Abstract

A confocal fluorescence spectroscopy system is instrumented to study depth-resolved autofluorescence in biological tissue. The system provides the capability of optical sectioning with the maximal detection depth up to 120 µm in the examined tissue samples. It was found that the topmost keratinizing epithelial layer produces strong fluorescence similar to collagen. The fluorescence signal from epithelial tissue between the keratinizing layer and stroma can be well resolved. The study results show that depth-resolved fluorescence spectroscopy has the potential to provide more accurate information for the diagnosis of tissue pathology.

© 2004 Optical Society of America

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References

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  1. R. Richards-Kortum and E. Sevick-Muraca, ???Quantitative optical spectroscopy for tissue diagnosis,??? Ann. Rev. Phy. Chem. 47 555-606 (1996).
    [CrossRef]
  2. G. A. Wagnieres, W. M. Star and B. C. Wilson, ???In vivo fluorescence spectroscopy and imaging for oncological applications,??? Photochem. Photobiol. 68, 603-32 (1998).
    [PubMed]
  3. N. Ramanujam, ???Fluorescence spectroscopy of neoplastic and non-neoplastic tissues,??? Neoplasia 2, 89-117 (2000).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  5. M. Kobayashi, K. Shibuya, H. Hoshino, and T. Fujisawa, ???Spectroscopic analysis of the autofluorescence from human bronchus using an ultraviolet laser diode,??? J. Biomed. Opt., 7, 603-608 (2002)
    [CrossRef] [PubMed]
  6. I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locker, C. P. Crum, C. W. Boone, R. R. Dasari, J. V. Dam, and M. S. Feld, ???NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,??? Cancer Res. 62, 682-687 (2002).
    [PubMed]
  7. N. Ghosh, S. K. Majumder and P. K. Gupta, ???Polarized fluorescence spectroscopy of human tissues,??? Opt. Lett. 22, 2007-2009 (2002).
    [CrossRef]
  8. M. G. Skala, G. M. Palmer, C. Zhu, Q. Liu, K. M. Vrotsos, C. L. Marshek-stone, A. Gendron-Fitzpatrick, and N. Ramanujam, ???Investigation of fiber-optic probe designs for optical spectroscopic diagnosis of epithelial pre-cancers,??? Lasers Surg. Med. 34, 25-38 (2004).
    [CrossRef] [PubMed]
  9. C. E. Bigelow, D. L. Conover and T. H. Foster, ???Confocal fluorescence spectroscopy and anisotropy imaging system,??? Opt. Lett. 28, 695-697 (2003).
    [CrossRef] [PubMed]
  10. G. M. Palmer, C. L. Marshek, K. M. Vrotsos and N. Ramanujam, ???Optimal methods for fluorescence and diffuse reflectance measurements of tissue biopsy samples,??? Lasers Surg. Med. 30, 191-200 (2002).
    [CrossRef] [PubMed]
  11. R. Drezek K. Sokolov, U. Utzinger, I. Boiko, A. Malpica, M. Follen, and R. Richards-Kortum, ???Understanding the contribution of NADH and collagen to cervical tissue fluorescence spectra: modeling, measurements and implications,??? J. Biomed. Opt. 6, 385-396 (2001).
    [CrossRef] [PubMed]
  12. J. A. Kiernan, Histological and histochemical methods: theory and practice (Butterworth Heinemann, 1999), Chap. 8

Ann. Rev. Phy. Chem. (1)

R. Richards-Kortum and E. Sevick-Muraca, ???Quantitative optical spectroscopy for tissue diagnosis,??? Ann. Rev. Phy. Chem. 47 555-606 (1996).
[CrossRef]

Cancer Res. (1)

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locker, C. P. Crum, C. W. Boone, R. R. Dasari, J. V. Dam, and M. S. Feld, ???NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,??? Cancer Res. 62, 682-687 (2002).
[PubMed]

J. Biomed. Opt. (2)

M. Kobayashi, K. Shibuya, H. Hoshino, and T. Fujisawa, ???Spectroscopic analysis of the autofluorescence from human bronchus using an ultraviolet laser diode,??? J. Biomed. Opt., 7, 603-608 (2002)
[CrossRef] [PubMed]

R. Drezek K. Sokolov, U. Utzinger, I. Boiko, A. Malpica, M. Follen, and R. Richards-Kortum, ???Understanding the contribution of NADH and collagen to cervical tissue fluorescence spectra: modeling, measurements and implications,??? J. Biomed. Opt. 6, 385-396 (2001).
[CrossRef] [PubMed]

Lasers Surg. Med. (2)

G. M. Palmer, C. L. Marshek, K. M. Vrotsos and N. Ramanujam, ???Optimal methods for fluorescence and diffuse reflectance measurements of tissue biopsy samples,??? Lasers Surg. Med. 30, 191-200 (2002).
[CrossRef] [PubMed]

M. G. Skala, G. M. Palmer, C. Zhu, Q. Liu, K. M. Vrotsos, C. L. Marshek-stone, A. Gendron-Fitzpatrick, and N. Ramanujam, ???Investigation of fiber-optic probe designs for optical spectroscopic diagnosis of epithelial pre-cancers,??? Lasers Surg. Med. 34, 25-38 (2004).
[CrossRef] [PubMed]

Neoplasia (1)

N. Ramanujam, ???Fluorescence spectroscopy of neoplastic and non-neoplastic tissues,??? Neoplasia 2, 89-117 (2000).
[CrossRef] [PubMed]

Opt. Lett. (2)

C. E. Bigelow, D. L. Conover and T. H. Foster, ???Confocal fluorescence spectroscopy and anisotropy imaging system,??? Opt. Lett. 28, 695-697 (2003).
[CrossRef] [PubMed]

N. Ghosh, S. K. Majumder and P. K. Gupta, ???Polarized fluorescence spectroscopy of human tissues,??? Opt. Lett. 22, 2007-2009 (2002).
[CrossRef]

Photochem. Photobiol. (2)

R. Drezek, C. Brookner, I. Pavlova, I. Boiko, A. Malpica, R. Lotan, M. Follen and R. Richards-Kortum, ???Autofluoresecence microscopy of fresh cervical-tissue sections reveals alterations in tissue biochemistry with dysplasia,??? Photochem. Photobiol. 73, 636-641 (2001).
[CrossRef] [PubMed]

G. A. Wagnieres, W. M. Star and B. C. Wilson, ???In vivo fluorescence spectroscopy and imaging for oncological applications,??? Photochem. Photobiol. 68, 603-32 (1998).
[PubMed]

Other (1)

J. A. Kiernan, Histological and histochemical methods: theory and practice (Butterworth Heinemann, 1999), Chap. 8

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Figures (5)

Fig. 1.
Fig. 1.

Normalized depth-resolved fluorescence spectra excited at 349 nm and 457 nm, respectively. The inlet of each figure shows the raw fluorescence intensity as a function of depth. a, b & c: Fluorescence of esophageal tissue and the corresponding histology. d, e & f: Fluorescence of highly keratinizing oral tissue and the corresponding histology. g, h & i: Fluorescence of non-keratinizing oral tissue and the corresponding histology. Scale bar: 100µm.

Fig. 2.
Fig. 2.

Masson and Verhoff-Van Geison stained sections. a & b: highly keratinizing rabbit oral tissue. c & d: non-keratinizing rabbit oral tissue. Scale bar: 100µm.

Fig. 3.
Fig. 3.

Peak normalized fluorescence spectra of collagen, elastin and keratin excited at 349 nm (solid lines) and 457 nm (dash lines).

Fig. 4.
Fig. 4.

Peak normalized bulk autofluorescence spectra. a: Rabbit esophageal tissues, b. Rabbit oral tissues.

Fig. 5.
Fig. 5.

Peak normalized depth-resolved fluorescence spectra excited at 349 nm from normal cervical squamous tissue (a) and the tissues with HPV infection (b, c). The inlet of each figure shows the raw fluorescence intensity as a function of depth.

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