Resolution of mixtures of fluorophores in biological media using fluorescence spectroscopy and Monte Carlo simulation
Résumé
In excitation–emission fluorescence spectroscopy, the simultaneous
quantitative prediction and qualitative resolution of mixtures
of fluorophores using chemometrics is a major challenge because
of the scattering and reabsorption effects (turbidity) presented
mainly in biomaterials. The measured fluorescence spectra are
distorted by multiple scattering and reabsorption events in the
surrounding medium, thereby diminishing the performance of the
commonly used three-way resolution methods such as parallel
factor (PARAFAC) analysis or multivariate curve resolutionalternating
least squares (MCR-ALS). In this work we show that
spectral loadings and concentration profiles from model mixtures
provided using PARAFAC and MCR-ALS are severely distorted by
reabsorption and scattering phenomena, although both models fit
rather well the experimental data in terms of percentage of the
explained variance. The method to correct the fluorescence
excitation–emission matrix (EEM) consisted in measuring the
optical properties (absorption parameter la , scattering parameter
ls, and anisotropy factor g) of samples and calculating the
corresponding transfer function by means of the Monte Carlo
simulation method. By applying this transfer function to the
measured EEM, it was possible to compensate for reabsorption
and scattering effects and to restore the ideal EEM, i.e., the EEM
that is due only to fluorophores, without distortions from the
absorbers and scatterers that are present. The PARAFAC and MCRALS
decomposition of the resulting ideal EEMs provided spectral
loadings and concentration profiles that matched the true profiles.
Domaines
Sciences du Vivant [q-bio]
Origine : Fichiers produits par l'(les) auteur(s)