## 3 Results and Discussion
### SERS Spectral Difference Among Various Encephialic Regions  
To investigate the differences in chemical composition in different encephalic regions, more than 20 SERS spectral lines were acquired from each compartment. Figure 6a showed the mean SERS spectra of four different encephalic regions (cereb cortex (CTX), hypothalamus (HYP), thalamus (TH) and hippocampus (Hi)) in the left and right hemispheres. It could be seen that the enhanced Raman signals of brain tissues were evidently varied among the major brain structures. The molecular components in CTX and TH demonstrated the most enhanced SERS signals, which might be ascribed to the higher neurocyte density in these encephalic regions, as the perineuronal nets were intensely positively charged that could closely be bonded with the negatively charged SERS substrate [33]. The Raman signals of brain tissues in HYP experienced a weak increase that could be ascribed to the fewer cells in HYP and weak affinity of the biochemical components and BP-AuNSs. To compare the biochemical deviations between the left and right hemispheres, the normalized SERS spectra as well as the difference spectrum of left versus right hemisphere were obtained (Figure 6b). The mean SERS spectra of brain tissues in left and right hemispheres illustrated the basically consistent pattern, indicating that the distribution of biochemical components were relatively uniform in left and right hemispheres. However, a certain degree of diversity in the content of biochemical molecules existed, as a fluctuation was observed in the SERS difference spectral line. The major Raman bands in the difference spectrum and their biochemical assignments were listed in Table 1[6; 34; 35; 36], where a differential distribution of the main biomacromolecules (protein, nucleic acid,  
Figure 5: Accurate SERS texture analysis of brain tissue. **(a)** Brightfield image; **(b)** SERS image induced by BP-AuNSs using the empty modelling approach. **(c)** Hematoxylin and eosin (H&E) staining of brain tissue section. Enlarged images showed the hippocampus (DG, dentate gyrus; CA, Ammon’s horn), medial habenular nucleus (MHb) and corpus callosum (cc) regions. The asterisk (*) showed the loss and displacement of tissue during section preparation process. Scale bar: 2 mm. lipid and carbohydrates) were observed between left hemisphere and right hemisphere. The dominant bands in right hemisphere were located at 1050-1300 cm\({}^{-1}\), which represented the amide III, C-N stretching in protein and DNA/RNA, thymine and guanine, respectively. More molecular information was excavated in the left hemisphere in comparison with that in right hemisphere, especially in the Raman shift ranged from 1300 to 1650 cm\({}^{-1}\). The Raman signals of protein, DNA/RNA and lipid were more plentiful in left hemisphere, which might reflect the inequitable division of labor between the two hemispheres. \begin{table}
\begin{tabular}{c c} \hline \hline
**Raman Shift (cm\({}^{-1}\))** & **Tentative Assignment** \\ \hline
645 L & Tyr, COO- bend or C-S str (p) \\
730 L & A (d) \\
747 L & T (d); Trp ring breath (p) \\
860 R & Ribose: C-C str, ring breath, C-O-C str (c) \\
964 L & C-C bk str (p) \\
1025 L & C-H in-plane Phe (p); glycogen \\
1109 R & C-N str (p) \\
1176 R & T, G, C-N str (d) \\
1230 R & Amide III (p) \\
1343 L & CH\({}_{2}\) def (p); A,G (d) \\
1410 L & COO- sym str (p) \\
1445–1470 L & CH\({}_{2}\)/CH\({}_{3}\) def (p, l) \\
1508–1530 L & A, C, G (d) \\
1546 L & Amide II, Trp (p) \\
1590 L & Amide II, Phe, Tyr (p); G, A (d) \\
1611 L & Tyr, The C=C bend (p) \\ \hline \hline \end{tabular} Abbreviations: L, left hemisphere; R, right hemisphere; p, protein; l, lipid; d, DNA/RNA; c, carbohydrates; Tyr, tyrosine; Trp, Tryptophan; Phe, phenylalanine; A, adenine; T, thymine; C, cytosine; G, guanine; bend, bending; str, stretching; def, deformation; breath, breathing; sym, symmetrical; bk, backbone.  
\end{table} 
Table 1: The assignments of the Raman bands in SERS difference spectrum of left versus right hemisphere.