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Background: The antioxidant status of the human skin provides protection against the destructive action of free radicals. Most antioxidants cannot be synthesized by the human organism itself, but have to be ingested with a healthy nutrition rich in fruit and vegetables. The Korean cuisine is known to be one of the healthiest worldwide. This binational study investigated the cutaneous carotenoid concentrations in German subjects, South Korean subjects and immigrant Korean subjects resident in Germany and examined whether dietary- and lifestyle-related differences are reflected in the cutaneous carotenoid concentrations. Methods: Measurements of the carotenoid concentrations of 714 healthy volunteers were performed using a non-invasive spectroscopic measurement system based on reflectance spectroscopy. Results: In the present study South Korean residents showed a significantly higher antioxidant status than both native German residents and Korean immigrants living in Germany (p < 0.001). The first generation of Korean immigrants to Germany over the age of 50 mostly preserved Korean dietary habits, showing significantly higher concentrations (p < 0.001) than the German-born second and third Korean generations under the age of 50. Conclusion: The results of the study indicate that a healthy nutrition alone does not provide a high antioxidant status unless the stress exposure can be reduced simultaneously.

Background: Oxidative stress plays an important role in the pathogenesis of various skin diseases. Thus, the antioxidant network of the skin relies on the uptake of exogenous antioxidants to ensure cell protection against radical formation. Green tea is one of the main sources of polyphenolic antioxidant compounds, but only few data are available on its cutaneous antioxidant effects. Methods: The radical scavenging properties of Benifuuki and Yabukita green tea were investigated in the human skin. Thirty-two participants who met the inclusion criteria were randomized to consume 3 cups per day of either Benifuuki tea, Yabukita tea, or water (control group) for 2 weeks. Electron paramagnetic resonance (EPR) spectroscopy was applied to measure the radical scavenging capacity of the skin in vivo before and after the intervention. Results: Both Yabukita and Benifuuki tea led to an increase in the radical scavenging activity of the skin by 28 and 29%, respectively, and the difference was significant when compared to the control group. Benifuuki tea, previously reported to be superior in bioavailability due to a highly absorbable methylated catechin, did not prove to be more effective than the common Yabukita tea. Conclusion: The results show that green tea enhances the radical scavenging capacity of the skin and support the hypothesis that green tea may offer protection against cutaneous oxidative stress.

Background/Aim: The main reason for extrinsic skin aging is the negative action of free radicals. The formation of free radicals in the skin has been associated with ultraviolet (UV) exposure and also to visible (VIS) and near-infrared (NIR) irradiations. The aim of the present study was to evaluate the efficacy of a sunscreen in the whole solar range. Methods: The radical-scavenging activity of a sunscreen in the UV, VIS, and NIR ranges was evaluated using electron paramagnetic resonance spectroscopy. Ex vivo penetration profiles were determined using confocal Raman microscopy on porcine ear skin at different time points after application. Results: Compared to the untreated skin, the sunscreen decreased the skin radical formation in the UV and VIS regions. Additional protection in the VIS and NIR ranges was observed for the sunscreen containing antioxidants (AO). The penetration depth of the cream was less than 11.2 ± 3.0 µm for all time points. Conclusion: A sunscreen containing AO improved the photoprotection in the VIS and NIR ranges. The sunscreen was retained in the stratum corneum. Therefore, these results show the possibility of the development of effective and safer sunscreen products.

Confocal Raman microscopy has been used to measure depth-dependent profiles of human SC in vivo in the high wavenumber (HWN) region. In order to keep the linearity of HWN region boundaries and to not remove an informative signal from Raman spectra, a new baseline subtraction procedure has been introduced. After baseline subtraction, the HWN spectrum was deconvoluted using 10 Gaussian functions with individual chemical meanings. The results show that the hydrogen bound water molecule types contributed differently to the water diffusion process in the SC. The most concentrated double donor-double acceptor (DDAA) and single donor-single acceptor (DA) water molecule types in the SC represent more than 90% of the SC's water and mostly contribute to the water flux in the skin. Single donor-double acceptor (DAA) and weakly-bound water molecule types represent less than 10% of the SC's water content. The most tightly hydrogen bound water molecule type, DAA, reaches its maximum concentration near the skin surface and does not take part in the water diffusion process via the SC. The results show that the hydrogen bonding state of water (DA/DDAA water molecule type ratio) reaches its maximum at the depth of approx. 30% of the SC thickness, which correlates well with the maximum lateral packing order of intercellular lipids (ICL) and the natural moisturizing factor (NMF), and does not coincide with the folding/unfolding state of keratin. The NMF's contribution to the bonding of water in the SC is supposed to dominate over that of ICL.

Blood cell analysis is one of the standard clinical tests. Despite the widespread use of exogenous markers for blood cell quantification, label-free optical methods are still of high demand due to their possibility for in vivo application and signal specific to the biochemical state of the cell provided by native fluorophores. Here we report the results of blood cell characterization using label-free fluorescence imaging techniques and flow-cytometry. Autofluorescence parameters of different cell types – white blood cells, red blood cells, erythrophagocytic cells – are assessed and analyzed in terms of molecular heterogeneity and possibilities of differentiation between different cell types in vitro and in vivo.

Fluorescent dye Thioflavin T (ThT) is a widely used probe for the detection of amyloid fibrils, which are protein aggregates involved in the pathogenesis of neurodegenerative disorders. Upon the formation of a complex with amyloids, the fluorescence quantum yield of ThT increases 1000-fold due to a dramatic reduction of the nonradiative decay rate. This is accompanied by a remarkable change of ThT fluorescence lifetime τ from ~1 to ~1000 ps, thus making it possible to assess ThT binding to different systems using τ as an indicator. However, when measuring ThT interaction with proteins, one can observe that the binding affinity determined from the ThT fluorescence intensity's dependence on protein concentration may be orders of magnitude lower than that determined using τ. Here we show that this discrepancy at least partly originates from a limited temporal resolution when determining the fluorescence lifetime of ThT in the ThT-protein system using the time-correlated single photon counting technique (TCSPC), which is usually characterized by a ~100 ps instrument response function. This results in the situation when a small fraction (~1%) of ThT molecules with a relatively slow decay (τ ~ 1000 ps) completely disguises the impact of ThT molecules with an ultrafast decay (τ ~ 1 ps) to the overall measured fluorescence decay curve. Moreover, using the femtosecond-resolved fluorescence up-conversion technique, we demonstrate that not only free ThT molecules but also a subpopulation of protein-bound ThT molecules exhibits fluorescence decay on a 1 ps timescale. The obtained results are of critical importance for a reliable interpretation of protein binding and aggregation experiments when using a ThT assay with a fluorescence lifetime determined by the TCSPC as an indicator.

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)

Actinic keratosis (AK) is considered as squamous cell carcinoma in situ (SCC), which is developing after a cumulative exposition to ultraviolet radiation [1]. The non-invasive in vivo imaging of healthy human skin and skin affected by AK and SCC is presented using two-photon tomography (TPT). Figure 1 shows images obtained in vivo at different depths.

An improvement of the penetration efficiency combined with the controlled release of actives in the skin can facilitate the medical treatment of skin diseases immensely. Dexamethasone (Dx), a synthetic glucocorticoid, is frequently used for the treatment of inflammatory skin diseases. To investigate the penetration of nano-sized lipid particles (NLP) loaded with Dx in comparison to a commercially available base cream, different techniques were applied. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the penetration of Dx, which was covalently labeled with the spin probe 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA). The penetration into hair follicles was studied using confocal laser scanning microscopy (CLSM) with curcumin-loaded NLP. The penetration of the vehicle was followed by confocal Raman microscopy (CRM). Penetration studies using excised porcine skin revealed a more than twofold higher penetration efficiency for DxPCA into the stratum corneum (SC) after 24h incubation compared to 4h incubation when loaded to the NLP, whereas when applied in the base cream, almost no further penetration was observed beyond 4h. The distribution of DxPCA within the SC was investigated by consecutive tape stripping. The release of DxPCA from the base cream after 24h in deeper SC layers and the viable epidermis was shown by EPR. For NLP, no release from the carrier was observed, although DxPCA was detectable in the skin after the complete SC was removed. This phenomenon can be explained by the penetration of the NLP into the hair follicles. However, penetration profiles measured by CRM indicate that NLP did not penetrate as deeply into the SC as the base cream formulation. In conclusion, NLP can improve the accumulation of Dx in the skin and provide a reservoir within the SC and in the follicular infundibula.

Background In humans, topically applied nanocarriers penetrate effectively into the hair follicles where they can be exploited for the localized and targeted treatment of skin disorders. Objective The objective of the present study was to examine the applicability of particle-based systems for follicular drug delivery in companion animals and livestock, which have a large follicular reservoir. Animals Skin samples from 10 beagle dogs, 14 Wistar rats and four ears from freshly slaughtered cross-bred pigs were used. Methods Fluoresceinamine labelled poly (L-lactide-co-glycolide) nanocarriers (256 or 430 nm) were applied on the different skin samples. After penetration, skin biopsies were removed and cryohistological cross sections prepared and investigated with regard to the follicular penetration depths (in μm ± standard deviation) of the nanocarriers using confocal laser scanning microscopy. Results In canine, rat and porcine hair follicles, the smaller nanoparticles were detected at mean follicular penetration depths of 630.16 ± 135.75 μm, 253.55 ± 47.36 μm and 653.40 ± 94.71 μm, respectively. The larger particles were observed at average follicular depths of 604.79 ± 132.42 μm; 262.87 ± 55.25 μm and 786.81 ± 121.73 μm, respectively, in canine, rat and porcine hair follicles. Statistically significant differences (P < 0.05) in the mean follicular penetration depths of the differently sized nanocarriers could be determined for the canine and porcine skin samples. Conclusion The mean follicular penetration depths of the differently sized nanocarriers were mostly significantly different between the different species, which might be due to different species-specific follicular dimensions. This issue needs to be addressed specifically in further studies.

The intercellular lipid structure of the stratum corneum (SC) plays a key role in skin barrier function. A depth profile of the intercellular lipid conformation and the lipid lateral packing order were measured in vivo in the human SC using confocal Raman microscopy. The depth profiles of the 2880 cm(-1)/2850 cm(-1) peak ratio intensity, which represent the C-H stretching and lateral packing order of lipids, and the 1080 cm(-1)/(1130 cm(-1) + 1060 cm(-1)) peak ratio, which represents the C-C skeleton vibration and trans-gauche conformation order of lipids, were investigated. The influence of keratin on the lipid peaks at 2850 cm(-1) and 2880 cm(-1) was excluded by the developed mathematical algorithm. The results show that the trans-conformation and lateral packing order of the intercellular lipids reach their maximum value in the SC at 20-40% of its depth and then decrease towards the stratum granulosum. These results show that at a depth of 20-40% (normally corresponding to a depth of 4-8 μm) the SC exhibits the most ordered lipids and therefore the highest skin barrier function. The lateral packing of lipids is more disordered on the surface and in the deeper parts of the SC, which may be associated with a reduced skin barrier function.

Die Dermatologie ist ein geeignetes Fachgebiet, um optische und spektroskopische Untersuchungen zur Analyse der Haut, aber auch zur Penetration topisch applizierter Substanzen durchzufuhren. Der Penetration topisch applizierter Substanzen in Form von Arzneimitteln und Pflegeprodukten kommt hierbei eine besondere Bedeutung zu. Wahrend laserscanmikroskopische Untersuchungen hauptsachlich dazu dienen, die zellulare Struktur abzubilden, wird mit Hilfe der Raman-Spektroskopie die inelastische Streuung von Licht an Molekulen oder Festkorpern erfasst. Damit ermoglicht die Raman-Spektroskopie den Nachweis von Stoffen in und auf der Haut. Hierbei wird Laserlicht in die Haut eingestrahlt. Neben der eingestrahlten Frequenz werden noch weitere Frequenzen beobachtet, deren Unterschiede zur Frequenz des eingestrahlten Lichts Informationen uber die Rotationsschwingungs- und Photonen- oder Spin-Flip-Prozesse liefern. Mit den erhaltenen Spektren konnen Substanzen selektiv nachgewiesen werden.

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).

Background/Aims: Inconsistent evidence is available that different ethnic groups exhibit differences in skin physiological parameters. Recently, variations in hair follicle morphology have been described, although the influence of such variations on the follicular penetration process has not been investigated until now. Methods: The aim of the present study was thus to investigate skin physiological parameters, follicle morphology and the penetration process in different ethnic groups. Results and Conclusion: Whereas no significant differences with regard to skin physiological parameters could be observed, morphological analysis of the hair follicles revealed, inter alia, that Caucasians had significantly larger terminal hair follicles than Asians and Africans. The surface of the hair follicle infundibulum was shown to be 3% in Caucasians, 2.6% in Africans and 2.4% in Asians. The investigations into penetration revealed no significant differences after a 30-min penetration time, whereas after 24 h, the Asian volunteers presented significantly larger amounts of sodium fluorescein in the hair follicles and the stratum corneum, which may be explained by cultural habits.

The increased ability of TMX-202 (derivative of imiquimod) to penetrate the intact stratum corneum (SC) and the follicular orifices of porcine ear skin was shown ex vivo using confocal Raman microscopy and laser scanning microscopy. Moreover, to assess whether TMX-202 is able to reach the immune cells, Langerhans cells extracted from pretreated human skin were investigated ex vivo using confocal Raman microscopy combined with multivariate statistical methods. Tracking the Raman peak of dimethyl sulfoxide centered at 690 cm(−1), the absorption of TMX-202 containing formulation by Langerhans cells was shown. To answer the question whether the TMX-202 active ingredient is able to reach Langerhans cells, the attraction of immune cells to TMX-202 containing formulation treated skin was measured in the in vivo rodent model Mastomys coucha. The results show that TMX-202 active ingredient is able to reach Langerhans cells after penetrating through the intact skin and subsequently attract immune cells. Both the intercellular/transcellular as well as the follicular pathways allow the penetration through the intact barrier of the SC.

We propose a fluorescence method for protein content assessment in fine house dust, which can be used as an indicator of the hygienic state of occupied rooms. The results of the measurements performed with 30 house dust samples, including ultrafiltration experiments, strongly suggest that the fluorescence emission of house dust extracts excited at 350 nm is mainly due to protein fragments, which are presumably keratin hydrolysates. This suggestion is supported by several facts: (i) Spectral band shapes for all the samples under investigation are close and correspond to that of keratin; (ii) fluorescence intensity correlates with the total protein content as provided by Lowry assay; (iii) treatment of the samples with proteinase K, which induces keratin hydrolysis, results in fluorescence enhancement without changing fluorescence band shape; and (iv) Raman spectra of keratin and fine house dust samples exhibit a very similar structure. Based on the obtained results and literature data, we propose a hypothesis that keratin is a major substrate for fluorescence species in fine house dust, which are responsible for emission at 350-nm excitation.

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 human skin, as the boundary organ between the human body and the environment, is under the constant influence of free radicals (FR), both from the outside in and from the inside out. Carotenoids are known to be powerful antioxidant substances playing an essential role in the reactions of neutralization of FR (mainly reactive oxygen species ROS). Carotenoid molecules present in the tissue are capable of neutralizing several attacks of FR, especially ROS, and are then destroyed. Human skin contains carotenoids, such as α-, γ-, β-carotene, lutein, zeaxanthin, lycopene and their isomers, which serve the living cells as a protection against oxidation. Recent studies have reported the possibility to investigate carotenoids in human skin quickly and non-invasively by spectroscopic means. Results obtained from in-vivo studies on human skin have shown that carotenoids are vital components of the antioxidative protective system of the human skin and could serve as marker substances for the overall antioxidative status. Reflecting the nutritional and stress situation of volunteers, carotenoids must be administered by means of antioxidant-rich products, e.g., in the form of fruit and vegetables. Carotenoids are degraded by stress factors of any type, inter alia, sun radiation, contact with environmental hazards, illness, etc. The kinetics of the accumulation and degradation of carotenoids in the skin have been investigated.

Background High doses of sun-emitted UV-radiation induce reactive oxygen species (ROS) as major pro-oxidants thus inducing premature skin aging. The best prevention of the destructive action of free radicals in human skin is textile coverings, topical sunscreens and the development of a high antioxidative protective network. Objective The effects of topical, systemic and combined application of antioxidants (AO) were investigated on human skin in vivo. Methods Topical application of creams and systemic incorporation of tablets both containing AO was investigated in vivo by resonance Raman spectroscopy. Results Topical, systemic and combined AO-treatments induced a statistically significant increase of AO levels in human skin while placebo did not show any changes. The highest accumulation was induced by the combination of topical and systemic AO. Carotenoid-tablets combined with placebo-cream induced less carotenoid accumulation than carotenoid-tablets alone. Carotenoid levelling after the end of treatment lasted for around 2 weeks following the topical application of AOs, and up to 5 weeks after systemic administration, depending on the BMI of volunteers. Conclusion Topically applied AO are stored in the SC for a short time only due to the rapid AO-depletion by desquamation, textile contact, washing and environmental stress. In contrast to topical application, the systemically applied carotenoids are stored in the body fat tissue and slowly released onto the skin surface with sweat and sebum. The combined topical and systemic application of AO represents an optimal form of protection of the AO-network.

Aiming at the development of strategies to prevent the hand-foot-syndrome, we propose to evaluate the amount of chemotherapeutics in the human skin together with carotenoids the latter serving as marker substances for the dermal antioxidative status. This approach is demonstrated by applying two-color Raman spectroscopy at 785 and 532 nm excitation for selective detection of chemotherapeutics and carotenoids, respectively. Porcine ear skin has proven to be suited as a model for corresponding spectroscopic basic in-vitro investigations.

Introduction: Intakes of fruit and vegetables rich in carotenoids are associated with a lower risk for a variety of chronic diseases. Therefore, the carotenoid levels in the skin have been measured in various studies to investigate the effects of nutrition and lifestyle. However, statistically clear data regarding the influence of age, gender, body mass index, and smoking behaviour were not documented. Methods: Thus, non-invasive resonance Raman measurements were performed on 151 healthy volunteers in Berlin, Germany. Results: The investigations have shown significantly enhanced total carotenoid values in the skin for women and non-smokers; individuals with a BMI lower than 30 also showed a trend to higher values. In the case of lycopene, significantly enhanced values were found in people younger than 40 and nonsmokers. Conclusion: Influences of gender, and smoking or non-smoking must be taken into account when carrying out a study with respect to the carotenoids. Age does not play any role for total carotenoids but for lycopene.

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.

Skin functions and structure are significantly influenced by nutrients. Antioxidants protect the supportive layer of the skin against any damaging irradiation effects and the action of free radicals. A lack of suitable methods means that the pharmacokinetic properties of systemically applied carotenoids transferred into the skin remain poorly understood. In this study, a natural kale extract or placebo oil were given orally to 22 healthy volunteers for 4 weeks. Carotenoid bioaccessibility was evaluated using non-invasive resonance Raman spectroscopy on the palm and forehead skin. For the analysis of the blood serum, the standard HPLC method was used. The blood and skin levels of the carotenoids increased significantly during the study but compared to the blood serum values, increases in skin were delayed and depended on the dermal area as well as on the carotenoid. Lycopene, measured as being low in the extract, increases more in the skin compared to the blood indicating that the natural mixture of the extract stabilizes the antioxidative network in the skin. After supplementation had ended, the carotenoids decreased much faster in the blood than in the skin. The delayed decrease in the skin may indicate a peripheral buffer function of the skin for carotenoids.

A system for shifted excitation resonance Raman spectroscopy (SERRDS) suitable for the application in medical practice for the in vivo detection of carotenoids in human skin is presented. This system comprises a miniaturized (150 mm × 27 mm × 12 mm) handheld probe and a wavelength-tunable diode laser-based 488 nm SHG light source. The diode laser provides two closely spaced excitation wavelengths. In parallel with the resonance excitation of carotenoids in the skin, SERRDS separates the fluorescence background from the Raman peaks. Inhomogeneities of human skin are averaged by the applied spot diameter of 3 mm. The implemented optics are designed for a detection of carotenoids over the whole excitation spot area. The system was calibrated using skin phantoms, resulting in a detection limit of 0.03 nmol g−1 (beta-carotene per gram of skin/tissue) which is more than one order of magnitude below the average beta-carotene concentration in human skin.

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.

Two-photon excited fluorescence of red blood cells (RBC) has been reported to be applicable for their assessment in vitro and in vivo. The corresponding fluorescence emission was ascribed to hemoglobin (Hb), however, as Hb is essentially non-fluorescent at single-photon excitation, the mechanism of two-photon excited fluorescence of RBC remains debatable. Here we show that a fluorescent photoproduct, characterized by an ultrafast decay of excitation, is formed after irradiation of Hb with femtosecond laser pulses with ca. 8 10−5 quantum yield, and that it is also fluorescent at single-photon excitation. The formation of a similar photoproduct was also shown for Hb continuous wave irradiation with blue light with ca. 10−5 formation quantum yield. The kinetics of the Hb photoproduct formation and its spectral properties were investigated. The obtained results clarify the processes responsible for RBC fluorescence observed in two-photon microscopy experiments.

Animal skin is widely used in dermatological free radical research. Porcine ear skin is a well-studied substitute for human skin. The use of bovine udder skin is rare but its high carotenoid content makes it particularly appropriate for studying the redox state of the skin. Yet, information on the suitability of animal skin for the study of external hazard effects on the redox state of human skin has been lacking. In this study, we investigated the activity of the antioxidant enzyme catalase and the carotenoid content defining the redox status as well as UV-induced radical formation of human, porcine ear and bovine udder skin ex vivo. In human skin only low levels of radical formation were detected following UV irradiation, whereas bovine skin contains the highest amount of carotenoids but the lowest amount of catalase. Porcine ear skin does not exhibit a carotenoid signal but its catalase activity is close to human skin. Therefore, radical formation can neither be correlated to the amount of catalase nor to the amount of carotenoids in the skin. All skin types can be used for electron paramagnetic resonance-based detection of radicals, but porcine skin was found to be the most suitable type.

A surface determination method for the application of 3D confocal Raman microscopy on inhomogeneous skin sections has been presented, which is based on depth profiles of the keratin contribution of the acquired Raman spectra. The method was compared to two similar auto-focusing methods that are based on the intensity of the reflected excitation light and Raman spectra, respectively. The measurements were performed on hair follicles containing skin sections of porcine ears ex vivo. The surface determination on such samples is especially challenging due to their different molecular composition and surface inhomogeneity. An advantage of this method is molecular sensitivity, whereby only the surface of the sample will be detected and not the substrate of the microscope slide, in the case of disruptions during the processing of samples. A disadvantage of the method is the increased overall acquisition time if only the surface spectra are to be applied for 2D mapping.

Resonance Raman spectroscopy was used for the fast in vivo detection of the concentration of carotenoid antioxidant substances such as beta-carotene and lycopene in human skin and for the measurement of their degradation dynamics, subsequent to infrared (IR) irradiation emitted by two different IR radiators applied at the same power density. One of the radiators was equipped with a water filter in front of the radiation source (WIRA) and the other was a usual broadband system without a water filter (standard IR radiator – SIR). It was found that the SIR exerted a higher influence on the degradation of carotenoids in the skin than the WIRA. Furthermore, all twelve volunteers who participated in the study felt that the irradiation with the SIR was disagreeably warmer on the skin surface compared to the WIRA, in spite of the same power density values for both radiators on the skin surface. The average degradation magnitude of the carotenoids in the skin of all volunteers after an IR irradiation was determined at 23% for WIRA and 33% for the SIR. A correlation (R2 ~ 0.6) was found between the individual level of carotenoids in the skin of the volunteers and the magnitude of degradation of the carotenoids for both IR radiators. Taking the previous investigations into consideration, which clearly showed production of free radicals in the skin subsequent to IR irradiation, it can be concluded that during the application of WIRA irradiation on the skin, fewer radicals are produced in comparison to the SIR.

In the present study, the distribution of the carotenoids as a marker for the complete antioxidative potential in human skin was investigated before and after the topical application of carotenoids by in vivo Raman spectroscopy with an excitation wavelength of 785 nm. The carotenoid profile was assessed after a short term topical application in 4 healthy volunteers. In the untreated skin, the highest concentration of natural carotenoids was detected in different layers of the stratum corneum (SC) close to the skin surface. After topical application of carotenoids, an increase in the antioxidative potential in the skin could be observed. Topically applied carotenoids penetrate deep into the epidermis down to approximately 24 μm. This study supports the hypothesis that antioxidative substances are secreted via eccrine sweat glands and/or sebaceous glands to the skin surface. Subsequently they penetrate into the different layers of the SC. A graph is presented. Typical Raman spectrum of untreated skin (forearm, 10 μm depth) under the excitation wavelength of 785 nm.

Human skin is exposed to infrared radiation (IR) from natural and artificial sources. In previous studies, near IR radiation (IRA; 760-1,440 nm) was shown to elicit a retrograde mitochondrial signaling response leading to induction of matrix metalloproteinase-1 (MMP-1) expression. These studies, however, have exclusively employed cultured human skin fibroblasts ex vivo. Here, we have assessed the in vivo relevance of these observations by exposing healthy human skin in vivo to physiologically relevant doses of IRA. Eighty percent of the tested individuals responded to IRA radiation by upregulating of MMP-1 expression. Specifically, IRA irradiation caused increased expression of MMP-1 in the dermis, but not in the epidermis. Raman spectroscopy revealed that IRA radiation also caused a significant decrease in the antioxidant content of human skin. In vitro studies had previously shown that IRA-induced MMP-1 expression was mediated through an oxidative stress response, which originates from the mitochondrial electron transport chain. We now report that incubation of cultured human dermal fibroblasts or treatment of human skin with specific antioxidants prevented IRA radiation-induced MMP-1 expression in vitro and in vivo. Thus, IRA irradiation most likely promotes premature skin aging and topical application of appropriate antioxidants represents an effective photoprotective strategy.

Antioxidant substances in the skin are expected to slow down photo ageing. We therefore developed the hypothesis that high levels of antioxidant substances may be correlated to lower levels of skin roughness. By utilizing modern optical non-invasive in vivo methods, the structures of the furrows and wrinkles as well as the concentration of lycopene were analyzed quantitatively on the forehead skin of 20 volunteers aged between 40 and 50 years. In a first step, the age of the volunteers was correlated to their skin roughness. Here, no significant correlation was found. In a second step, a significant correlation was obtained between the skin roughness and the lycopene concentration (R=0.843). These findings indicate that higher levels of antioxidants in the skin effectively lead to lower levels of skin roughness, and therefore support our hypothesis.

In the present paper the application of optical non-invasive methods in dermatology and cosmetology is discussed. Laser scanning microscopy (LSM) and optical coherent tomography (OCT) are the most promising methods for this application. Using these methods, the analysis of different skin parameters like dryness and oiliness of the skin, the barrier function and the structure of furrows and wrinkles are discussed. Additionally the homogeneity of distribution of topically applied creams, as well as their penetration into the skin were investigated. It is shown that these methods are highly valuable in dermatology for diagnostic and therapy control and for basic research, for instance in the field of structure analysis of hair follicles and sweat glands. The vertical images of the tissue produced by OCT can be easily compared with histological sections. Unfortunately, the resolution of the OCT technique is not high enough to carry out measurements on a cellular level, as is possible by LSM. LSM has the advantage that it can be used for the investigation of penetration and storage processes of topically applied substances, if these substances have fluorescent properties or if they are fluorescent-labelled.

Various fruit, vegetables and plants contain a high amount of carotenoids such as beta-carotene, lycopene, lutein, zeaxanthin, and other antioxidants in different concentrations and combinations. These substances used by nature are an effective defense mechanism against the destructive action of free radicals, which are the result of the negative influence of the environment. It was found that the peel of fruit and vegetables usually contains a higher amount of carotenoids than the pulp. Humans cannot synthesize carotenoids by itself, thus, they should be applied systemically with a supplementation rich in these substances. The diet, based on the consumption of a high amount of carotenoid rich products, increased the concentration of carotenoids in the human skin. Taking into consideration the results of previous studies, there is a strong correlation between the appearance of the skin concerning furrows and wrinkles and the level of carotenoid lycopene obtained in the skin. The present results demonstrate that a diet rich in products, which contain a high amount of carotenoids, can be an efficient strategy to reduce skin aging. In the present study, resonance Raman spectroscopy was used as a fast non-invasive optical method for the determination of the carotenoid antioxidant substances in fruit, vegetables and for the determination of the influence of a carotenoid rich diet on the antioxidant level of the human skin.

Resonance Raman spectroscopy was used as a fast and non-invasive optical method for the determination of the influence of IR radiation on the degradation of carotenoid antioxidant substances beta-carotene and lycopene in the human skin. It was found that the beta-carotene and lycopene concentration in the skin are decreased almost immediately subsequent to IR irradiation. The magnitude of degradation of carotenoids in the skin was determined at 27% for beta-carotene and 38% for lycopene on average. A strong correlation between the individual level of beta-carotene and lycopene in the skin of volunteers and the magnitude of destruction of carotenoids beta-carotene and lycopene in the skin was found to be R 2=0.78 and R 2=0.89 correspondingly. Taking into consideration the absence of absorption in the infrared range of spectra and the relative heat stability of carotenoids up to 50° C, the action of free radicals, which can be produced subsequent to IR irradiation in the skin, can be the main purpose for the degradation of carotenoid substances in the skin.

Propylene glycol is one of the known substances added in cosmetic formulations as a penetration enhancer. Recently, nanocrystals have been employed also to increase the skin penetration of active components. Caffeine is a component with many applications and its penetration into the epidermis is controversially discussed in the literature. In the present study, the penetration ability of two components - caffeine nanocrystals and propylene glycol, applied topically on porcine ear skin in the form of a gel, was investigated ex vivo using two confocal Raman microscopes operated at different excitation wavelengths (785nm and 633nm). Several depth profiles were acquired in the fingerprint region and different spectral ranges, i.e., 526-600cm(-1) and 810-880cm(-1) were chosen for independent analysis of caffeine and propylene glycol penetration into the skin, respectively. Multivariate statistical methods such as principal component analysis (PCA) and linear discriminant analysis (LDA) combined with Student's t-test were employed to calculate the maximum penetration depths of each substance (caffeine and propylene glycol). The results show that propylene glycol penetrates significantly deeper than caffeine (20.7-22.0μm versus 12.3-13.0μm) without any penetration enhancement effect on caffeine. The results confirm that different substances, even if applied onto the skin as a mixture, can penetrate differently. The penetration depths of caffeine and propylene glycol obtained using two different confocal Raman microscopes are comparable showing that both types of microscopes are well suited for such investigations and that multivariate statistical PCA-LDA methods combined with Student's t-test are very useful for analyzing the penetration of different substances into the skin.

The objective of the study was to investigate the influence of a food supplement rich in antioxidants on the antioxidant status of the skin. For this reason, the blue-green algae Spirulina platensis powder was used for oral application during eight weeks. The effect of oral application of the antioxidant-containing Spirulina platensis on characteristic skin aging parameters, e.g., concentration of cutaneous carotenoids and the collagen/elastin index (SAAID), was investigated in vivo. A significant average increase from 2.67 ± 0.86 arb. units to 3.25 ± 0.93 arb. units (p &lt, 0.001) in the cutaneous carotenoid concentration was detected subsequent to oral application of the carotenoid-containing Spirulina platensis powder, showing a significant improvement of the antioxidant status of the skin. A slight but not significant increase (p = 0.33) in the dermal SAAID mean values was measured from −0.54 ± 0.11 to −0.51 ± 0.11 subsequent to oral intake of Spirulina platensis powder.

The understanding of transdermal substance penetration pathways remains an important field for the development of future topical drugs and cosmetics. Laser Doppler flowmetry is a well-established method for evaluating cutaneous perfusion. In a study on 6 healthy male volunteers, we topically applied the vasoactive substance benzyl nicotinate on two test areas with open and obturated hair follicles and measured changes in the blood flow by Doppler flowmetry. Contrary to occluded follicles, the application onto the test area with open follicles led to a statistically significant perfusion increase within the first 5 minutes, emphasizing the importance of the follicular pathway for epidermal penetration.

Background: The formation of free radicals in human skin by solar ultraviolet radiation is considered to be the main reason for extrinsic skin aging. The antioxidants in human tissue represent an efficient protection system against the destructive action of these reactive free radicals. In this study, the parameters of the skin, epidermal thickness, stratum corneum moisture, elasticity and wrinkle volume, were determined before and after the treatment with antioxidant- or placebo-containing tablets and creams. Methods: The study included 5 groups of 15 volunteers each, who were treated for 2 months with antioxidant-containing or placebo tablets, creams or a combination of antioxidant-containing tablets and cream. The skin parameters were measured at time point 0 and at week 8 utilizing ultrasound for the determination of epidermal thickness, a corneometer for stratum corneum moisture measurements, skin profilometry for quantifying the wrinkle volume and a cutometer for determining the elasticity. Results: The verum cream had a positive influence on epidermal thickness, elasticity and skin moisture, but the verum tablets improved the epidermal thickness only. The combined application of verum tablets and creams led to a significant improvement of all investigated skin parameters, whereas the application of placebo tablets or cream did not influence any parameters. Conclusion: The topical and oral supplementation of antioxidants can be an instrument to improve several skin parameters and potentially counteract or decelerate the process of extrinsic skin aging.

Skin aging is determined by genetic aspects and by the action of free radicals. At small concentrations, these reactive molecules contribute essentially to signaling processes in the human organism. If their concentration exceeds a critical threshold, the free radicals can destroy cells and cell compartments. The human organism has developed a protective antioxidant system against the detrimental action of free radicals. Most of these antioxidants cannot be produced in the human body but have to be ingested with food rich in antioxidants, for instance with fruit and vegetables. Resonance Raman and reflectance spectroscopy was used to detect carotenoids, which represent marker substances for the whole antioxidative potential in human skin. It could be demonstrated that the antioxidant status of the skin reflects the nutritional and stress behavior of human subjects. Increasing the concentration of cutaneous antioxidants by topical or systemic application could be an appropriate way to reduce the destructive action of the free radicals. Consequently, high antioxidant concentrations in human skin might be an efficient mechanism to prevent skin aging.

A non-invasive optical method forin vivodetermination of the concentration of carotenoid antioxidant substance lycopene in the skin, based on resonance Raman spectroscopy, is presented. The Ar+laser with an excitation wavelength at 514.5 nm was used, which corresponds to the maximum of the absorption of lycopene. This wavelength excited more lycopene than other carotenoids, because of the different absorption spectra of the carotenoids in the green spectral range. The absence of the reabsorption of carotenoids at the present wavelength and the elimination of the influences of heterogeneities of the skin, such as microstructure and pigmentation on Raman measurements by the use of a broad excitation laser beam, allows lycopene to be measured at a high accuracy. The utilization of the measurements of back reflected light from the skin allowed measurements to be performed on volunteers with all skin types.

It is well known from the literature that carotenoid antioxidant substances decrease after the irradiation of the skin with UV light. Available literature has shown no information about the response time and total dynamics of degradation of carotenoid antioxidants after UV irradiation. The measurements were made with the HPLC method, which is time-consuming and does not give relevant information about the dynamics of degradation of carotenoids in the skin after UV irradiation. With the introduction of new noninvasive spectroscopic methods, it became possible to measure in vivo the behavior of carotenoid antioxidant substances, such as betacarotene and lycopene in the skin after UV light exposure. In the present study, the resonance Raman spectroscopy method was used as a fast and noninvasive optical method to measure the dynamics of degradation of beta-carotene and lycopene in living human skin after UV exposure. It was found that the beta-carotene and lycopene concentration in the skin does not decrease immediately after UV irradiation. There is a time delay, which varies from 30 up to 90 min for beta-carotene and from 0 up to 30 min for lycopene. A strong nonlinear correlation between the individual antioxidant level of volunteers and the magnitude of destruction of antioxidants in the skin was found.