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The main components of the stratum corneum (SC), water, lipids, and proteins, are non-homogeneously distributed throughout the depth. The quantitative determination of their concentration profiles and penetration depth of topically applied substances are urgent topics of dermatological and cosmetic research. Confocal Raman micro-spectroscopy has distinct advantages when determining semi-quantitative concentrations of SC components and topically applied substances non-invasively and in vivo. In this work, we applied a tailored multivariate curve resolution-alternating least squares (tMCR-ALS) method to analyze Raman spectra of the SC in the 2000-4000 cm

Background Solar radiation causes skin damage through the generation of reactive oxygen species (ROS). While UV filters effectively reduce UV‐induced ROS, they cannot prevent VIS‐induced (400‐760 nm) oxidative stress. Therefore, potent antioxidants are needed as additives to sunscreen products. Methods We investigated VIS‐induced ROS formation and the photoprotective effects of the Nrf2 inducer Licochalcone A (LicA). Results Visible spectrum of 400‐500 nm dose‐dependently induced ROS in cultured human fibroblasts at doses equivalent to 1 hour of sunshine on a sunny summer day (150 J/cm2). A pretreatment for 24 hours with 1 µmol/L LicA reduced ROS formation to the level of unirradiated cells while UV filters alone were ineffective, even at SPF50+. In vivo, topical treatment with a LicA‐containing SPF50 + formulation significantly prevented the depletion of intradermal carotenoids by VIS irradiation while SPF50 + control did not protect. Conclusion LicA may be a useful additive antioxidant for sunscreens.

The human stratum corneum (SC) contains an abundant amount of carotenoid antioxidants, quenching free radicals and thereby protecting the skin. For the precise measurements of the depth-dependent carotenoid concentration, confocal Raman microscopy is a suitable method. The quantitative concentration can be determined by the carotenoid-related peak intensity of a Gaussian function approached at ≈1524 cm-1 using non-linear regression. Results show that the carotenoid concentration is higher at the superficial layers of the SC then decreases to a minimum at 20% SC depth and increases again towards the bottom of the SC. In the present work, two carotenoid penetration pathways into the SC are postulated. The first pathway is from the stratum granulosum to the bottom of the SC, while in the second pathway, the carotenoids are delivered to the skin surface by sweat and/or sebum secretion and penetrate from outside. The carotenoids are aggregated at the superficial layers, which are shown by high correlation between the aggregation states of carotenoids and the lateral organization of lipids. At the 30%-40% SC depths, the ordered and dense lipid molecules intensify the lipid-carotenoid interactions and weaken the carotenoid-carotenoid interaction and thus exhibit the disaggregation of carotenoids. At 90%-100% SC depths, the carotenoid-lipid interaction is weakened and the carotenoids have a tendency to be aggregated. Thus, the molecular structural correlation of carotenoid and SC lipid might be reserved in the intercellular space of the SC and also serves as the skeleton of the intercellular lipids.

Background Skin diseases can develop upon disadvantageous microclimate in relation to skin contact with textiles of supporting devices. Increased temperature, moisture, mechanical fracture, pressure, and inflammatory processes often occur mutually and enhance each other in their adverse effects. Therefore, the early prevention of skin irritations by improvement of microclimatic properties of skin in contact with supporting devices is important. Materials and methods In this study, the microclimate under occlusion with polyester, cotton, chloroprene rubber, and silicone textiles, used for supporting devices, was analyzed by determining several characteristic physiologic skin parameters in vivo, including temperature, moisture, and transepidermal water loss (TEWL). This is achieved by comparing a miniaturized in vivo detection device with several established optical and sensory methods in vivo. Results A highly significant TEWL decrease was found after polyester, chloroprene rubber, and silicone application. The application of all materials showed highly significant decrease in skin surface temperature, with chloroprene rubber showing the lowest. Similarly, all materials showed highly significant increase in relative moisture, where the highest increase was found for chloroprene rubber and silicone and the lowest increase for cotton. The cutaneous carotenoid concentration of chloroprene rubber, silicone, and polyester decreased. A manipulation of the surface structure of the stratum corneum was recognized for all materials except for cotton by laser scanning microscopy. Conclusion The skin parameters temperature, relative moisture, antioxidant status, and TEWL can effectively characterize the microclimatic environment during occlusion with medical supporting materials. These parameters could potentially be used to develop standardized testing procedures for material evaluation.

Background A nutrition rich in fruit and vegetables and a healthy lifestyle become more and more important in the industrial countries to counteract oxidative stress and promote health. For many years, it has been possible to control human cutaneous carotenoids noninvasively by resonance Raman spectroscopic systems and by spatially resolved reflectance spectroscopy. Methods Ten volunteers took a commercially available fruit and vegetable extract daily for a time period of 5 weeks. A second group served as control group and did not take any supplements (10 volunteers). To monitor the status of the cutaneous carotenoids noninvasively, an optical sensor based on multiple spatially resolved reflectance spectroscopy was applied once a week. Results The study could demonstrate that the intake of the supplement significantly increase the cutaneous carotenoid values of the young adults by 50%. The control group without any supplementation showed also significantly increased values, ie, by 10%, which might be due to the fact that their lifestyle was controlled. Conclusion The results illustrate that a biofeedback by measuring the skin carotenoids could improve the lifestyle of young adults and that a regular consumption of fruit and vegetables directly or as a drink can increase the concentration of cutaneous carotenoids significantly.

Multiphoton tomography (MPT) is a prospective tool for imaging the skin structure. Aiming to increase the probing depth, a comparative ex vivo study of optical clearing of porcine ear skin was performed by using two optical clearing agents (OCAs), i.e., glycerol and iohexol (OmnipaqueTM ) at different concentrations, which exhibit different osmotic properties. The results show that a topical application of glycerol or OmnipaqueTM solutions onto the skin for 60 min significantly improved the depth and contrast of the MPT signals. By utilizing 40%, 60% and 100% glycerol, and 60% and 100% OmnipaqueTM it was demonstrated that both agents improve autofluorescence and SHG (second harmonic generation) signals from the skin. At the applied concentrations and agent time exposure, glycerol is more effective than OmnipaqueTM . However, tissue shrinkage and cell morphology changes were found for highly concentrated glycerol solutions. OmnipaqueTM , on the contrary, increases the safety and has no or minimal tissue shrinkage during the optical clearing process. Moreover OmnipaqueTM allows for robust multimodal optical/X-ray imaging with automatically matched optically cleared and X-ray contrasted tissue volumes. These findings make OmnipaqueTM more prospective than glycerol for some particular application.

The routine diagnostic procedure of actinic keratosis (AK) and invasive squamous cell carcinoma (SCC) is a histological examination after taking a biopsy. In the past decades, non-invasive optical methods for skin examination have been developed. Patients with clinical diagnosis of AK or SCC were examined. The morphological criteria were determined for healthy, AK and SCC skin and compared for statistically significant differences. In this study, the applicability of multiphoton tomography (MPT) as an in vivo diagnostic tool for AK and SCC was evaluated. Changes in the morphology of the keratinocytes such as broadened epidermis, large intercellular spaces, enlarged nucleus and a large variance in cell shape could easily be recognized. The cell nuclei of AK and SCC were significantly larger compared to healthy skin cells in all cell layers. The nucleus-cytoplasm ratio was also significantly higher for AK and SCC than for the healthy skin cells. It was even higher in SCC compared to spinous and basal cell layer of AK. The cell density in AK and SCC was significantly lower than in the basal and spinous cell layers of healthy skin. In SCC, the cell density was significantly lower than in AK. Concerning the intercellular spaces, significant differences were found for AK and healthy skin in spinous and basal cell layer and for SCC compared to AK and healthy skin. In this study, MPT proved to be a valuable non-invasive imaging method for in vivo detection and discrimination of AK and SCC from healthy skin.

It is known that the collagen and elastin fibre structures are changing with age. There is little knowledge about the influence of body area as these investigations have been limited for ethical reasons, so far. Thus, modified non-invasive two-photon microscopy was used providing vertical optical sections of second harmonic generation and autofluorescence to calculate the collagen-to-elastin ratio and its alterations depending on the investigated body site in vivo. The results of this study indicate that the impact of different body areas could be higher than the influence of age and should be considered in future studies.

Background The in vivo multiphoton tomography has evolved into a useful tool for the non-invasive investigation of morphological and biophysical characteristics of human skin. Until now, changes of skin have been evaluated mainly by clinical and histological techniques. The current study addresses the effects of a changed acquisition time for single scans in a Z-stack on the directly related qualitative and quantitative interpretability of the data. Methods A test area of the skin was used for scanning 12 Z-stacks of 10 volunteers aged between 25 and 34 years. The stacks were taken up to a depth of 220 μm at increments of 10 μm at four different times, 1, 3, 7, 13 s, per scan. Subsequently, the second harmonic generation (SHG)-to-autofluorescence aging index of dermis (SAAID) was evaluated at three different measuring depths, i.e. at the maximum of SHG as well as at depths of 60 and 150 μm. Results The evaluation did not reveal any significant differences in the SAAID behavior between the Z-stacks of each test area scanned at different acquisition times. However, the acquisition time of 1 s/frame increases the measurement stability without influencing the SAAID behavior. The resolution of subcellular structures decreases significantly at scan times ≤3 s, whereas the acquisition time from 7 to 13 s warrants a high image quality. Conclusion The study has shown that there are no significant differences between the scan speeds per scan in a Z-stack and the resulting SAAID. Acquisition times of 7 s are suitable for the morphological evaluation whereas a further extension to 13 s does not result in any benefits. A scan time per image of 1 s is sufficient for the quantitative evaluation of SAAID thus substantially reducing the possible influence of movement artifacts.

UV irradiation is one of the most harmful exogenous factors for the human skin. In addition to the development of erythema, free radicals, that is reactive oxygen species (ROS), are induced under its influence and promote the development of oxidative stress in the skin. Several techniques are available for determining the effect of UV irradiation. Resonance Raman spectroscopy (RRS) measures the reduction of the carotenoid concentration, while electron paramagnetic resonance (EPR) spectroscopy enables the analysis of the production of free radicals. Depending on the method, the skin parameters are analysed in vivo or ex vivo. This study provides a critical comparison between in vivo and ex vivo investigations on the ROS formation and carotenoid depletion caused by UV irradiation in human skin. The oxygen content of tissue was also determined. It was shown that the antioxidant status measured in the skin samples in vivo and ex vivo was different. The depletion in the carotenoid concentration in vivo exceeded the value determined ex vivo by a factor of about 1.5, and the radical formation after UV irradiation was significantly greater in vivo by a factor of 3.5 than that measured in excised human skin, which can be explained by the lack of oxygen ex vivo.

Resonance Raman spectroscopy and multi-photon tomography were used in vivo to analyse the influence of sun exposure on the cutaneous carotenoids and collagen/elastin fibers. Comparing Berlin (low sun exposure) and Monegasque (high sun exposure) volunteers, it could be demonstrated that extended sun exposure significantly reduces the cutaneous carotenoids and collagen/elastin concentration (p < 0.05). The tendency towards correlation (R(2) = 0.41) between the dermal collagen/elastin (SAAID) and carotenoids confirms the important role of antioxidants in the protection against sun-induced negative effects. The application of sunscreen was shown to be effective, protecting cutaneous carotenoids and collagen/elastin from being damaged subsequent to sun exposure.

The cutaneous carotenoid concentration correlates with the overall antioxidant status of a person and can be seen as biomarker for nutrition and lifestyle. 50 high school students were spectroscopically measured for their cutaneous carotenoid concentrations initially in a static phase, followed by an intervention phase with biofeedback of their measured values, living a healthy lifestyle and on healthy food this time. The volunteers showed higher carotenoid concentrations than found in previous studies. A significant correlation of healthy lifestyle habits and a high antioxidant status could be determined. Subjects improved their nutritional habits and significantly increased their carotenoid concentration during intervention. Follow-up five months later showed a consolidation of the increase. The investigations show that a healthy diet and a well-balanced lifestyle correlate with a high cutaneous antioxidant concentration and that spectroscopic biofeedback measurement of cutaneous carotenoids as part of an integrated prevention program is a feasible and effective means to raise the health awareness in adolescents. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

SynopsisThe efficacy of a drug is characterized by its action mechanismand its ability to pass the skin barrier. In this article, differentmethods are discussed, which permit this penetration process tobe analysed non-invasively. Providing qualitative and quantita-tive information, tape stripping is one of the oldest procedures forpenetration studies. Although single cell layers of corneocytes areremoved from the skin surface, this procedure is considered asnon-invasive and is applicable exclusively to the stratum corne-um. Recently, optical and spectroscopic methods have been usedto investigate the penetration process. Fluorescence-labelleddrugs can be easily detected in the skin by laser scanning micros-copy. This method has the disadvantage that the dye labellingchanges the molecular structures of the drug and consequentlymight influence the penetration properties. The penetrationprocess of non-fluorescent substances can be analysed by Ramanspectroscopy, electron paramagnetic resonance, CARS and mul-tiphoton microscopic measurements. Using these methods, theconcentration of the topically applied formulations in differentdepths of the stratum corneum can be detected by moving thelaser focus from the skin surface deeper into the stratum corne-um. The advantages and disadvantages of these methods will bediscussed in this article.Re´sume´L’efficacite´ d’un me´dicament se caracte´rise par son me´canismed’action et sa capacite´ a` passer la barrie`re de la peau. Dans le pre´sentdocument les diffe´rentes me´thodes sont discute´es, qui permettentd’analyser le processus de la pe´ne´tration de fac¸on non invasive. L’ar-rachage (tape stripping) est l’une des plus anciennes me´thodes pourles e´tudes de pe´ne´tration puisqu’elle fournit de l’information qualita-tive et quantitative. Bien que des cellules isole´es des couches de cor-ne´ocytes soient retire´es de la surface de la peau, cette proce´dure estconside´re´e comme non-invasive; elle est applicable exclusivement a` lacouche corne´e. Re´cemment, des me´thodes spectroscopiques etoptiques et ont e´te´ utilise´es pour e´tudier le processus de pe´ne´tration.Les me´dicaments marque´s par fluorescence peuvent eˆtre de´tecte´sfacil-ement dans la peau par la microscopie a` balayage laser. Cette me´thodepre´sente l’inconve´nient que le label colore´ change les structuresmole´culaires de la drogue et par conse´quent sont susceptibles d’influ-encer les proprie´te´sdepe´ne´tration. Le processus de pe´ne´tration dessubstances non-fluorescentes peut eˆtre analyse´ par spectroscopieRaman, par la re´sonance paramagne´tique e´lectronique, CARS et pardes mesures de microscopie multiphotonique. En utilisant ces me´th-odes, la concentration des formulations topiques a` des profondeursdiffe´rentes du stratum corneum peut eˆtre de´tecte´e par le de´placementdu focus laser a` partir de la surface de la peau vers les couches plusprofondes de la couche corne´e. Les avantages et les inconve´nients deces me´thodes seront discute´sdanslepre´sent document.IntroductionSubstances are often topically applied in dermatology and cosmetol-ogy to improve the skin barrier and to treat dermatological diseases[1–4]. Whereas in the first case, the action of the drug is related tothe uppermost cell layers of the skin – the stratum corneum, in thesecond case, it is related to the living cells. Penetration of the skinbarrier is, therefore, indispensable. The investigation of this penetra-tion process is essential for the development and optimization of newpharmaceutics and cosmetic products [5, 6]. Whereas in the past, ithad been assumed that topically applied substances pass the skin bar-rier via the intercellular route [7, 8], it could be demonstrated thatalso the follicular route can represent an important penetration path-way [9, 10]. It was found, for example, for caffeine [11] and minoxi-dil [12], that these substances penetrate the skin barrier veryefficiently via the follicular route. For these investigations, the orificesof the hair follicles were artificially closed [11]. When these orificeswere blocked, the penetration of both substances into the blood circu-lation was considerably delayed. Previously, penetration investiga-tions had been based inter alia on diffusion cells or attenuated totalreflectance Fourier transform infrared spectroscopy, which describeonly the intercellular penetration in vitro [13, 14]. Today, however,methods considering also the influence of the follicular penetrationare to be used. In this article, different methods for the analysis of thepenetration process are described and discussed in terms of theiradvantages and limitations.This article elucidates both conventional penetration in vivo pro-cedures and recent developments in the field of optical technologiesused in in vivo penetration research.

Based on compelling in vivo and in vitro studies on human skin, carotenoids are thought to be of great interest as powerful antioxidants acting to prevent free-radical-induced damages, including premature skin ageing and the development of skin diseases such as cancer. Among the available techniques that are suitable for noninvasive determination of carotenoids in human skin, are resonance Raman spectroscopy (RRS) and reflection spectroscopy (RS). For RS, a LED-based miniaturized spectroscopic system (MSS) was developed for noninvasive measurement of carotenoids in human skin. The optimization and subsequent calibration of the MSS was performed with the use of RRS. A strong correlation between the carotenoid concentration determined by the RS and for the RRS system was achieved for human skin in vivo (R = 0.88) and for bovine udder skin in vitro (R = 0.81).

The antimicrobial treatment of wounds is still a major problem. Tissue-tolerable electrical plasma (TTP) is a new approach for topical microbial disinfection of the skin surface. The aim of the present study was to investigate the influence of TTP on a carotenoid profile in relation to skin physiology parameters (epidermal barrier function, stratum corneum (SC) hydration, surface temperature and irritation parameters). We were interested in the interaction of TTP and the antioxidative network, as well as the consequences for skin physiology parameters. These parameters are also indicative of TTP safety in vivo. For plasma application, 'Kinpen 09' was used (surface exposure 30-43°C) for 3 s. Beta-carotene and water profiles were assessed by in vivo Raman microspectroscopy (skin composition analyzer 3510). Skin physiology parameters were measured with Tewameter TM 300, Corneometer CM 825, skin thermometer and Chromameter CR 300. All parameters were assessed non-invasively on seven healthy volunteers before and after plasma application in vivo. We could show that TTP application leads to a decrease in beta-carotene especially in the superficial SC. Skin-surface temperature increased by 1.74°C, while the transepidermal water loss (TEWL) increase indicated an impaired barrier function. SC hydration decreased as seen in water profile especially in the superficial layers and capacitance values. A slight increase in skin redness was measurable. The induction of reactive oxygen species is probably the major contributor of TTP efficacy in skin disinfection. Skin physiology parameters were influenced without damaging the skin or skin functions, indicating the safety of TTP under in vivo conditions.

The interaction of free radicals with antioxidants is a topic of increasing interest in the development of prevention strategies against skin ageing. Carotenoids can serve as marker substances for the complete antioxidative network of human skin. Recently, it has become possible to measure the carotenoids non-invasively and online using resonance Raman spectroscopy. This method has been used in various studies to investigate the interaction of carotenoid antioxidants and free radicals in human skin. In this review, the results of the selected studies are summarized and compared. It could be demonstrated that the carotenoid concentration of the skin reflects the lifestyle of individuals. A high level of carotenoids can be achieved with a healthy diet rich, for instance, in fruit and vegetables. Stress factors such as illness, UV and IR radiation of the sun, and smoking and alcohol consumption reduce the concentration of the carotenoids in the skin. It could be demonstrated that premature skin ageing was less in people with a high level of antioxidants in their tissue. Consequently, the furrows and wrinkles were not so deep and dense as in the skin of individuals with a low antioxidant level. The measurements are highly suited for the development of anti-ageing strategies and can be efficiently used in the medical diagnostics and therapy control.

Background: Non-invasive measurements are of major interest for investigating the effects of stress, nutrition, diseases or pharmaceuticals on the antioxidative capacity of the human skin. However, only a few non-invasive methods are available. Material and Methods: The resonance Raman spectroscopy is well established to monitor carotenoids in the skin, but correlations with other antioxidants have not yet been described. Electron paramagnetic resonance spectroscopy used for measurements of free radicals has already been used elsewhere to investigate the reduction of applied long-living nitroxide radicals, caused by skin antioxidants and UV irradiation, but only a single or up to four volunteers were included in these studies. Therefore, in this study, the two methods were applied in parallel on 17 volunteers, and the rate constant of the nitroxide decrease was correlated with the cutaneous carotenoid concentration. Results and Discussion: A correlation with R = 0.65 was found, supporting the thesis that different antioxidants protect each other and build an antioxidative network in the skin. The results also give first indications that the carotenoids serve as marker substances for the antioxidative capacity, if the nutrition is well balanced.

The influence of stress factors on human skin induces the production of free radicals. Free radicals react immediately with antioxidants contained in the skin, giving rise to their depletion and with the surrounding molecules, resulting in their damage, disorganization and even destruction. High amounts of free radicals are produced in the upper skin layers, i.e. mainly in the epidermis, subsequent to sun irradiation. Irradiation of the skin in the infra-red (IR) range of the spectra, applied at physiological doses, can produce free radicals. The magnitude of destruction of antioxidants, such as carotenoids, can serve as a marker of the extent of the stress factor, characterized by the quantity of produced free radicals. In this study, measurements on the degradation of cutaneous carotenoids following IR skin irradiation of 12 healthy volunteers (skin type II), with two IR sources (standard infrared radiator = SIR and water filter infrared = wIRA) were taken using resonance Raman spectroscopy. Topical application of the antioxidant beta-carotene (2 mg/cm(2) ) provided protection for the human skin when exposed to IR radiation. The magnitude of the degradation of dermal carotenoids after IR irradiation was significantly higher for SIR than for wIRA irradiation, for both non-treated and cream-treated skin areas. The amount of destroyed carotenoids after IR irradiation was higher in the case of pretreatment with beta-carotene than for the untreated skin, indicating that the superficial part of antioxidants is most important for protecting against external stressors. The direct comparison of beta-carotene content was significantly higher for the cream-treated compared to untreated areas for all pairs: baseline, wIRA, after wIRA, baseline SIR and after SIR. Additionally, topically applied carotenoids as a single antioxidant component are less stable than the carotenoids in the skin incorporated by nutrition and accumulated in a mixture with different antioxidant substances. Resonance Raman spectroscopy can be used for the non-invasive measurements of carotenoids, which can be rated as marker substances of redox processes.

Heart failure (HF) is among the socially significant diseases, involving over 2% of the adult population in the developed countries. Diagnostics of the HF severity remains complicated due to the absence of specific symptoms and objective criteria. Here, we present an indicator of the HF severity based on the imaging of tissue parameters around the nailfold capillaries. High resolution nailfold video capillaroscopy was performed to determine the perivascular zone (PZ) size around nailfold capillaries, and 2-photon tomography with fluorescence lifetime imaging was used to investigate PZ composition. We found that the size of PZ around the nailfold capillaries strongly correlates with HF severity. Further investigations using 2-photon tomography demonstrated that PZ corresponds to the border of viable epidermis and it was suggested that the PZ size variations were due to the different amounts of interstitial fluid that potentially further translates in clinically significant oedema. The obtained results allow for the development of a quantitative indicator of oedematous syndrome, which can be used in various applications to monitor the dynamics of interstitial fluid retention. We therefore suggest PZ size measured with nailfold video capillaroscopy as a novel quantitative sensitive non-invasive marker of HF severity.