The applicability of Fourier transform infrared microspectroscopy for correction against matrix effects in X-ray fluorescence microimaging of tissues

Abstract

X-ray fluorescence (XRF) and Fourier transform infrared (FTIR) microscopy techniques are now considered popular for rapid and label-free complementary spectrochemical analysis of chemical elements and molecular systems in biological specimens. The morphological heterogeneity but also the inhomogeneities associated with the thickness/density of biological samples demonstrate challenges for the quantitative XRF microimaging. Therefore, in the present work, we proposed for the first time the application of the total absorbance under the FTIR spectra as a mass surface correction procedure for two-dimensional (2D) XRF microimaging of tissues. We also evaluated the equivalence of the developed correction method based on total absorbance of FTIR spectra with the proposed approaches based on incoherent scattering of primary X-rays as well as on the membrane Si-Kα transmission signal, on the example of selected rat organ tissues. Thin cryo-sections taken from various organs of Wistar rats were deposited on silicon nitride membranes (Si₃N₄). The FTIR microscopy studies were performed to collect infrared absorption spectra, used then for the determination of total absorbance values in the selected areas of tissue samples. In turn, hard X-ray imaging based on synchrotron radiation allowed the determination of characteristic radiation intensities of the elements detectable from the tissue, as well as the characteristic radiation of the membrane Si and incoherently scattered X-ray photons (Compton scattering). The latter served as correction factors for the surface mass of the sample alongside the FTIR total absorbance. The qualitative and quantitative analyses showed a high agreement between the results of elemental surface mass correction using total absorbance under FTIR spectra of tissues with those obtained using surface mass correction factors determined directly from XRF spectra. Therefore, the proposed procedure is a good alternative in cases where the surface mass effect of the sample cannot be eliminated based on the information provided directly by the XRF spectrum, as in the case of using polymer films as sample support. We have also proposed a procedure for synchronizing SRXRF and FTIR images, not limited to visual inspection of imaging/mapping data, but also enabling quantitative analysis. We found that the total absorbance determined from FTIR spectra can be successfully used as a correction factor for eliminating the surface mass effect in XRF microimaging of thin freeze-fried tissues and therefore to obtain the surface mass-independent elemental quantities. The proposed approach for 2D-FTIR-XRF analysis can also be a powerful and versatile tool for fostering a correlation and co-localization analysis to search for common distribution patterns between molecular arrangements and chemical elements.

Keywords: Fourier transform infrared microspectroscopy; Surface mass correction; Synchrotron radiation X-ray fluorescence; Tissues.