Abstract

Fluorescence from fluorophores embedded in a turbid medium like biological tissue gets strongly modulated by the wavelength dependent absorption and scattering properties of tissue. This makes it extremely difficult to extract valuable biochemical information from tissue which is present in the intrinsic line shape and intensity of fluorescence from tissue fluorophores. We present an experimental approach to remove the distorting effect of scattering and absorption on intrinsic fluorescence of fluorophores embedded in a turbid medium like tissue. The method is based on simultaneous measurement of polarized fluorescence and polarized elastic scattering spectra from a turbid medium. The polarized fluorescence normalized by the polarized elastic scattering spectra (in the wavelength range of fluorescence emission) was found to be free from the distorting effect of absorption and scattering properties of the medium. The applicability range of this technique to recover intensity and line shape information of intrinsic fluorescence has been investigated by carrying out studies on a variety of tissue phantoms having different absorption and scattering properties. The results obtained show that this technique can be used to recover intrinsic line shape and intensity information of fluorescence from fluorophores embedded in a scattering medium for the range of optical transport parameters typically found in biological tissue.

© 2003 Optical Society of America

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References

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    [CrossRef]
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    [CrossRef] [PubMed]
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    [PubMed]
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    [CrossRef] [PubMed]
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Ann. Rev. Phys. Chem. (1)

R. Richards Kortum, E. Sevick-Muraca, �??Quantitative optical spectroscopy for tissue diagnosis,�?? Ann. Rev. Phys. Chem. 47, 556 - 606 (1996).

Appl. Opt. (10)

G.C. Tang, A. Pradhan, W. Sha, J. Chen, C.H. Liu, S.J. Wahl, R.R. Alfano, �??Pulsed and CW laser fluorescence spectra from cancerous, normal, and chemically treated normal human breast and lung tissues,�?? Appl. Opt. 28, 2337 �?? 2342 (1989).
[CrossRef] [PubMed]

M. Keijzer, R.R. 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]

A.J. Durkin, S. Jaikumar, N. Ramanujam, R.R. Kortum, �??Relation between fluorescence spectra of dilute and turbid samples,�?? Appl. Opt. 33, 414 �?? 423 (1994).
[CrossRef] [PubMed]

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]

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]

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]

M.G. Muller, I. Gergakoudi, Q. Zhang, J. Wu, M.S. Feld, �??Intrinsic fluorescence spectroscopy in turbid media: disentangling effects of scattering and absorption,�?? Appl. Opt. 40, 4633 �?? 4646 (2001).
[CrossRef]

M.S. Nair, N. Ghosh, N.S. Raju, A. Pradhan, �??Propagation of fluorescence in human breast tissues: a diffusion theory model,�?? Appl. Opt. 41, 4024 �?? 4035 (2002).
[CrossRef] [PubMed]

J.M. Schmitt, A.H. Gandjbakhche, R.F. Bonner, �??Use of polarized light to discriminate short-path photons in a multiply scattering medium,�?? Appl. Opt. 32, 6535 - 6546 (1992).
[CrossRef]

N. Ghosh, S.K. Mohanty, S.K. Majumder, P.K. Gupta, �??Measurement of optical transport properties of normal and malignant human breast tissue,�?? Appl. Opt. 40, 176 -184 (2001).
[CrossRef]

IEEE J. Quantum Electron. (2)

W.F. Cheong, S.A. Prahl, A.J. Welch, �??A review of the optical properties of tissues,�?? IEEE J. Quantum Electron. 26, 2166 �?? 2185 (1990).
[CrossRef]

R.R.Alfano, G.C.Tang, A.Pradhan, W.Lam, D.S.J. Choy, E.Opher, �??Fluorescence spectra from cancerous and normal human breast and lung tissues,�?? IEEE J. Quantum Electron. 23, 1806-1811 (1987).
[CrossRef]

J. Biomed. Opt. (1)

N. Zhadin, R.R. Alfano, �??Correction of the internal absorption effect in fluorescence emission and excitation spectra from absorbing and highly scattering media,�?? J. Biomed. Opt. 3, 171 �?? 186 (1998).
[CrossRef] [PubMed]

Lasers Surg. Med. (1)

G. C. Tang, A. Pradhan, R. R. Alfano, �??Spectroscopic studies of human cancer and normal lung and breast tissues,�?? Lasers Surg. Med. 9, 290-295 (1989).
[CrossRef] [PubMed]

Opt. Lett. (1)

Photochem. Photobiol. (1)

G. A. Wagnieres, W. M. Star, and B. C. Wilson, �??In vivo fluorescence spectroscopy and imaging for oncological applications,�?? Photochem. Photobiol. 68, 603-632 (1998).
[PubMed]

Phys. Rev. E (2)

D. Bicout, C. Brosseu, 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]

N. Ghosh, S.K. Majumder, P.K. Gupta, �??Fluorescence depolarization in a scattering medium: Effect of size parameter of scatterer,�?? Phys. Rev. E 65, 0266081-0266086 (2002).
[CrossRef]

Other (2)

C. F. Bohren, D. R. Hoffman, Absorption and scattering of light by small particles, (Wiley, New York, 1983) Chapter 4, pp 82-129.

J. Lackowicz, Principles of Fluorescence Spectroscopy, Plenum Press, (New York, 1983) Chapter 5, pp 111-150.
[CrossRef]

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Figures (8)

Fig. 1.
Fig. 1.

440 nm excited unpolarized fluorescence spectra recorded from four different tissue phantoms having a fixed FAD concentration of 20 µM. The inset of the figure shows the fluorescence spectra normalized by intensity at 520 nm. The values of the reduced scattering coefficient µs/ and absorption coefficient (µa) at 440 nm and 540 nm for the four samples are listed in table 1. (F.I. stands for Fluorescence Intensity in all the subsequent figures).

Fig. 2. (a)
Fig. 2. (a)

440 nm excited polarized fluorescence spectra recorded from four different tissue phantoms having a fixed FAD concentration of 20 µM. The inset 2a shows the fluorescence spectra normalized by intensity at 520nm.

Fig. 2. (b)
Fig. 2. (b)

Polarized elastic scattering spectra recorded from the same samples.

Fig. 2. (c)
Fig. 2. (c)

Polarized fluorescence spectra normalized by polarized elastic scattering spectra and 2 (d) the peak-normalized spectral line shapes (normalized by intensity at 520 nm) for the same spectra from the four samples. The values of the reduced scattering coefficients (µs/) and absorption coefficients (µa) at 440 nm and 540 nm for the four samples are listed in Table 1.

Fig. 3. (a)
Fig. 3. (a)

440 nm excited unpolarized fluorescence spectra recorded from different tissue phantoms with varying concentration of FAD. The inset of the figure shows the fluorescence spectra normalized by intensity at 520 nm.

Fig. 3. (b)
Fig. 3. (b)

440 nm excited polarized fluorescence spectra recorded from the same samples. The inset of the figure shows the polarized elastic scattering spectra recorded from the same phantoms A, C, D, E, F, G, H, I.

Fig. 3. (c)
Fig. 3. (c)

Polarized fluorescence spectra normalized by polarized elastic scattering spectra for the same samples. The inset of the figure shows peak normalized spectral line shape (normalized by intensity at 520 nm). The values of the reduced scattering coefficients (µs/) and absorption coefficients (µa) at 440 nm and 540 nm for the samples and the concentrations of FAD used in the samples are listed in Table 2.

Fig. 4. (a)
Fig. 4. (a)

The normalized fluorescence spectrum of pure FAD with extract ed intrinsic fluorescence from phantom. Fig. 4 (b) Variation of measured and intrinsic fluorescence intensities at 520 nm with varying FAD concentration

Tables (2)

Tables Icon

Table 1. Values of absorption and reduced scattering coefficients at different wavelengths

Tables Icon

Table 2 Values of absorption and reduced scattering coefficients at different wavelengths for different set of phantoms

Equations (2)

Equations on this page are rendered with MathJax. Learn more.

I pol ( λ ) = [ I ( λ ) G × I ( λ ) ]
IF = [ I ( λ ) G × I ( λ ) ] FL / [ I ( λ ) G × I ( λ ) ] ESS

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