# Assessment of Skin Deep Layer Biochemical Profile Using Spatially Offset Raman Spectroscopy
## 4 Discussion  
Previous studies suggested that it is feasible to spectrally separate the top layer of epidermis (stratum corneum) from the dermis by means of confocal Raman spectroscopy combined with K-means cluster analysis [40]. In this study we concluded that although some spectral signature (1671 cm\({}^{-1}\)) from the stratum corneum was evident in our measurements, a discrimination between epidermis and dermis (including all sublayers) was not possible with a defocused Raman system. Furthermore, it was difficult to observe clear spectral changes in the transition from epidermis to dermis. This is due to: (a) the absence of major biochemical differences between the two layers, (b) the low confocality and large sampling volume of the employed setup and (c) the weak signal-to-noise ratio when probing deeper layers. Although the insufficient spatial resolution of our system did not allow for specifically probing the most lipid-rich and therefore spectrally-distinct sublayer of stratum corneum, such a possibility would not provide any advantage over a potential cancer diagnosis since not all malignant lesions develop superficially. Through our investigation of spatially offset measurements, we concluded that a defocused Raman system configured in a zero offset mode is able to capture biochemical information from deeper skin layers and specifically between the skin surface and the epidermis-dermis junction, where the lesions mainly develop and therefore the sampling volume for future cancer screening would lay. Our results are not conclusive, as human skin is subjected to great variability (such as age and race) [51], affecting the recorded Raman signal. However, they provide a strong indication of the benefits of a defocused Raman system applied to a potential in vivo skin cancer screening. In any case, a defocused Raman modality would allow for the employment of higher laser power for in vivo skin measurements within the suggested ANSI MPE limits (0.06-0.1 mW/cm\({}^{2}\)) (ANSI Z136.1-2014 American National Standard for the Safe Use of Lasers) Additionally, the relatively large sampling volume (compared to a confocal measurement) of defocused Raman spectroscopy can reduce the number and time of measurements required and render the implementation of the technique into clinics faster and simpler. There are currently a number of studies focusing on the diagnostic potential of skin cancer using either a multimodal [18] or fiber optic Raman probes [17]. A few of those have been reported to reach a depth of up to 200 \(\upmu\)m [16], considering the fact that non-melanoma skin cancers invade the lower dermis layers of the skin. Although our study does not include any malignant samples, it clearly shows that a SORS approach would be able to adequately sample the biochemical profile of deeper skin layers similarly to measurements demonstrated on healthy samples. The novelty of our work is that the SORS modality is not just able to look deeper inside the skin tissue but most importantly to collect Raman photons with a bias towards inner skin layers. In that respect, this study employs deep Raman spectroscopy to assess the deep layer biochemical profile of skin, paving the way for non-invasive skin cancer screening. The following are available online at [[https://www.mdpi.com/article/10.3390/app11209498/s1](https://www.mdpi.com/article/10.3390/app11209498/s1)]([https://www.mdpi.com/article/10.3390/app11209498/s1](https://www.mdpi.com/article/10.3390/app11209498/s1)), Table S1: Raman band assignments of healthy eyelid and scalp human skin layers. Conceptualization, M.Z.V., K.S., G.G., I.D.B. and N.K. ; methodology, M.Z.V. and N.K. ; validation, M.Z.V., K.S. and N.K. ; formal analysis, M.Z.V. and N.K. ; investigation, M.Z.V., K.S. and N.K. ; data curation, M.Z.V. and N.K. ; writing--original draft preparation, M.Z.V. ; writing--review and editing, M.Z.V., K.S., G.G., I.D.B. and N.K. ; supervision, N.K. ; project administration, M.Z.V. and N.K. ; funding acquisition, M.Z.V. and N.K.