ ) Δ θ 2 π + 1 2 I 0 ( λ ) M ( λ ) Δ θ 2 π
I S ( λ ) = 1 2 I 0 ( λ ) M ( λ ) Δ θ 2 π
S ( λ ) + M ( λ ) = 1
I IIB ( λ ) = I 0 ( λ ) B ( λ ) Cl + Δ θ 2 π I 0 ( λ ) F ( λ ) S ( λ ) Cl ( 1 Cl σ ( λ ) )
+ Δ θ 2 π I 0 ( λ ) S ( λ ) F ( λ ) Cl ( 1 Cl σ ( λ ) ) + Δ θ 2 π I 0 ( λ ) S ( λ ) ( 1 Cl σ ( λ ) )
+ 1 2 I 0 ( λ ) M ( λ ) Δ θ 2 π ( 1 Cl σ ( λ ) )
F ( λ ) = Δ θ f P ( λ , θ ) sin θ d θ
B ( λ ) = Δ θ b P ( λ , θ ) sin θ d θ
I B ( λ ) = 1 2 I 0 ( λ ) M ( λ ) Δ θ 2 π ( 1 Cl σ ( λ ) )
I IIB ( λ ) I B ( λ ) I IIB ( λ ) + I B ( λ ) = 2 π Cl Δ θ B ( λ ) + ClS ( λ ) F ( λ ) + ClS ( λ ) [ F ( λ ) σ ( λ ) ] + S ( λ ) 1 + 2 π Cl Δ θ B ( λ ) + Cl [ 2 S ( λ ) F ( λ ) σ ( λ ) ]
1 + 2 π Cl Δ θ B ( λ ) + Cl [ 2 S ( λ ) F ( λ ) σ ( λ ) ] 1
I IIB ( λ ) I B ( λ ) I IIB ( λ ) + I B ( λ ) 2 π Cl Δ θ B ( λ ) + ClS ( λ ) F ( λ ) + ClS ( λ ) [ F ( λ ) σ ( λ ) ] + S ( λ )
I IIB ( λ ) I B ( λ ) I IIB ( λ ) + I B ( λ ) 2 π Cl Δ θ B ( λ ) + ClS ( λ ) F ( λ ) + S ( λ ) =
= k 1 B ( λ ) + k 2 F ( λ ) + DC

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