Influence of the reference spectrum used as input to the RMieS-EMSC algorithm for correction of spectral distortions induced by Mie scattering in FTIR hyperspectral images

Abstract

Fourier transform infrared (FTIR) microspectroscopy has shown great promise as a tool to assess the histological architecture of tissue samples by providing morphochemical maps that enable the evaluation of both spatial and compositional information based on the signatures of molecular vibrations. However, FTIR datasets collected in transmission and transflection modes are subjected to undesired spectral contributions non-related to the phenomenon of light absorption that must be corrected prior any analysis. The algorithm so-called “Resonant Mie Scattering – Extended Multiplicative Scatter Correction (RMieS-EMSC)” has been well succeeded in vibrational spectroscopy in removing spectral distortions. The main idea of the algorithm is to reconstruct the datasets that present undesired spectral contributions based on a scatter-free reference spectrum. In this study, FTIR hyperspectral images acquired from cutaneous malignant lesions were submitted to RMieS-EMSC protocol using Matrigel and average spectrum in order to evaluate the influence of the reference spectrum in the final correction. False-color maps obtained after segmentation were compared in terms of image quality and consistency with standard histopathology in order to evaluate the ability of each method to reproduce the histological structures of the specimen. Our findings indicate that the choice of reference spectrum has very little difference on the outcome of image analysis. The images obtained by both spectra used as reference may not have resulted in identical false-color maps, but the differences between the morphochemical maps are relatively small.