## 2 Materials and Methods
### Fourier-Transform Infrared Imaging Measurements and Data Analysis  
FTIRI measurements were performed using a Bruker INVENIO-R interferometer coupled with a Hyperion 3000 Vis-IR microscope and equipped with a Focal Plane Array (FPA) detector operating at liquid nitrogen temperature (Bruker Optics, Ettlingen, Germany). First, to identify the areas of interest, the microphotograph of each section was acquired using a television camera. Then, on each section, IR maps were acquired in transmission mode with a 15\(\times\) condenser/objective. Each IR image was 164 \(\times\) 164 \(\upmu\)m size and was composed of 4096 pixel/spectra; the spatial resolution of each pixel was 2.56 \(\times\) 2.56 \(\upmu\)m. IR maps were acquired in the 4000-900 cm\({}^{-1}\) spectral range, with a spectral resolution of 4 cm\({}^{-1}\). Each pixel/spectrum was the result of 256 scans. Before starting acquisition of each sample, the background spectrum was acquired with the same parameters on a clean portion of the CaF\({}_{2}\) optical window. All IR maps were opportunely pre-processed (OPUS 7.5 software package, Bruker Optics, Ettlingen, Germany); more in detail, they were submitted to the Atmospheric Compensation routine, to avoid carbon dioxide and water vapor atmospheric contributions, and then to Vector Normalization routine in the 4000-900 cm\({}^{-1}\) spectral range, to correct differences in sample quantity; in fact, it is known that the intensity of the bands in absorption spectra is proportional not only to the concentration of the sample but also to the optical length of the light within it. To pinpoint the topographical distribution of meaningful macromolecules, false color images were generated by integrating pre-processed IR maps under the following regions of interest (ROI): 3030-3000 cm\({}^{-1}\) (stretching vibrations of =CH groups in unsaturated lipid alkyl chains; UNSAT); 3000-2800 cm\({}^{-1}\) (stretching vibrations of CH\({}_{2}\) and CH\({}_{3}\) groups in saturated lipid alkyl chains; SAT), and 1700-1480 cm\({}^{-1}\) (Amide I and II bands, representative of the vibrational modes of the peptide linkage in proteins; PRT). Different scales were adopted due to different IR absorbances displayed by the investigated macromolecules: black/blue colors indicated areas with the lowest absorbance values, while pink/white colors indicated areas with the highest ones (OPUS 7.5 software package, Bruker Optics, Ettlingen, Germany). For each experimental group, 500 spectra were randomly extracted from each IR map and used for both multivariate and univariate analyses. Firstly, extracted IR spectra were converted in second derivative mode (Savitzky-Golay filter, 9 points of smoothing) and submitted to principal component analysis in the 3030-900 cm\({}^{-1}\) spectral range with no further preprocessing. PCA scores plots, and corresponding PC loadings were obtained (Origin PRO 2018 software). Then, the above-described extracted spectra were integrated under the following regions of interest: 3030-2800 cm\({}^{-1}\) (representative of saturated and unsaturated alkyl chains in lipids; LIP), 1780-1700 cm\({}^{-1}\) (representative of fatty acids; FA), and 1700-1480 cm\({}^{-1}\) (representative of proteins; PRT) [39; 40]. Finally, for each experimental group, the average spectrum (centroids) and the average spectrum \(\pm\) standard deviation (S.D.) spectra were also calculated (Averaging routine, OPUS 7.5), and submitted to a peak-fitting procedure in the 3030-2800 cm\({}^{-1}\) and 1700-1480 cm\({}^{-1}\) spectral regions, containing the vibrational modes of lipids and proteins, respectively. The number and position of all the underlying bands were evaluated using second derivative minima analysis and fixed during fitting procedure with Gaussian functions (GRAMS/AI 9.1, Galactic Industries, Inc., Salem, NH, USA). For each underlying peak, the exact position (expressed as wavenumbers) and the integrated area were obtained. Peaks assignment was performed according to the scientific literature.