Correction of the Internal Absorption Effect in Fluorescence Emission and Excitation Spectra from Absorbing and Highly Scattering Media: Theory and Experiment

Abstract

Fluorescence spectra measured from biological samples, such as tissues or cell suspensions, are usually distorted due to the light absorption by intrinsic chromophores. These distortions are aggravated by strong scattering of light inside the samples. A new method is described for a fast correction of these spectral distortions, using only steady-state spectroscopic measurements. The method is based on the formulas derived from a simplified photon diffusion model, in the isotropic one-dimensional approximation applied to a semi-infinite, highly scattering, and moderately absorbing medium with a refractive-index-matched boundary. The formulas describe the spectral distortions of the fluorescence emission and excitation spectra, together with the diffuse reflectance spectrum, as the functions of one spectral characteristic of the medium, the darkness, which is the ratio of absorption coefficient and reduced scattering coefficient. The algorithm does not involve any iterative procedures, and offers a direct, simple, and fast method for real-time spectral correction. The true fluorescence emission or excitation spectrum is directly calculated from a pair of experimental spectra: the fluorescence emission or excitation spectrum and the diffuse reflectance spectrum, measured from the same position on a sample. The correction produces the profile of the true fluorescence spectrum, the same as the one measured from the corresponding sample with an infinitely low absorption and no scattering. The restoration of the spectral profiles of true fluorescence emission and excitation spectra was tested experimentally, using highly scattering phantoms with a fluorescent dye and a deliberately added nonfluorescent dye producing strong inner-filter distortions. © 1998 Society of Photo-Optical Instrumentation Engineers.

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

“…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.…”

“…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.…”

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.…”

Noninvasive Glucose Sensing with Raman Spectroscopy

“…[54][55][56][57][58] In fluorescence spectroscopy, however, diffuse reflectance correction of spectral distortions in biological media has been studied extensively. Analytical models based on photon migration theory 43 , diffusion theory 45,59,60 , as well as empirical models 61 , have been reported to obtain "intrinsic fluorescence." In the following, we will review a particular correction method based on photon migration theory for fluorescence spectroscopy, and introduce its Raman counterpart.…”