## 4 Discussion  
Although the experimental parameters and data processing differed between 1064 nm and 785 nm Raman instruments, the main spectral findings were consistent, as shown by the summarized assignments in Table 1. Lipid and control tissues were detected in non-tumor specimen 5, and tumor was detected in tumor specimen 3, both in this work using the 1064 nm Raman probe system and in the previous work using a 785 nm Raman microscope [17]. Overall, the results from both instruments agree better for non-tumor specimens, according to Table 1, whereas discrepancies are more prevalent in tumor  
Figure 4: Centroid Raman spectra of tumor-assigned clusters in tumor specimens (**A**) and control-assigned clusters in control specimens (**B**). Figure 3: Dendrogram of hierarchical cluster analysis with k-means cluster centroids of Raman spectra as input: tumor (red), lipid (orange), necrosis (green), and control (purple) in the spectral region 800-1150 cm\({}^{-1}\). The samples are labeled as non-tumor (N) and tumor (T) according to Table 1.  
specimens. A difference in non-tumor 3 is the detection of necrosis at 1064 nm excitation and tumor at 785 nm. Differences in tumor 5 are related to the detection of necrosis at 1064 nm excitation and tumor, lipid, and necrosis at 785 nm, and differences in tumor 7 are the detection of control at 1064 nm and tumor at 785 nm. The discrepancies may originate from variations in sample orientations and heterogeneities, which occurred when the sample was returned to the vial after 785 nm experiments, frozen, thawed, and remounted again into the holder for the 1064 nm experiments. During intraoperative frozen section analysis, ink spots are commonly employed to mark the sample's orientation within the resection area, minimizing ambiguities. However, the spectral contributions of the ink may interfere with the Raman results. As a main difference between results from 1064 nm and 785 nm instruments, more tissue types are identified at 785 nm excitation. For simplicity, in Table 1, epithelial tissue, plastic, and pigment particles were not included, and they were detected at 785 nm excitation but not at 1064 nm excitation. Possible explanations are the better spectral resolution of 4 cm\({}^{-1}\) at 785 nm (instead of 10 cm\({}^{-1}\) here), better lateral resolution at a step size of 250 um (instead of 400 to 500 um here), and the vertex component analysis (VCA) algorithm. The superior discriminatory power of VCA was already demonstrated to visualize individual cell nuclei in Raman images of lung tissue [27] and 3D cell cultures [28]. KMC that was applied here failed to resolve cell nuclei in the same data sets. Another factor for the reduced discrimination ability could be the ca. twofold deeper penetration of 1064 nm light than 785 nm light that was recently quantitatively assessed [29]. Whereas deeper light penetration is beneficial for higher Raman intensities, signal detection from deeper tissue layers may result in \"spectral dilution\". This term was introduced in the context of infrared imaging, where lower-resolved images failed to resolve spectral features [30]. Here, the deeper penetration of 1064 nm light tends to probe more lipids in control tissue, as evident from more intense lipid bands near 1076 and 1452 cm\({}^{-1}\) (Figure 2E), and vice versa, more proteins in lipid-rich tissue, as evident from more intense collagen bands near 860, 928, 1269 in amide III region, and 1655 cm\({}^{-1}\) in amide I region (Figure 2G). The dendrograms of the HCA show for 1064 and 785 nm data that tumor forms a separate group and lipid-rich tissues are well separated from necrosis, tumor, and control. Necrosis was arranged between the tumor and lipids here, whereas necrosis overlaps with the control at 785 nm excitation. This can be explained by the selected spectral range from 800 to 1150 cm\({}^{-1}\) here and from 800 to 1380 cm\({}^{-1}\) in reference [17].