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

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

Keratin-water-NMF interaction as a three layer model in the human stratum corneum using in vivo confocal Raman microscopy

The secondary and tertiary structure of keratin and natural moisturizing factor (NMF) are of great importance regarding the water regulating functions in the stratum corneum (SC). In this in vivo study, the depth-dependent keratin conformation and its relationship to the hydrogen bonding states of water and its content in the SC, are investigated using confocal Raman microscopy. Based on the obtained depth-profiles for the β-sheet/α-helix ratio, the stability of disulphide bonds, the amount of cysteine forming disulphide bonds, the buried/exposed tyrosine and the folding/unfolding states of keratin, a “three layer model” of the SC, regarding the keratin-water-NMF interaction is proposed. At the uppermost layers (30–0% SC depth), the keratin filaments are highly folded, entailing limited water binding sites, and NMF is mostly responsible for binding water. At the intermediate layers (70–30% SC depth), the keratin filaments are unfolded, have the most water binding sites and are prone to swelling. At the bottom layers (100–80% SC depth), the water binding sites are already occupied with water and cannot swell substantially. The hydrogen bonding states of water molecules can only be explained by considering both, the molecular structure of keratin and the contribution of NMF as a holistic system

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

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 approximate to 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

Response to comment by Puppels et al. on “A modification for the calculation of water depth profiles in oil‐treated skin by in vivo Raman microscopy”

The presence of penetrated oils in the stratum corneum (SC), oil-induced occlusion of the SC and formation of occluding homogeneous film on the skin surface are discussed in relation to their influence on results of water profile calculations using conventional and newly proposed extended methods. It is shown that the conventional method does not determine the water profiles in treated skin correctly due to the superposition of Raman bands of SC's proteins and penetrated and remnant oils

A modification for the calculation of water depth profiles in oil‐treated skin by in vivo confocal Raman microscopy

In this study, an extended calculation method for the determination of the water profiles in oil-treated skin is proposed, which is based on the calculation of the ratio between the Raman band intensities of water (3350-3550 cm−1) and keratin Amide I at 1650 cm−1. The proposed method is compared with the conventional method based on the ratio of the Raman band intensities of water (3350-3550 cm−1) and keratin at 2930 cm−1. The conventional method creates artifacts in the depth profiles of the water concentration in oil-treated skin, showing a lower amount of water in the upper and intermediate layers of the stratum corneum, which is due to the superposition of oil- and keratin-related Raman bands at 2930 cm−1. The proposed extended method shows no artifacts and has the potential to determine the water depth profiles after topical application of formulations on the skin

Stratum corneum occlusion induces water transformation towards lower bonding state: a molecular levelin vivostudy by confocal Raman microspectroscopy

Objective: It is conventionally understood that occlusive effects are the retention of excessive water in the stratum corneum (SC), the increase of SC thickness (swelling) and a decrease of the transepidermal water loss. However, the influence of occlusion on water binding properties in the SC is unknown. Methods: The action of plant-derived jojoba and almond oils, as well as mineral-derived paraffin oil and petrolatum topically applied on human skin, is investigated in vivo using confocal Raman microspectroscopy. To understand the oils’ influence on the SC on the molecular level, the depth-dependent hydrogen bonding states of water in the SC and their relationship to the conformation of keratin, concentration of natural moisturizing factor (NMF) molecules and lipid organization were investigated. Results: A significant SC swelling was observed only in petrolatum-treated skin. The water concentration was increased in oil-treated skin in the intermediate SC region (40–70% SC depth). Meanwhile, the amount of free, weakly and tightly bound water increased, and strongly bound water decreased in the uppermost SC region (0–30% SC depth). The NMF concentration of oil-treated skin was significantly lower at 50–70% SC depth. The lateral organization of lipids in oil-treated skin was lower at 0–30% SC depth. The secondary structure of keratin was changed towards an increase of β-sheet content in mineral-derived oil-treated skin and changed towards an increase of α-helix content in plant-derived oil-treated skin. Conclusion: The occlusive properties can be summarized as the increase of free water and the transformation of water from a more strongly to a more weakly hydrogen bonding state in the uppermost SC, although some oils cause insignificant changes of the SC thickness. The accompanied changes in the keratin conformation at the intermediate swelling region of the SC also emphasize the role of keratin in the SC’s water-transporting system, that is the water in the SC transports intercellularly and intracellularly in the intermediate swelling region and only intercellularly in the uppermost non-swelling region. Bearing this in mind, almond, jojoba and paraffin oils, which are not occlusive from the conventional viewpoint, have an occlusion effect similar to petrolatum on the SC

Electrohydrodynamic spray applicator for homogenous application and reduced overspray of sunscreen

Background: The recommended amount of sunscreen by hand application (2 mg/cm(2)) is in reality not achieved, which decreases the homogeneity and thereby the effective sun protection factor (SPF). Materials and Methods: The homogeneity of sunscreen applied by a newly developed spray applicator using an electrostatically charged aerosol, for which a hand rubbing of the formulation is not necessary, is evaluated. In vivo experiments were performed on the volar forearms of human volunteers using the spray applicator compared to the standardized hand application according to ISO 24444. Results: The distribution homogeneity was assessed qualitatively using in vivo laser scanning microscopy and quantitatively by absorption spectroscopy after tape stripping and by the standard deviation of multiple spatially displaced reflectance measurements for non-invasive SPF determination below the minimal erythemal dose, which showed a significantly higher homogeneity by 20.9% after spray application compared to hand application. Conclusion: Non-invasive SPF determination of multiple spatially displaced reflectance measurements was proven to be a suitable method for the non-invasive determination of the sunscreen distribution homogeneity. Electrostatically charged spray application increased the sunscreen distribution homogeneity on the skin and can reduce the amount of overspray

In vivo detection of changes in cutaneous carotenoids after chemotherapy using shifted excitation resonance Raman difference and fluorescence spectroscopy

Background: Various cutaneous toxicities under chemotherapy indicate a local effect of chemotherapy by secretion after systemic application. Here, changes in the fluorescence and Raman spectral properties of the stratum corneum subsequent to intravenous chemotherapy were assessed. Methods: Twenty healthy subjects and 20 cancer patients undergoing chemotherapy were included. Measurement time points in cancer patients were before the first cycle of chemotherapy (Tbase) and immediately after intravenous application of the chemotherapy (T1). Healthy subjects were measured once without any further intervention. Measurements were conducted using an individually manufactured system consisting of a handheld probe and a wavelength-tunable diode laser-based 488 nm SHG light source. Hereby, changes in both skin fluorescence and shifted excitation resonance Raman difference spectroscopy (SERRDS) carotenoid signals were assessed. Results: Healthy subjects showed significantly (P <.001) higher mean concentrations of carotenoids compared to cancer subjects at Tbase. An increase in fluorescence intensity was detected in almost all patients after chemotherapy, especially after doxorubicin infusion. Furthermore, a decrease in the carotenoid concentration in the skin after chemotherapy was found. Conclusion: The SERRDS based noninvasive detection can be used as an indirect quantitative assessment of fluorescent chemotherapeutics. The lower carotenoid SERRDS intensities at Tbase might be due to cancerous diseases and co-medication. © 2020 The Authors. Skin Research and Technology Published by John Wiley & Sons Ltd

Red- and Near-Infrared-Excited Autofluorescence as a Marker for Acute Oxidative Stress in Skin Exposed to Cigarette Smoke Ex Vivo and In Vivo

Air pollution is increasing worldwide and skin is exposed to high levels of pollution daily, causing oxidative stress and other negative consequences. The methods used to determine oxidative stress in the skin are invasive and non-invasive label-free in vivo methods, which are severely limited. Here, a non-invasive and label-free method to determine the effect of cigarette smoke (CS) exposure on skin ex vivo (porcine) and in vivo (human) was established. The method is based on the measurement of significant CS-exposure-induced enhancement in red- and near-infrared (NIR)-excited autofluorescence (AF) intensities in the skin. To understand the origin of red- and NIR-excited skin AF, the skin was exposed to several doses of CS in a smoking chamber. UVA irradiation was used as a positive control of oxidative stress in the skin. The skin was measured with confocal Raman microspectroscopy before CS exposure, immediately after CS exposure, and after skin cleaning. CS exposure significantly increased the intensity of red- and NIR-excited skin AF in a dose-dependent manner in the epidermis, as confirmed by laser scanning microscopy AF imaging and fluorescence spectroscopy measurements. UVA irradiation enhanced the intensity of AF, but to a lower extent than CS exposure. We concluded that the increase in red- and NIR-excited AF intensities of the skin after CS exposure could clearly be related to the induction of oxidative stress in skin, where skin surface lipids are mainly oxidized