Fluorescence spectroscopy of tissue: recovery of intrinsic fluorescence from measured fluorescence

Abstract

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.

Diagnosis of breast cancer using fluorescence and diffuse reflectance spectroscopy: a Monte-Carlo-model-based approach

“…For fluorescence, a number of studies [19][20][21][22][23][24][25] have been carried out to disentangle the effects of absorption and scattering from the measured fluorescence spectrum to recover the intrinsic fluorescence of the tissue. The intrinsic fluorescence is due only to fluorophores present in the medium, from which fluorophore concentrations can be extracted using simple analytical models.…”

“…A majority of these studies used analytical methods to derive mathematical expressions for the intrinsic fluorescence, from which the intrinsic fluorescence spectrum can be calculated. [20][21][22]24,25 These methods are based on the diffusion equation and thus are valid for a limited range of absorption and scattering, not flexible in their applicability to various probe geometries or require extensive empirical calibration. Biswal et al 23 proposed a method that was based on simultaneous measurements of polarized fluorescence and polarized elastic scattering spectra, and turbidity-free fluorescence was obtained by normalizing the polarized fluorescence spectrum by the polarized elastic scattering spectrum.…”

Fluorescence measurements for evaluating the application of multivariate analysis techniques to optically thick environments.

“…Accounting for the effect of scattering to obtain "scattering-free" fluorescence spectra has been of interest to the research community for some time [27][28][29][30][31][32][33][34][35]. Light scattering could be incorporated into our experiment by adding a scattering matrix to the cuvette-contained samples.…”

A review of attenuation correction techniques for tissue fluorescence

“…An attenuation correction technique has been subsequently presented (Gardner et al 1996a) which utilizes the equations to calculate the fluorescence transfer function. The technique is based on measurements of diffuse reflectance at excitation and emission wavelengths, a prerequisite being that the effective penetration depths at all wavelengths either need to be known or estimated (since they may prove difficult to measure in practice).…”