Deconvolution of multicomponent fluorescence spectra in the presence of absorption inner-filter effects

Abstract

Spectral correction factors required for correction of multicomponent fluorescence spectra are developed for a right-angle cuvette geometry and an open-ended fiber-optic probe geometry derived from the right-angle geometry by use of fiber-optic waveguides. The correction factors are simple in form as well as usage, which consists of multiplying each observed fluorescence intensity by the appropriate correction factor before deconvolution. The spectral correction factors correct for the masking of fluorescence intensities by absorption inner-filter effects, which lead to nonlinearities in fluorescence signals. Thus, after correction, any quantitative or qualitative mathematical technique based on the linear-combination assumption may be used for deconvolution. The correction factors were developed from approximate light-intensity models which account for excitation and emission absorption and the physical parameters of the measurement geometries. Hence, spectral correction requires two procedures which are not standard practice in fluorescence spectroscopy, namely (i) measurement of excitation and emission absorbance and (ii) characterization of the physical parameters. For the latter, model calibration methods are presented for each measurement geometry. Finally, a method for the open-ended geometry, accounting for the open-ended geometry, accounting for the transmission properties of the fiber-optic probe, is presented for deconvolution by multiple linear regression using a reference database of single-component fluorescence spectra acquired with the right-angle cuvette geometry.