## 4 Discussion
Despite the polarity of the water molecule, those Raman peaks are primarily assigned to water [79]. Moreover, Kirsch et al. [79] recognized lower water content in WM compared to GM of normal brain. This caused Kochan et al. [46] to suggest that variation in tissue thickness during tissue drying could falsify the results. Due to higher water content, GM would become thinner than WM. As a consequence of this height difference, higher intensity values would be recorded within WM areas [46]. To exclude that this assumption applies to the presented results, the height profile of a tissue section containing the smaller ROI of the local scan was determined using the digital microscope VHX-7000 (Keyence Deutschland GmbH, Neu-Isenburg, Germany), see Figure A1 (Appendix A). The color map does not show a correlation between the height and the tissue type. Therefore, the determined reflectivity within the VIS and NIR spectral range is a characteristic of the tissue itself rather than its physical property. For the same reason, the chemical information based on Raman spectroscopy is rated as reliable. The color coding within the created Raman images (Figure 3(2c-d,4c-d)) confirms the observations of Mizuno et al. [33], Kast et al. [34], and Kochan et al. [46], who detected the higher intensity peak ratio and the sum of intensity peak ratios within the WM. The peak positions of the measured Raman peaks within WM and GM were in good accordance with the peak positions assigned in the literature (see Table 3). A significant shift of the Raman peak positions is observed for OCT. Below 1400 cm\({}^{-1}\), a shift to higher wavenumbers was determined, and vice versa, a shift to lower wavenumbers occurred above 1400 cm\({}^{-1}\). This is an indicator for the detection of the broad fluorescence background of the glass slide. Since this trend in shift was not recognized within the Raman spectra of GM and WM, it is assumed that the laser light is primarily scattered and absorbed by the optical properties of the tissue. However, a prolongation of the laser beam and a subsequent decrease in depth resolution due to the mismatch of refractive indices was studied by Everall et al. [80; 81; 82; 83]. The replacement of the dry objective by a water immersion objective for Raman measurements would be a possibility to increase the depth resolution and to reduce the background signal. Bonnier et al. [39] analyzed human skin by Raman spectroscopy at various excitation wavelengths with a water immersion objective. Besides the reduction of the background signal, Bonnier et al. observed a decrease in photodamage at lower laser wavelength due to thermal heat sink. Anyhow, a switch in immersion medium might result in an adaption of the Raman's excitation light path because of variation in the pupil's lens diameter for different objectives. Nevertheless, the discrimination of GM and WM based on the differences of the chemical compositions was possible with the presented Raman path. Especially the sum of two peak ratios in the high-wavenumber region led to good results for the local scan. The high values that were indicated by the yellow and reddish pixels can be explained by previous observations of Mizuno et al. [33]. Already in 1992, he reported an increase in the peaks at 2852 cm\({}^{-1}\) and 2885 cm\({}^{-1}\) in the WM and myelin fraction, whereas the peak at 2938 cm\({}^{-1}\) was high in all brain tissues. In 2018, Ji et al. [84] identified the CH stretch region as an indicator to visualize the myelinated axons. Especially the peak around 2850 cm\({}^{-1}\), which is assigned to the CH\({}_{2}\) stretch modes of lipids. Since the location of the yellow pixels within the WM of the local scan were in accordance with the blue pigmentation in the polarization image with the activated WWC, the blue color suggests a higher density of myelinated axons. Lower densities, which are primarily present in the internal capsule, were indicated in orange. The polarization microscopy without the WWC did not show a color difference of the various structure within WM.