COMPARISON OF METHODS TO DETERMINE CHROMOPHORE CONCENTRATIONS FROM FLUORESCENCE-SPECTRA OF TURBID SAMPLES

Background and Objective: This paper compares the ability of three ana lytic techniques to predict chromophore concentration from fluorescenc e emission spectra of homogenous, turbid samples with optical properti es similar to human tissue. Study Design, Materials and Methods: Two m odels of light propagation were implemented (exponential attenuation, two flux Kubelka-Munk theory); a priori information about sample optic al properties was used to analyze data with the two flux Kubelka-Munk model. The third data analysis technique utilizes the method of partia l least squares (PLS) to develop an empirical, Linear model of sample fluorescence from a training set with optical properties and known con centrations representative of those to be predicted. This model can be applied to predict chromophore concentrations in the unknown samples. Results: Of the three methods, PLS achieved the most accurate results and was able to predict fluorophore concentration to within +/- 6% of true values. Conclusion: We investigated conditions under which PLS p redictions were most accurate and find that best results are achieved when predictions are based on fluorescence emission spectra at more th an one excitation wavelength with inclusion of the tail of Rayleigh sc attering at the excitation wavelength. (C) 1996 Wiley-Liss, Inc.