Craig M. Gardner, Steven L. Jacques, and Ashley J. Welch, "Fluorescence spectroscopy of tissue: recovery of intrinsic fluorescence from measured fluorescence," Appl. Opt. 35, 1780-1792 (1996)
We present a method for recovering the intrinsic fluorescence coefficient, defined as the product of the fluorophore absorption coefficient and the fluorescence energy yield, of an optically thick, homogeneous, turbid medium from a surface measurement of fluorescence and from knowledge of medium optical properties. The measured fluorescence signal is related to the intrinsic fluorescence coefficient by an optical property dependent path-length factor. A simple expression was developed for the path-length factor, which characterizes the penetration of excitation light and the escape of fluorescence from the medium. Experiments with fluorescent tissue phantoms demonstrated that intrinsic fluorescence line shape could be recovered and that fluorophore concentration could be estimated within ±15%, over a wide range of optical properties.
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These are valid for an air–tissue interface for tissues with anisotropy in the range 0.7−0.9.
These are valid for an air–phantom interface for water-based tissue phantoms with anisotropy in the range 0.8–0.9.
Tissue phantoms were composed of 0.5 μM of Rhodamine 6G and varying amounts of hemoglobin (Hb/HbO2) and polystyrene microspheres in PBS.
Included in the last two columns are approximate values for the reduced scattering coefficient at 550 nm,
, and the absorption coefficient at 514 and 550 nm for each sample.
Table 3.
Sensitivity Analysis of the Calculated Intrinsic Fluorescence Coefficient, β, to Errors in Optical Parameters
Relative errors in one, two, or all four optical parameters were used to calculate errors in the one-dimensional correction factor, using the relationship in Eq. (13).
Path-length factor errors were used to calculate errors in the recovered value of the intrinsic fluorescence coefficient. For this set of error analyses, the true optical properties were Rd(λex) = 0.1, δ(λex) = 0.03 cm, Rd(λem) = 0.2, and δ(λem) = 0.05 cm.
Table 4.
Results of Least-Squares Estimation of Fluorophore Concentrationa
A least-squares regression was performed on intrinsic fluorescence coefficient data between 530 and 650 nm, with fluorophore concentration as the free parameter [Eq. (25)].
Each phantom contained 0.5 μM of Rhodamine 6G.
Tables (4)
Table 1.
Empirical Expressions for How the Six Parameters of Eq. (13) Depend on Diffuse Reflectance (Rd)
These are valid for an air–tissue interface for tissues with anisotropy in the range 0.7−0.9.
These are valid for an air–phantom interface for water-based tissue phantoms with anisotropy in the range 0.8–0.9.
Tissue phantoms were composed of 0.5 μM of Rhodamine 6G and varying amounts of hemoglobin (Hb/HbO2) and polystyrene microspheres in PBS.
Included in the last two columns are approximate values for the reduced scattering coefficient at 550 nm,
, and the absorption coefficient at 514 and 550 nm for each sample.
Table 3.
Sensitivity Analysis of the Calculated Intrinsic Fluorescence Coefficient, β, to Errors in Optical Parameters
Relative errors in one, two, or all four optical parameters were used to calculate errors in the one-dimensional correction factor, using the relationship in Eq. (13).
Path-length factor errors were used to calculate errors in the recovered value of the intrinsic fluorescence coefficient. For this set of error analyses, the true optical properties were Rd(λex) = 0.1, δ(λex) = 0.03 cm, Rd(λem) = 0.2, and δ(λem) = 0.05 cm.
Table 4.
Results of Least-Squares Estimation of Fluorophore Concentrationa
A least-squares regression was performed on intrinsic fluorescence coefficient data between 530 and 650 nm, with fluorophore concentration as the free parameter [Eq. (25)].
Each phantom contained 0.5 μM of Rhodamine 6G.