# Assessment of Skin Deep Layer Biochemical Profile Using Spatially Offset Raman Spectroscopy
## 3 Results
### Human Skin Tissue
#### 3.2.2 SORS Measurements  
The reference spectra acquired from the individual skin layers provided the basis for the analysis of SORS measurements on samples from the scalp (thickest epidermis) and eyelid (thinnest epidermis) prior to dissection. Figure 5 shows the general spectral patterns for a representative healthy eyelid as measurement offset increases. The spatial offset range employed in SORS measurements of human skin was limited between 0-2 mm, as values close to zero were shown to exhibit the highest SNR in tissue phantoms of similar optical properties and geometry. In Figure 5 we can see that as the offset increases the spectra gain features of the subcutaneous fat. Although it is difficult to discern changing trends in the transition from dermis to epidermis, we notice that collagen spectral features such as the most obvious 872 cm\({}^{-1}\) band (hydroxyproline), start showing up at around 100 \(\upmu\)m spatial offset which corresponds to less than 100 \(\upmu\)m of probing depth, as suggested by previous studies [48]. This is in line with the dermis depth in the human eyelid where the thinnest epidermis (0.05 mm thickness) on the body can be found. Another characteristic band which is more dominant in spectra up to 100 \(\upmu\)m is the vibration at 1242 cm\({}^{-1}\), corresponding to the \(\alpha\)-helix secondary structure of keratin. This band starts diminishing after 120 \(\upmu\)m of offset and is eventually replaced by the 1258 cm\({}^{-1}\) lipid band in fat tissue. Raman spectra recorded with a spatial offset larger than 160-180 \(\upmu\)m show increasing evidence of subcutaneous fat signal contribution with vibrations at 972, 1063, 1083, 1123 and 1746 cm\({}^{-1}\)which are characteristic to lipids vibration modes (Table S1) and become more prominent with increasing sampling depth  
Due to a lack of distinct and unique biochemical signals from each skin layer, we were unable to employ SORS and SNR ratios to assess the individual layer signal contribution in human samples. Yet, the highly similar Raman spectra in Figure 5 suggest that in the case of a skin lesion within that depth range, a non-interfering unambiguous set of Raman signals will arise in a similar way to the phantoms study. To evaluate the spectral features recorded in the measuring volume with increasing offset we used the Principal Component Analysis (PCA) method as an unsupervised classification approach. PCA was run on human skin tissue data originating from scalp (Figure 6) and eyelid (Figure 7) separately, to rule out dependence on epidermis thickness. In the PCA scatter plot of scalp samples (Figure 6a), Sample 3 and 2A consistently separate from the rest across Principal Component 1 (PC 1), whose loading explains 96.4% of the spectral variance. Similarly, Sample 5A separates from the eyelid samples across PC 1 (97.1%) in the PCA scatter plot (Figure 7a). In the case of Sample 3 and 2A, this separation also occurs across PC 2, for which the corresponding loading is positively associated with bands assigned to the fat tissue. Those bands include 1081, 1301, 1437, 1525, 1656 and 1744 cm\({}^{-1}\) and are moving in the opposite direction from 1003, 1247 and 1670 cm\({}^{-1}\), which are assigned to skin typical bands negatively associated with the loading for PC2 (Figure 8). This is expected for Sample 3 as it is the thickest one (6 mm) from those measured and therefore a thick bottom layer of fat is expected in the measuring volume. In the case of Sample 2A, the strong presence of a fat layer in the collected Raman signal is due to the thin epidermis compared to the total sample volume (Table 1). Figure 5: Spatially offset Raman spectra from a representative healthy eyelid sample. All spectra are background corrected, smoothed, normalized to phenylalanine residue (1004 cm\({}^{-1}\)) and vertically shifted for clear presentation and comparison. The perpendicular line represents the position of the 872 cm\({}^{-1}\) band (hydroxyproline), whereas solid line boxes highlight areas of expected spectral changes with offset.