Characterizing stratum corneum structure, barrier function, and chemical content of human skin with coherent Raman scattering imaging

Abstract

The most superficial layer of the epidermis, the stratum corneum, plays a crucial role in retaining hydration; if its structure or composition is compromised, dry skin may result as a consequence of poor water retention. Dry skin is typically treated with topical application of humectant agents that attract water into the skin. Corneometry, the industry standard for measuring skin hydration, works by assessing the bulk electrical properties of skin. However, this technique samples a large volume of tissue and thus does not resolve the biochemical changes that occur at the cellular level that may underlie mechanisms of dry skin. These limitations can be addressed using coherent Raman scattering (CRS) microscopy to probe the intrinsic vibrational modes of chemical groups such as lipids and water. In the present study, ex vivo human skin explants undergoing dehydration and humectant-induced rehydration were measured via CRS imaging and corneometry. Corneometry data and chemically specific images were obtained from the stratum corneum of each patient sample at each timepoint. The resulting data was statistically analyzed using linear mixed effect model regression analysis. The cellular imaging data revealed water loss in the stratum corneum during dehydration that was correlated with corneometer readings. Interestingly, the imaging data and corneometer readings show differences under the experimental rehydration conditions. The rehydration results suggest that hydration restored by the humectant agents may not be retained by the corneocytes in the ex vivo model system. Given the complementary nature of corneometry, a bulk assessment tool, and CRS microscopy, a modality with subcellular resolution implemented here in an en-face tissue imaging setup, these techniques can be used to measure uptake and efficacy of topical compounds in order to better understand their mode of action and improve therapeutic applications.

Fig. 1

CRS images of human stratum corneum acquired from ex vivo skin explants. (a–c) CARS and (d–f) SRS images of stratum corneum showing (a,d) lipid-weighted content; (b,e) protein-weighted content; and (c,f) water-weighted content. The NRB in the CARS data manifests itself as a homogeneous and unspecific haze distributed across the field of view.

Fig. 2

Manual segmentation of SRS lipid content image to distinguish intracellular and extracellular spaces for subsequent analysis. (a) Unlabeled image. (b) Manually segmented image, showing corneocytes identified by indices 1 through 22, and extracellular space corresponding to the surrounding region identified by index number 23.

Fig. 3

Corneometer measurements obtained from ex vivo human skin throughout the dehydration time course on a plastic substrate (i.e. rapid dehydration) and a gel substrate (i.e. slow dehydration). Data points correspond to the mean of the triplicate corneometer measurements with error bars indicating the standard error of the mean. Statistically significant deviations from the corresponding initial timepoint are denoted by asterisks and determined via Student’s t-test (*: p < 0.05/N ; **: p < 0.01/N ; ***: p < 0.001/N, adjusted using Bonferroni correction with N = 4 pairwise comparisons).

Fig. 4

Corneometry-based assessment of ex vivo human skin hydration dynamics during rehydration under various environmental and treatment conditions. Data points correspond to the rate of change of corneometer measurements per hour, with error bars showing the 95% confidence interval. For the ambient condition without treatment (control), asterisks denote rates of change significantly different from zero; for all other conditions, they denote rates of change that are significantly different from the control (*: p < 0.05; **: p < 0.01; ***: p < 0.001, where p-values are adjusted using Holm-Bonferroni correction with N = 36 metrics).

Fig. 5

Chemical content dynamics of ex vivo human skin during rehydration under various environmental and treatment conditions. Data points correspond to the rate of change of CRS imaging metrics per hour, with error bars showing the 95% confidence interval. For the ambient condition without treatment (control), asterisks denote rates of change significantly different from zero; for all other conditions, they denote rates of change that are significantly different from the control (*: p < 0.05; **: p < 0.01; ***: p < 0.001, where p-values are adjusted using Holm-Bonferroni correction with N = 36 metrics).

Fig. 6

Morphological dynamics of ex vivo human corneocytes during rehydration under various environmental and treatment conditions. Data points correspond to the rate of change of each spatial metric in microns per hour with error bars showing the 95% confidence interval. Nearest neighbor distances are computed between corneocyte cell centers (NNDCenters) as well as between cell walls (NNDWalls). For the ambient condition without treatment (control), asterisks denote rates of change significantly different from zero; for all other conditions, they denote rates of change that are significantly different from the control (*: p < 0.05; **: p < 0.01; ***: p < 0.001, where p-values are adjusted using Holm-Bonferroni correction with N = 36 metrics).