# Assessment of Skin Deep Layer Biochemical Profile Using Spatially Offset Raman Spectroscopy
## 3 Results
### Human Skin Tissue  
#### 3.2.1 Reference Spectra  
Contrary to the simple two-layer system used in phantom measurements, skin tissue comprises of several heterogeneous layers which lack distinct biochemical Raman signals. This was observed in literature before [38], but also shown in our reference spectra acquired from dissected tissues using confocal Raman spectroscopy (Figure 4). As shown in Figure 4,  
Figure 3: (i) Representative Raman signal of top and bottom layer with offset for Phantoms type A and B (Panel **A(i)** and **B(i)** respectively). (ii) SNR versus offset for Phantoms type A and SORS and SNR versus offset for Phantoms type B (Panel **A(ii)** and **B(ii)** respectively). SORS ratio values were normalized to the respective zero offset value for convenience. rather than exhibiting unique Raman features, the three main skin layers (epidermis, dermis and subcutaneous tissue) inherently share common bands but in different ratios. In Figure 4 we observe that Raman bands of epidermis and dermis exhibit a great degree of similarity. Given the age of the donors in this study (70 and 62 years old), this observation is in agreement with previous studies [39] where the two skin layers in older donors show very few spectral differences. The main spectral features present in the layers of epidermis and dermis are Raman bands at 852, 872 and 939 cm\({}^{-1}\) assigned to the amino acid side chain vibrations of proline and hydroxyproline (collagen type I) as well as to a C-C stretching vibration of the collagen backbone [40]. As collagen is the main constituent of the human dermis layer (70% of the dry weight and 90% of the total protein content) [41], we notice that those spectral features are more prominent there compared to the epidermis layer, which is in line with the literature [40]. Other bands observed are the ones associated with vibrations of amide bonds in polypeptide chains: Amide I at 1656-1668 cm\({}^{-1}\) (predominantly C=O stretching), amide II at 1554 cm\({}^{-1}\) (\(\
u\)(C-N) and \(\delta\)(N-H)) and amide III at 1242-1268 cm\({}^{-1}\) (C-N stretching and N-H bending vibrations) [39]. Amide I and III consist of underlying bands whose strength is related to  
Figure 4: Typical Raman spectra of separate skin layers of (**a**) healthy human eyelid and (**b**) healthy human scalp. All spectra are baseline corrected and normalized to phenylalanine content, to help visualize changes between skin layers by enabling spectral comparison. Raman band assignments can be found in Table S1. Dashed boxes highlight areas of multiple bands assigned to vibrations of similar biomolecules. the secondary structure of the proteins. In those bands, maximums at 1242 and 1656 cm\({}^{-1}\), respectively, reveal the predominance of \(\alpha\)-helix structure, whereas maximums at 1268 and 1668 cm\({}^{-1}\) are related to an unordered \(\beta\)-sheet structure [42; 43]. Although previous studies agree on the predominance of the \(\alpha\)-helix conformation in the epidermis [44], our study shows the epidermis to exhibit a slightly shifted to the right amide I band. This may relate to an underlying lipid band at 1671 cm\({}^{-1}\) assigned to ceramides in the stratum corneum [45]. Other Raman bands include CH\({}_{2}\) deformation (1450 cm\({}^{-1}\)) assigned to both protein and lipid content and CH in-plane bending and stretching (1031 cm\({}^{-1}\) and 1170 cm\({}^{-1}\), respectively) assigned to phenylalanine [40; 43]. Both of those bands are similar in morphology and height between dermis and epidermis. A closer look at the lower wavelength area, reveals vibrations of phenylalanine (620 cm\({}^{-1}\)) and cysteine (642 cm\({}^{-1}\)), with the latter to be stronger in the case of epidermis as cysteine is a major amino acid of keratin fibers [46]. This is also confirmed by the stronger epidermis band at around 700 cm\({}^{-1}\) where a C-S cysteine vibration also manifests [43].