OSA's Digital Library

Applied Optics

Applied Optics


  • Vol. 40, Iss. 25 — Sep. 1, 2001
  • pp: 4633–4646

Intrinsic fluorescence spectroscopy in turbid media: disentangling effects of scattering and absorption

Markus G. Müller, Irene Georgakoudi, Qingguo Zhang, Jun Wu, and Michael S. Feld  »View Author Affiliations

Applied Optics, Vol. 40, Issue 25, pp. 4633-4646 (2001)

View Full Text Article

Enhanced HTML    Acrobat PDF (283 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



The fluorescence from a turbid medium such as biologic tissue contains information about scattering and absorption, as well as the intrinsic fluorescence, i.e., the fluorescence from an optically thin sample of pure fluorophores. The interplay of scattering and absorption can result in severe distortion of the intrinsic spectral features. These distortions can be removed by use of a photon-migration-based picture and information from simultaneously acquired fluorescence and reflectance spectra. We present experimental evidence demonstrating the validity of such an approach for extracting the intrinsic fluorescence for a wide range of scatterer and absorber concentrations in tissue models, ex vivo and in vivo tissues. We show that variations in line shape and intensity in intrinsic tissue fluorescence are significantly reduced compared with the corresponding measured fluorescence.

© 2001 Optical Society of America

OCIS Codes
(170.5280) Medical optics and biotechnology : Photon migration
(170.7050) Medical optics and biotechnology : Turbid media
(290.4210) Scattering : Multiple scattering
(300.1030) Spectroscopy : Absorption
(300.2530) Spectroscopy : Fluorescence, laser-induced

Original Manuscript: January 9, 2001
Revised Manuscript: April 23, 2001
Published: September 1, 2001

Markus G. Müller, Irene Georgakoudi, Qingguo Zhang, Jun Wu, and Michael S. Feld, "Intrinsic fluorescence spectroscopy in turbid media: disentangling effects of scattering and absorption," Appl. Opt. 40, 4633-4646 (2001)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. K. T. Schomacker, J. K. Frisoli, C. C. Compton, T. J. Flotte, J. M. Richter, N. S. Nishioka, T. F. Deutsch, “Ultraviolet laser-induced fluorescence of colonic tissue basic biology and diagnostic potential,” Lasers Surg. Med. 12, 63–78 (1992). [CrossRef]
  2. C. H. Liu, B. B. Das, W. L. S. Glassman, G. C. Tang, K. M. Yoo, H. R. Zhu, D. L. Akins, S. S. Lubicz, J. Cleary, R. Prudente, E. Celmer, A. Caron, R. R. Alfano, “Raman, fluorescence, and time-resolved light-scattering as optical diagnostic-techniques to separate diseased and normal biomedical media,” J. Photochem. Photobiol. B 16(2), 187–209(1992). [CrossRef]
  3. L. I. Laifer, K. M. O’Brien, M. L. Stetz, G. R. Gindi, T. J. Garrand, L. I. Deckelbaum, “Biochemical basis for the difference between normal and atherosclerotic arterial fluorescence,” Circulation 80, 1893–1901 (1989). [CrossRef] [PubMed]
  4. P. K. Gupta, S. K. Majumder, A. Uppal, “Breast cancer diagnosis using nitrogen laser excited autofluorescence spectroscopy,” Lasers Surg. Med. 21, 417–422 (1997). [CrossRef]
  5. C.-T. Chen, H. K. Chiang, S.-N. Chow, C.-Y. Wang, Y.-S. Lee, J.-C. Tsai, C.-P. Chiang, “Autofluorescence in normal and malignant human oral tissues and in DMBA-induced hamster buccal pouch carcinogenesis,” J. Oral Pathol. Med. 27, 470–474 (1998). [CrossRef] [PubMed]
  6. M. Anidjar, O. Cussenot, S. Avrillier, D. Ettori, M. J. Villette, J. Fiet, P. Teillac, A. Le Duc, “Ultraviolet laser-induced autofluorescence distinction between malignant and normal urothelial cells and tissues,” J. Biomed. Opt. 1, 335–341 (1996). [CrossRef] [PubMed]
  7. G. I. Zonios, R. M. Cothren, J. T. Arendt, J. Wu, J. VanDam, J. M. Crawford, R. Manoharan, M. S. Feld, “Morphological model of human colon tissue fluorescence,” IEEE Trans. Biomed. Eng. 43, 113–122 (1996). [CrossRef] [PubMed]
  8. N. Ramanujam, M. F. Mitchell, A. Mahadevan, S. Warren, S. Thomsen, E. Silva, R. Richards-Kortum, “In vivo diagnosis of cervical intraepithelial neoplasia using 337-nm-excited laser-induced fluorescence,” Proc. Natl. Acad. Sci. USA 91, 10193–10197 (1994). [CrossRef]
  9. R. Richards-Kortum, E. Sevick-Muraca, “Quantitative optical spectroscopy for tissue diagnosis,” Ann. Rev. Phys. Chem. 47, 555–606 (1996). [CrossRef]
  10. M. Keijzer, R. R. Richards-Kortum, S. L. Jacques, M. S. Feld, “Fluorescence spectroscopy of turbid media—autofluorescence of the human aorta,” Appl. Opt. 28, 4286–4292 (1989). [CrossRef] [PubMed]
  11. M. Kriegmair, H. Stepp, P. Steinbach, W. Lumper, A. Ehsan, H. G. Stepp, K. Rick, R. Knuchel, R. Baumgartner, A. Hofstetter, “Fluorescence cystoscopy following intravesical instillation of 5-Aminolevulinic acid—a new procedure with high sensitivity for detection of hardly visible urothelial neoplasias,” Urologia Internationalis 55, 190–196 (1995). [CrossRef]
  12. G. A. Wagnières, W. M. Star, B. C. Wilson, “In vivo fluorescence spectroscopy and imaging for oncological applications,” Photochem. Photobiol. 68, 603–632 (1998). [PubMed]
  13. A. E. Profio, D. R. Doiron, J. Sarnaik, “Fluorometer for endoscopic diagnosis of tumors,” Med. Phys. 11, 516–520 (1984). [CrossRef] [PubMed]
  14. P. Lenz, “Endoscopic fluorescence detector,” Rev. Sci. Instrum. 59, 930–933 (1988). [CrossRef]
  15. T. J. Farrell, R. P. Hawkes, M. S. Patterson, B. C. Wilson, “Modeling of photosensitizer fluorescence emission and photobleaching for photodynamic therapy dosimetry,” Appl. Opt. 37, 7168–7183 (1998). [CrossRef]
  16. A. J. L. Jongen, H. J. C. M. Sterenborg, “Mathematical description of photobleaching in vivo describing the influence of tissue optics on measured fluorescence signals,” Phys. Med. Biol. 42, 1701–1716 (1997). [CrossRef] [PubMed]
  17. J. R. Mourant, I. J. Bigio, J. Boyer, R. L. Conn, T. Johnson, T. Shimada, “Spectroscopic diagnosis of bladder cancer elastic light scattering,” Lasers Surg. Med. 17, 350–357 (1995). [CrossRef]
  18. F. Koenig, R. Larne, H. Enquist, F. J. McGovern, K. T. Schomacker, N. Kollias, T. F. Deutsch, “Spectroscopic measurement of diffuse reflectance for enhanced detection of bladder carcinoma,” Urology 51, 342–345 (1998). [CrossRef] [PubMed]
  19. G. Zonios, L. T. Perelman, V. Backman, R. Manoharan, M. Fitzmaurice, J. Van Dam, M. S. Feld, “Diffuse reflectance spectroscopy of human adenomatous colon polyps in vivo,” Appl. Opt. 38, 6628–6637 (1999). [CrossRef]
  20. J. B. Fishkin, O. Coquoz, E. R. Anderson, M. Brenner, B. J. Tromberg, “Frequency-domain photon migration measurements of normal and malignant tissue optical properties in a human subject,” Appl. Opt. 36, 10–20 (1997). [CrossRef] [PubMed]
  21. F. F. Jöbsis, M. O’Connor, A. Vitale, H. Vreman, “Intracellular redox changes in functioning cerebral cortex. I. Metabolic effects of epileptiform activity,” J. Neurophysiol. 34, 735–749 (1971). [PubMed]
  22. A. Mayevsky, B. Chance, “Repetitive patterns of metabolic changes during cortical spreading depression of the awake rat,” Brain Res. 65, 529–533 (1974). [CrossRef] [PubMed]
  23. A. Mayevsky, B. Chance, “Intracellular oxidation-reduction state measured in situ by a multichannel fiber-optic surface fluorometer,” Science 217, 537–540 (1982). [CrossRef] [PubMed]
  24. C. Ince, J. M. C. C. Coremans, H. A. Bruining, “In vivo NADH fluorescence,” in Oxygen Transport to Tissue XIV, W. Erdmann, D. F. Bruley, eds. (Plenum, New York, 1992).
  25. J. Wu, M. S. Feld, R. P. Rava, “Analytical model for extracting intrinsic fluorescence in turbid media,” Appl. Opt. 32, 3585–3595 (1993). [CrossRef] [PubMed]
  26. A. J. Durkin, S. Jaikumar, N. Ramanujam, R. Richards-Kortum, “Relation between fluorescence-spectra of dilute and turbid samples,” Appl. Opt. 33, 414–423 (1994). [CrossRef] [PubMed]
  27. N. N. Zhadin, R. R. Alfano, “Correction of the internal absorption effect in fluorescence emission and excitation spectra from absorbing and highly scattering media: theory and experiment,” J. Biomed. Opt. 3, 171–186 (1998). [CrossRef] [PubMed]
  28. C. M. Gardner, S. L. Jacques, A. J. Welch, “Fluorescence spectroscopy of tissue: recovery of intrinsic fluorescence from measured fluorescence,” Appl. Opt. 35, 1780–1792 (1996). [CrossRef] [PubMed]
  29. M. S. Patterson, B. W. Pogue, “Mathematical model for time-resolved and frequency-domain fluorescence spectroscopy in biological tissues,” Appl. Opt. 33, 1963–1974 (1994). [CrossRef] [PubMed]
  30. S. K. Majumder, P. K. Gupta, A. Uppal, “Autofluorescence spectroscopy of tissues from human oral cavity for discriminating malignant from normal,” Lasers Life Sci. 8, 211–227 (1999).
  31. Q. Zhang, M. G. Müller, J. Wu, M. S. Feld, “Turbidity-free fluorescence spectroscopy of biological tissue,” Opt. Lett. 25, 1451–1453 (2000). [CrossRef]
  32. In the current version we do not need to consider the effective albedo, aeff, introduced in Ref. 25, because the new form of the escape probability [Eq. 2(b)] takes into account differences in optical properties at the excitation and the emission wavelengths. Additionally, we find that the previously introduced effective anisotropy coefficient, geff, does not significantly affect the resulting intrinsic fluorescence spectrum.
  33. R. A. Zangaro, L. Silveira, R. Manoharan, G. Zonios, I. Itzkan, R. R. Dasari, J. Van Dam, M. S. Feld, “Rapid multiexcitation fluorescence spectroscopy system for in vivo tissue diagnosis,” Appl. Opt. 35, 5211–5219 (1996). [CrossRef] [PubMed]
  34. R. M. Cothren, G. B. Hayes, J. R. Kramer, B. A. Sacks, C. Kittrell, M. S. Feld, “A multifiber catheter with an optical shield for laser angiosurgery,” Lasers Life Sci. 1, 1–12 (1986).
  35. A. J. Durkin, S. Jaikumar, R. Richards-Kortum, “Optically dilute, absorbing and turbid phantoms for fluorescence spectroscopy of homogenous and inhomogenous samples,” Appl. Spectrosc. 47, 2114–2121 (1993). [CrossRef]
  36. U. Brackmann, Lambdachrome Laser Dyes (Lambda Physik GmbH, Goettingen, Germany, 1997).
  37. O. W. van Assendelft, Spectrophotometry of Haemoglobin Derivatives (Thomas, Springfield, Ill., 1970).
  38. H. C. van de Hulst, Light Scattering by Small Particles (Dover, New York, 1957).
  39. G. I. Zonios, “Diffuse reflectance spectroscopy of human colon tissue,” Ph.D. dissertation (MIT, Cambridge, Mass., 1998).
  40. J. R. Mourant, J. P. Freyer, A. H. Hielscher, A. A. Eick, D. Shen, T. M. Johnson, “Mechanisms of light scattering from biological cells relevant to noninvasive optical-tissue diagnostics,” Appl. Opt. 37, 3586–3593 (1998). [CrossRef]
  41. S. W. E. van de Poll, M. G. Müller, Q. Zhang, J. Myles, J. R. Kramer, M. S. Feld, “Combined fluorescence and reflectance spectroscopy of arterial wall improves the identification of atherosclerosis,” Circulation 100, 2349, Suppl. SNov.2 (1999).
  42. H. Zeng, C. MacAulay, D. I. McLean, B. Palcic, “Spectroscopic and microscopic characteristics of human skin autofluorescence emission,” Photochem. Photobiol. 61, 639–645 (1995). [CrossRef] [PubMed]
  43. G. Renault, E. Raynal, M. Sinet, M. Muffat-Joly, J.-P. Berthier, J. Cornillault, B. Godard, J.-J. Pocidalo, “In situ double-beam NADH laser fluorimetry: a choice of a reference wavelength,” Am. J. Physiol. 246, H491–H499 (1984). [PubMed]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article

OSA is a member of CrossRef.

CrossCheck Deposited