Skin Res Technol. 2019;1–9. wileyonlinelibrary.com/journal/srt | 1 © 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Received: 19 December 2018 | Accepted: 14 February 2019 DOI: 10.1111/srt.12698 ORIGINAL ARTICLE Novel confocal Raman microscopy method to investigate hydration mechanisms in human skin Hequn Wang 1 | Qihong Zhang 2 | Guangru Mao 1 | Oscar Conroy 1 | Yelena Pyatski 2 | Michael J. Fevola 1 | Gabriela Oana Cula 1 | Prithwiraj Maitra 1 | Richard Mendelsohn 2 | Carol R. Flach 2 Hequn Wang and Qihong Zhang contributed equally to this work. 1 Johnson and Johnson Consumer Companies, Inc., Skillman, New Jersey 2 Department of Chemistry, Rutgers University, Newark, New Jersey Correspondence Carol R. Flach, Department of Chemistry, Rutgers University, Newark, NJ. Email: flach@rutgers.edu Funding information Johnson and Johnson Consumer Companies, Inc. Abstract Background: Skin hydration is essential for maintaining stratum corneum (SC) flexi- bility and facilitating maturation events. Moisturizers contain multiple ingredients to maintain and improve skin hydration although a complete understanding of hydration mechanisms is lacking. The ability to differentiate the source of the hydration (water from the environment or deeper skin regions) upon application of product will aid in designing more efficacious formulations. Materials and Methods: Novel confocal Raman microscopy (CRM) experiments allow us to investigate mechanisms and levels of hydration in the SC. Using deuterium oxide (D 2 O) as a probe permits the differentiation of endogenous water (H 2 O) from exogenous D 2 O. Following topical application of D 2 O, we first compare in vivo skin depth profiles with those obtained using ex vivo skin. Additional ex vivo experiments are conducted to quantify the kinetics of D 2 O diffusion in the epidermis by introduc- ing D 2 O under the dermis. Results: Relative D 2 O depth profiles from in vivo and ex vivo measurements compare well considering procedural and instrumental differences. Additional in vivo experi- ments where D 2 O was applied following topical glycerin application increased the longevity of D 2 O in the SC. Reproducible rates of D 2 O diffusion as a function of depth have been established for experiments where D 2 O is introduced under ex vivo skin. Conclusion: Unique information regarding hydration mechanisms are obtained from CRM experiments using D 2 O as a probe. The source and relative rates of hydration can be delineated using ex vivo skin with D 2 O underneath. One can envision compar - ing these depth-dependent rates in the presence and absence of topically applied hydrating agents to obtain mechanistic information. KEYWORDS confocal Raman microscopy, epidermis, skin barrier, skin hydration, stratum corneum, water content