# Assessment of Skin Deep Layer Biochemical Profile Using Spatially Offset Raman Spectroscopy
## 3 Results  
### Tissue Phantoms  
In order to explore the signal contribution of the PF layer in the collected Raman spectra, when the latter was located in different positions in tissue phantoms (on the top of Phantoms A and buried in a 3 mm depth in Phantoms B), we employed the SORS configuration of the optical setup. SORS measurements were acquired after decoupling the excitation and collection optical paths in different degrees, by changing their relative distance, resulting in a series of different offsets (0-10 mm with an interval of 0.5 mm). The individual contributions of gelatin and the PF layer in the collected Raman spectra were assessed in correlation with the offset in both types of tissue phantoms (Figure 3). As expected, the top layer signal in both cases diminishes faster with offset compared to the bottom layer signal (Figure 3A(i),B(i)). This is expected as in two-layer phantoms the Raman photons that are generated deeper inside the phantom are more likely to migrate laterally before they are emitted from the sample surface [33]. However, we also notice that in Phantoms A (Figure 3A) the bottom layer (gelatin) signal significantly deteriorates with increasing offset compared to the bottom layer (PF) in Phantoms B (Figure 3B). As the bottom layer thickness in Phantoms A is significantly larger compared to Phantoms B, our observation is in line with previous studies which demonstrate a dramatic decrease in signal strength with bottom layer thickness [34]. It needs to be noted that although Phantoms B consist in reality three-layer systems and the laser beam dimensions are considerable compared to the PF layer thickness, the respective top and bottom signal contributions (Figure 3B(i)) exhibit a similar trend as previously observed in 2-layer samples studies [34]. The offset measurements in Phantoms type B were assessed in terms of: (a) the ratio of signal from the bottom layer (PF) to the signal from the top layer (gelatin) (SORS ratio) and (b) the normalized bottom layer SNR (Figure 3B(ii)). Although Phantoms B are not typical 2-layer phantoms, the SORS ratio can also be applied to them through the definition of the normalized ratio of signal from the intermediate (PF) layer to the signal from the top (gelatin) layer [34]. Raman signal variation with spatial offset was studied in three distinct layer samples before, showing that the SORS ratio varies in a nonlinear fashion for the intermediate/top layer [34]. However, in our case the top and bottom layers exhibit identical chemical signature as the intermediate (PF) layer is essentially embedded in a gelatin matrix. For that reason, we would expect the gelatin Raman signal to be a combination of top and bottom layer contribution. Nevertheless, the total matrix signal is still diminishing faster compared to the intermediate (PF) layer signal up to a certain offset (7 mm) where the SORS ratio reaches a plateau and gelatin contribution overtakes (Figure 3B(ii)). Therefore the SORS ratio generally increases with offset, resembling the trend that has been demonstrated experimentally and through numerical simulations in 2-layer systems of similar optical properties [34]. For Phantoms A, the calculation of the SORS ratio would entail no physical meaning as the PF layer (representing the lesion) is located at the top of the gelatin matrix. The normalized SNR was instead calculated for different offsets (Figure 3A(ii)). The resulting plot indicates the signal generated at minimum offset (0.5 mm) measurement as the most efficient one. This is expected as the PF layer is placed at the top of the phantom and therefore the majority of Raman scattered photons generated at the surface would back-scatter towards the collection probe. Due to the defocused nature of the laser beam and the comparable PF thickness to the beam size, the optimum offset in our case was not zero. This is in line with previous studies also describing a modified SORS configuration, but for a different clinical application [35].