## 2 Materials and Methods
### Data Acquisition  
The proof-of-concept consisted of two measurement sets (global and local) for tissue analysis, while one set of measurement comprised sequential tissue scans with each modality. These modalities, as already mentioned in Section 2.2, were subdivided into incident light modes (Figure 1, red and blue light path) to measure the unstained sample and the transmitted light mode (Figure 1, green light path) to record brightfield photographs of the H&E-stained sample (gold standard). The measurements were started with the global scans in order to record the whole sample. Afterwards, local scans were carried out. The purpose of the local scans was to map ROI with smaller step sizes by achieving a higher spatial resolution of the hyperspectral data. The ROI was one tissue section with a boundary of GM (cortex) and WM (corpus callosum). First of all, the frozen and unstained tissue section was left to thaw at room temperature. Measurements were also carried out at room temperature. No immersion medium between the sample surface and the microscope objectives was used. The microscope stage, the camera, as well as the spectrometers, were triggered by an in-house developed LabVIEW program (National InstrumentsTM) during the scanning procedure. In the present case, the sample on the microscope stage was scanned along the spatial axes (x- and y-axes) while either photographs or spectra were recorded at each measurement point. This methodology is called point mapping. Consequently, three-dimensional (x, y, z) data sets were acquired where x and y were the image coordinate and z was the spectrum or the photograph, respectively. The step size between two neighboring measurement points depended on the modality. The photographs were consistently taken with an overlap of approximately 20%. This resulted in a step size of 720 m in the x-direction and 540 m in y-direction for the 5\(\times\) objective. With the 20\(\times\) objective, samples were scanned in intervals of 180 m along the x-axis and 135 m along the y-axis. Raman spectra as well as VIS/NIR reflectance spectra were recorded pointwise with step sizes of either 200 m (global scans) or 5 m (local scans). Prior to one set of measurement, a white balance of the camera was carried out in the incident light brightfield mode using the 5\(\times\) objective and a Zenith(r) Diffuse Reflectance Standard (Reflectivity \(\approx\) 99%, SG 3052, SphereOptics GmbH, Herrscribing, Germany) as a reference. Therefore, the voltage value of the halogen lamp was set to 5 V and the camera's integration time was 100 ms. Subsequently, data acquisition of the unstained tissue section was started with the incident light brightfield mode using the same operating voltage and magnification but at a shorter integration time of 10 ms. Those parameters were identical to the parameters of the following brightfield scan using the 20\(\times\) objective. The workflow of the data acquisition for one set of measurement, which includes several scanning runs, is shown in Table 2.  
Due to a change in the illumination technique for darkfield microscopy and the weak signal of tissue for polarization microscopy, the light intensity was increased by maximizing the lamp's voltage value (12 V) for the mentioned modalities. The integration time was adjusted so that neither an underexposure nor an overexposure of the photographs was recognized. The exact values are shown in Table 2.  
Afterwards, Raman map measurements were performed. The laser power was set to 100% resulting in 57 mW at the sample surface. Each spectrum was recorded for 4000 ms at a single accumulation. In advance, a dark signal measurement without laser light was executed to detect photon counts caused by the detector. This offset signal is a function of temperature and exposure time [58]. It was removed from each Raman spectrum in order to achieve raw spectra.