Skin hydration is a complex process that influences the physical and mechanical properties of skin. Various technologies have emerged over the years to assess this parameter, with the current standard being electrical probe-based instruments. Nevertheless, their inability to provide detailed information has prompted the use of sophisticated spectroscopic and imaging methodologies, which are capable of in-depth skin analysis that includes structural and composition details. Modern imaging and spectroscopic techniques have transformed skin research in the dermatological and cosmetics disciplines, and are now commonly employed in conjunction with traditional methods for comprehensive assessment of both healthy and pathological skin. This article reviews current techniques employed in measuring skin hydration, and gives an account on their principle of operation and applications in skin-related research.

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Review of Modern Techniques for the Assessment of Skin Hydration

Confocal Raman microspectroscopy is widely used in dermatology and cosmetology for analysis of the concentration of skin components (lipids, natural moisturizing factor molecules, water) and the penetration depth of cosmetic/medical formulations in the human stratum corneum (SC) in vivo. In recent years, it was shown that confocal Raman microspectroscopy can also be used for noninvasive in vivo depth-dependent determination of the physiological parameters of the SC, such as lamellar and lateral organization of intercellular lipids (ICLs), folding properties of keratin, water mobility, and hydrogen bonding states. The results showed that the strongest skin barrier function, which is primarily manifested by the orthorhombic organization of ICLs, is provided at ≈20–40% SC depth, which is related to the maximal bonding state of water with surrounding components in the SC. The secondary and tertiary structures of keratin determine water binding in the SC, which is depth-dependent. This paper shows the technical possibility and advantage of confocal Raman microspectroscopy in noninvasive investigation of the skin and summarizes recent results on in vivo investigation of the human SC.

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Current Views on Noninvasive in vivo Determination of Physiological Parameters of the Stratum Corneum Using Confocal Raman Microspectroscopy

Determination of the molecular composition of the skin is crucial for numerous tasks in medicine, pharmacology, dermatology and cosmetology. Confocal Raman microspectroscopy is a sensitive method for the evaluation of molecular depth profiles in the skin in vivo. Since the Raman spectra of most of the skin constituents significantly superimpose, a spectral decomposition by a set of predefined library components is usually performed to disentangle their contributions. However, the incorrect choice of the number and type of components or differences between the spectra of the basic components measured in vitro and in vivo can lead to incorrect results of the decomposition procedure. Here, we investigate an alternative data-driven approach based on a non-negative matrix factorization (NNMF) algorithm of depth-resolved Raman spectra of skin that does not require a priori information of spectral data for the analysis. Using the model and experimentally measured depth-resolved Raman spectra of the upper epidermis in vivo, we show that NNMF provides depth profiles of endogenous molecular components and exogenous agents penetrating through the upper epidermis for the spectra and concentration. Moreover, we demonstrate that this approach is capable of providing new information on the molecular profiles of the skin.

Blind source separation of molecular components of the human skin in vivo: non-negative matrix factorization of Raman microspectroscopy data.

Confocal Raman microscopy has a number of advantages in investigating the human stratum corneum (SC) in vivo and ex vivo. The penetration profiles of xenobiotics in the SC, as well as depth profiles of the physiological parameters of the SC, such as the concentration of water depending on the strength of hydrogen bonds, total water concentration, the hydrogen bonding state of water molecules, concentration of intercellular lipids, the lamellar and lateral packing order of intercellular lipids, the concentration of natural moisturizing factor molecules, carotenoids, and the secondary and tertiary structure properties of keratin are well investigated. To consider the depth-dependent Raman signal attenuation, in most cases a normalization procedure is needed, which uses the main SC’s protein keratin-related Raman peaks, based on the assumption that keratin is homogeneously distributed in the SC. We found that this assumption is not accurate for the bottom part of the SC, where the water concentration is considerably increased, thus, reducing the presence of keratin. Our results demonstrate that the bottom part of the SC depth profile should be multiplied by 0.94 in average in order to match this non-homogeneity, which result in a decrease of the uncorrected values in these depths. The correctly normalized depth profiles of the concentration of lipids, water, natural moisturizing factor and carotenoids are presented in this work. The obtained results should be taken into consideration in future skin research using confocal Raman microscopy.

/pdf/non-invasive-depth-profiling-of-the-stratum-corneum-in-vivo-3hd9nulfgm.pdf

Non-invasive depth profiling of the stratum corneum in vivo using confocal Raman microscopy considering the non-homogeneous distribution of keratin.

The skin barrier function is mostly provided by the stratum corneum (SC), the uppermost layer of the epidermis. To noninvasively analyze the physiological properties of the skin barrier functionin vivo, it is important to determine the SC thickness. Confocal Raman microscopy (CRM) is widely used for this task. In the present in vivo study, a new method based on the determination of the DNA concentration profile using CRM is introduced for determining the SC thickness. The obtained SC thickness values are compared with those obtained using other CRM-based methods determining the water and lipid depth profiles. The obtained results show almost no significant differences in SC thickness for the utilized methods. Therefore, the results indicate that it is possible to calculate the SC thickness by using the DNA profile in the fingerprint region, which is comparable with the SC thickness calculated by the water depth profiles (ANOVA test p = 0.77) and the lipid depth profile (ANOVA test p = 0.74). This provides the possibility to measure the SC thickness by using the DNA profile, in case the water or lipid profile analyses are influenced by a topically applied formulation. The increase in DNA concentration in the superficial SC (0-2 µm) is related to the DNA presence in the microbiome of the skin, which was not present in the SC depth below 4 µm.

/pdf/in-vivo-tracking-of-dna-for-precise-determination-of-the-4j2uj6qyq1.pdf

In vivo Tracking of DNA for Precise Determination of the Stratum Corneum Thickness and Superficial Microbiome Using Confocal Raman Microscopy.

Confocal Raman has been widely used for measuring the water concentration profile inside skin to calculate clinical end points, such as stratum corneum thickness. In this article, multivariate curve resolution was applied to resolve the pure components contained in high frequency (2500-4000 cm-1) in vivo confocal Raman data. Three components were identified by comparing with reference spectra of materials in skin. These three components are water, protein, and lipid. The score values associated with these three components were transformed to mass ratio by leveraging the response factors for protein and lipid in a calibration model utilizing the pure material spectra. The concentration profiles for protein and lipid as a function of depth across the stratum corneum are utilized as new clinical end points. Results from an in vivo study with individuals who experience atopic dermatitis symptoms successfully demonstrated a statistical difference between Raman spectra from nonlesion and lesion skin sites. Trends in the depth profiles of the skin components are consistent with previous literature reports.

MCR Approach Revealing Protein, Water, and Lipid Depth Profile in Atopic Dermatitis Patients' Stratum Corneum via in Vivo Confocal Raman Spectroscopy.

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in human subjects. This information is very important for skin-related diseases and characterizing skin care product performance. This protocol illustrates a method for confocal Raman spectra collection and the subsequent analysis of the spectral dataset leveraging chemometrics. The goal of this method is to establish a standard protocol for data collection and provide general guidance for data analysis. Preprocessing (e.g., removal of outlier spectra) is a critical step when processing large datasets from clinical studies. As an example, we provide guidance based on prior knowledge of a dataset to identify the types of outliers and develop specific strategies to remove them. A principal component analysis is performed, and the loading spectra are compared with spectra from reference materials to select the number of components used in the final multivariate curve resolution (MCR) analysis. This approach is successful for extracting meaningful information from a large spectral dataset.

Resolving Water, Proteins, and Lipids from In Vivo Confocal Raman Spectra of Stratum Corneum through a Chemometric Approach.

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MCR Approach Revealing Protein, Water, and Lipid Depth Profile in Atopic Dermatitis Patients' Stratum Corneum via in Vivo Confocal Raman Spectroscopy.

Resolving Water, Proteins, and Lipids from In Vivo Confocal Raman Spectra of Stratum Corneum through a Chemometric Approach.