## 4 Discussion  
The scanning results show that different information content is provided based on the modality. Brightfield and darkfield images are complementary techniques to visualize the morphological structure of the unstained tissue. The image obtained from the evaluation of the local scan's reflectance spectra in the VIS spectral region can be visually localized within the brightfield image of the local scan. However, the spatial resolution of the microscopic \begin{table}
\begin{tabular}{c c c c} \hline
**Peak Position/cm\({}^{-1}\)** & **Lipid Assignments** & **Protein Assignments** & **References** \\ \hline
1001–1008 & & C-C ring breathing & [34; 46; 72] \\  & & of phenylalanine & \\
1296–1308 & CH\({}_{2}\) twisting and & & [35; 55; 73] \\  & wagging & & \\
1337–1344 & & C-H deformation, & [35; 73] \\  & & CH\({}_{2}\) bending and & CH\({}_{2}\) bending and \\
1438–1452 & scissoring & scissoring & [34; 72; 74] \\  & CH\({}_{3}\) bending & CH\({}_{3}\) bending & \\
1659–1664 & C=C stretching & Amide I (C=O stretching) & [33; 46; 46] \\
2850–2860 & CH\({}_{2}\) symmetric & CH\({}_{2}\) symmetric & [46; 72; 73] \\  & stretching & stretching & \\
2880–2895 & CH\({}_{2}\) asymmetric & CH\({}_{2}\) asymmetric & [46; 72; 73] \\  & stretching & stretching & [46; 72; 73] \\
2929–2937 & CH\({}_{3}\) stretching & CH\({}_{3}\) stretching & [46; 72; 73] \\ \hline \end{tabular}
\end{table} 
Table 3: Raman peak position and assignment of functional groups to distinguish GM and WM. image is higher than the spatial resolution of the spectroscopic-based image. While the step size for spectra acquisition of the local scan is 5 \(\upmu\)m, the pixel size of a microscopic photograph using the 20\(\times\) magnification is 0.17 \(\upmu\)m. In contrast, common color cameras are based on three color channels (red, green, blue), whereas spectrometer record hyperspectral data where each intensity value is assigned to a wavelength. The calculated VIS reflectance spectra did not show absorption bands other than the absorption of the gold coating. The main absorber in the tissue below 600 nm is hemoglobin. Since neither the camera detected a reddish area nor the spectra showed the hemoglobin-specific reflectivity drops between 500 and 600 nm, it can be assumed that hemoglobin was no longer present in the tissue section due to a successful perfusion of the animal. Another indicator that confirms the quality of sample preparation was the absence of a Raman peak around 1585 cm\({}^{-1}\) that could be assigned to hemorrhage [34]. Similar to the reflectance spectra in the VIS wavelength range, NIR reflectance spectra did not contain molecular-specific information. Confocal imaging of deep mouse brain with long-wavelength (NIR) illumination was demonstrated by Xia et al. [77]. The researchers took advantage of the tissue's lower light absorption and scattering in the optical window around 1700 nm. An excitation light source in this NIR region combined with reflectance confocal microscopy enabled deep penetration into tissue with high spatial resolution. The results were images at various depths based on reflectivity in the NIR region. A prominent absorber in the NIR region is water with absorption bands at 1450 nm and 1900 nm [78]. Weak NIR absorption bands within tissue originate from biomolecules such as lipids, collagen, and proteins. Because of the biomolecules, poor light absorption in the NIR spectral range a sample thickness of 16 \(\upmu\)m was not sufficient to detect molecule-specific information in the NIR range. Additionally, absorption bands of these biomolecules are usually overlapped by the strong absorption properties of water [78]. A statement regarding the air-dried tissue's water content based on other modalities of the imaging system is not possible. The detection range of the Raman spectrometer does not cover the high-wavenumber range between 3200 cm\({}^{-1}\) and 3500 cm\({}^{-1}\). Otherwise, the detection of the OH bonds' vibrations near 3245 cm\({}^{-1}\) and 3425 cm\({}^{-1}\) would be possible.