## 3 Results  
The formalin-fixed paraffin-embedded brain tissue samples (serial sections of 10 \(\upmu\)m thickness), were collected from each rat for the FTIR, LA-ICP-MS, and the H&E analyses. The numerical comparisons of the lipid/protein ratio and the ratios of some specific lipid spectral of the regions of interest of this study, are listed in Table 1. Figure 1A shows a rostral section (Bregma 1.56), highlighting three other regions of interest--SSMC, CC, and CP; the corresponding FTIR image is shown in Figure 1B. Figure 1C-I shows the images of the biochemical components--Amide I, Amide II, \(\
u_{\text{as}}\)(CH\({}_{3}\)) asymmetric, \(\
u_{\text{s}}\)(CH\({}_{2}\)) symmetric, \(\
u_{\text{as}}\)(CH\({}_{2}\)) asymmetric stretching, olefin, and ester, respectively. These images were colored, according to the calculated biochemical content values, where red color corresponded to the highest bio-content and the blue color corresponded to the lowest bio-content, as shown on the color bars in the figure. The FTIR spectra of the SSMS, CC, and CP are shown in Figure 2A-C, respectively. Figure 2A represents the SSMC average FTIR spectrum. The spectrum revealed the ester absorption band that arose at \(\sim\)1730 cm\({}^{-1}\) from the \(\
u\)(C=O) ester (carbonyl) group, had weak absorption. The FTIR spectrum revealed that a robust olefinic=CH band arose at about 3012 cm\({}^{-1}\). This was interpreted as that the SSMC structure contains lipids with a high degree of unsaturation. The protein absorption bands, such as amide II, amide I, and the N-H bands, arose at 1545, 1653, and 3290 cm\({}^{-1}\), respectively. The amide I and amide II have strong absorption. The spectral data collected from the CC, in Figure 2B, revealed a strong peak which ascended from the \(\
u\)(C=O) at 1470 cm\({}^{-1}\), which was attributed to the phospholipid. The FTIR spectrum revealed that the olefinic=CH band was weaker in the CC, than in the SSMC. Whereas, the FTIR spectrum identified that the band at 1464 cm\({}^{-1}\), which arose from the CH\({}_{2}\) bending vibration, was robust. The bands at 2852 and 2955 cm\({}^{-1}\) that arose from the \(\
u_{\text{s}}\)(CH\({}_{2}\)) and \(\
u_{\text{as}}\)(CH\({}_{2}\)), showed stronger absorption than  
Figure 2: **(A–C)** Representative FTIR spectra of the SSMC, CC, and CP. **(D)** H&E images of the WM and the GM, and **(E)** FTIR image of the same region with a high-resolution, based on the lipid/protein ratio. **(F)** FTIR spectra of the WM and the GM. The color bar represents a high-concentration, in red, and a low-concentration, in blue. the SSMC spectrum. The band at 2955 cm\({}^{-1}\) which arose from the \(\
u_{\rm as}\)(CH\({}_{3}\)) of the lipids fatty acids, showed weaker absorption, in comparison to the SSMC spectrum. Figure 2C is a representative FTIR spectrum of the CP. Here, the spectral band of the carbonyl group \(\
u\)(C=O) of the ester component, had a low-absorption intensity, due to the low esterified-phospholipid content. The \(\
u_{\rm s}\)(CH\({}_{2}\)) and \(\
u_{\rm as}\)(CH\({}_{2}\)) spectral bands showed a weaker absorption, in comparison to the CC spectrum. The FTIR spectrum at the CP region exhibited strong olefinic=CH band, in comparison to the CC spectrum; indicating that the CP was rich in unsaturated lipids The FTIR spectrum was dominated by amide I and amide II spectral bands. Figure 2D is a representative H&E image of the WM and GM, with the corresponding FTIR image obtained with a higher resolution, based on the lipid/protein ratio presented in Figure 2E. The data revealed here, that the white matter showed a higher intensity in the lipid/protein ratio signals, which correlated well with the presence of the myelin sheaths around the nerve axons located in this region. The spectra collected from these regions were dominated by the protein, lipid, and the ester bands (Figure 2F). The WM spectrum had two strong absorption bands, at 2850 and 2920 cm\({}^{-1}\), compared to the spectrum collected from the gray matter.