Determination of the antioxidative capacity of the skin in vivo using resonance Raman and electron paramagnetic resonance spectroscopy

Abstract: 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.     

In vivo measurement of human dermis by 1064 nm-excited fiber Raman spectroscopy.

BACKGROUND/AIMS: Although chemical information on the dermis in vivo is highly important in skin research, an efficient method for gathering this information is yet to be developed. Here, we demonstrate that newly developed near-infrared (1064 nm) excited Raman spectroscopy is a powerful method for chemical analysis of human skin in vivo. METHODS: We used a laboratory-constructed Raman spectrometer equipped with a highly sensitive near-infrared detector (Hamamatsu Photonics), an optical fiber probe and a 1064 nm Nd:YAG laser. Raman spectra of porcine skin (in vitro) and human skin (in vivo) were measured with this spectrometer. RESULTS: The Raman spectrum of porcine skin measured from the outer side resembles that of the dermis more than that of the epidermis. The Raman spectra of human skin (cheek, forehead, inner forearm, outer forearm, palm) depend on the portion measured with the probe. The spectra of the forehead and inner forearm show larger lipid signals than that of the palm. CONCLUSIONS: The Raman spectrum of skin measured with the 1064 nm Raman system primarily reflects the chemical composition of the dermis. The 1064 nm excited Raman spectroscopy is useful for research of the dermis and skin appendages.     

The non‐homogenous distribution and aggregation of carotenoids in the stratum corneum correlates with the organization of intercellular lipids in vivo

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.     

Carotenoids in human skin

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.     

Non-invasive raman spectroscopic detection of carotenoids in human skin

Carotenoids are thought to play a significant part in the skin's anti-oxidant defense system, and may help prevent malignancy. Inability to measure skin carotenoid content readily has, however, made it difficult to establish the relationship between carotenoid concentration and the occurrence of cutaneous malignancy. We have measured in vivo carotenoid concentration using a noninvasive optical method, Raman spectroscopy. To validate our instrumentation, abdominoplasty skin was evaluated by both Raman spectroscopy and high-performance liquid chromatography determination for carotenoid content. Evaluation of the Raman signal in specific carotenoid solutions was also performed. Precision of Raman measurements within skin sites, within subjects, and between subjects was measured. Sensitivity of the method was evaluated as a function of anatomical region and the distribution of carotenoids within the stratum corneum. Lastly, we evaluated the Raman signal in actinic keratosis and basal cell carcinoma lesions and perilesional skin and compared this with region-matched sites in healthy subjects. Our results indicate that the Raman scattering method reflects the presence of carotenoids in human skin and is highly reproducible. Evaluation of five anatomical regions demonstrated significant differences in carotenoid concentration by body region with the highest carotenoid concentration noted in the palm. Comparison of carotenoid concentrations in basal cell carcinomas, actinic keratosis, and their perilesional skin demonstrate a significantly lower carotenoid concentration than in region-matched skin of healthy subjects. These results represent the first evidence that carotenoid concentration in the skin correlate with the presence or absence of skin cancer and precancerous lesions.     

Comparison of the depth profiles of water and water-binding substances in the stratum corneum determined in vivo by Raman spectroscopy between the cheek and volar forearm skin: effects of age, seasonal changes and artificial forced hydration

BACKGROUND: Dermatologists and cosmetic scientists are becoming increasingly interested in stratum corneum (SC) hydration because the SC plays an important role in keeping the skin surface soft and smooth. However, conventional in vivo noninvasive methods do not provide direct information about the depth profiles of water content or SC components that hold water. OBJECTIVES: To study the depth profiles of water and SC components in vivo by Raman spectroscopy, and to analyse the changes due to age, anatomical location, season and water application. METHODS: In vivo Raman spectra of the skin of the cheek and the volar forearm were obtained from 50 healthy Japanese volunteers of different ages (age range 22-76 years) with a confocal Raman spectrometer. The depth-dependent profiles of water and of SC water-binding components were calculated from the respective Raman spectra. RESULTS: The depth profile of the SC water content was observed in a pattern ranging from around 30% (water/wet tissue) at the outermost layer of the SC to about 70% at the deeper living layer. Although the water content at a depth of 10-30 microm in the forearm skin tended to be lower in older subjects than in younger subjects, no such difference was found in the much thinner SC of the cheek. Moreover, there was no seasonal difference in depth profile of water content from the mid part of the SC to an 80-microm depth from the skin surface both in the cheek and in the forearm. The water content of all the evaluated SC components showed a gradual decrease from the surface to deeper portions. The mean amounts of lactate in the forearm skin and cholesterol in the cheek skin were significantly higher in younger subjects than in older subjects. In contrast, the levels of free amino acids and trans-urocanic acid were higher in the forearm skin of older subjects than in younger subjects. The relative amounts of urea and lactate were the highest in summer, when that of trans-urocanic acid was the lowest. Prolonged water application on the forearm skin even for 90 min resulted in a remarkable increase in water content throughout the SC, even reaching the granular layer, which was only gradually released from the upper part of the SC after discontinuation of the hydration procedure. CONCLUSIONS: Our present findings suggest that changes in the concentration depth profiles of water, free amino acids and lipids in the skin depend on age, anatomical site and season. These findings indicate the important roles played by various water-holding substances in the SC in the regulation of SC water content.     

Millimeter wave reflectivity used for measurement of skin hydration with different moisturizers

Background/aims: A new non‐invasive method for determining the free water content in human skin has been developed. The method analyzes the reflection of millimeter (mm) wavelength electromagnetic waves. The amount of reflection of mm waves depends on an electrical property (namely, the permittivity) of the skin, and this depends upon the free water content of the various skin layers. The aim of the present study was to use the mm wave reflectometry method for determination of free water content in healthy skin treated with different hydrating substances. Methods: Skin lotion, pure water, glycerol, and petroleum jelly (an occlusive substance) were used for hydration of skin. The amount of free water was calculated using the permittivity values of skin layers found from fitting a three layer skin model to measured reflection data. The skin model consisted of (1) the stratum corneum (SC), (2) the viable epidermis plus the dermis, and (3) fat layers. Results: Mm wave reflection was significantly affected by the water content of the thick SC of the palm but not by the very thin SC of the forearm. Treatment of the forearm and palm skin with different hydrating substances produced notable changes of the free water content in the SC, but not in the viable epidermis or dermis. The greatest hydration was produced by pure water and skin lotion, and the lowest by petroleum jelly. However, petroleum jelly produced prolonged retention of water in the SC following its hydration by other moisturizers. The content of free water was found to return to its baseline value after removal of moisturizers in as short a time as 8.3 min. Conclusion: The study shows that mm wave reflectometry can be used as a sensitive technique for the non‐invasive determination of water content in living skin.     

Topical beta-carotene protects against infra-red-light-induced free radicals

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.     

In vivo evaluation of the protective capacity of sunscreen by monitoring urocanic acid isomer in the stratum corneum using Raman spectroscopy

Background/purpose: Urocanic acid (UCA) is a major ultraviolet (UV) ray‐absorbing component in the epidermis, and it isomerizes from the trans to the cis form upon exposure to UV radiation. Continuous measurement of UCA isomers in the skin at the same area is not available using conventional methods. This study aimed to evaluate the protective capacity of sunscreen by non‐invasively monitoring the trans‐UCA (t‐UCA) amount in the stratum corneum (SC) by confocal Raman spectroscopy. Methods: In vivo Raman spectra of the skin at the cheek or volar forearm were obtained from 27 healthy Japanese volunteers of different ages (age range, 22–53 years) throughout a whole year using confocal Raman spectroscopy. Eighteen healthy male Japanese volunteers (age range, 25–52 years) were enrolled for the evaluation of the protective capacity of sunscreen. The concentration depth profile of t‐UCA relative to keratin was calculated from the Raman spectra in the 400–2200 cm⁻¹ region. Then, the integrated amount within the depth of 0–12 μm was calculated, which represented the total amount of t‐UCA in the SC. Results: The integrated amount of t‐UCA in the cheek skin was significantly lower than that in the volar forearm skin throughout a year. The amount of it in the volar forearm skin was significantly the lowest in summer, but not in the cheek skin. The amount of t‐UCA decreased immediately after UV exposure even below 1 minimal erythema dose, remained low for 1 week, and gradually increased up to the initial level about 2 weeks after UV exposure. The decrease in the t‐UCA amount was hindered by the application of sunscreen on the skin surface. There were no statistical differences in response to UV exposure between the erythema‐positive and erythema‐negative groups. Conclusions: The monitoring of the amount of t‐UCA in the SC by confocal Raman spectroscopy is a good method to assess the efficacy of sun protective substances.     

Dermal carotenoid level and kinetics after topical and systemic administration of antioxidants: Enrichment strategies in a controlled in vivo study

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.     

Echographic assessment of age-dependent variations of skin thickness

Background/aims: Skin thickness relative to age is complex and may be influenced both by intrinsic changes, extrinsic changes related to sun exposure and constitution, including sex. Age-related changes may also differ in different anatomical sites. The object was to characterize skin aging relative to these influences.
Methods: A-mode and B-mode high-frequency (20 MHz) ultrasound examination was performed using the Dermascan C. 162 subjects were examined (91 women, 71 men) in the age range 27 to 90 years. 6 anatomical locations (forehead, cheek, volar forearm, dorsal forearm, upper abdomen, buttocks) were studied.
Results: Females have thinner skin than men. Subjects aged more than 70 years have significant thinning of the skin. Age-related thinning is more pronounced in women. Relative skin-thickness decrease is less at sun-exposed areas in comparison with protected areas.
Conclusion: Ultrasund examination allows characterization of skin aging, and the influences of sex and anatomical site with sun-induced changes were differently superimposed on intrinsic changes and constitution.     

Functional food and bioavailability in the target organ skin

Reactive free radicals can be produced in the skin by the action of environmental factors, such as sun radiation and toxins. These radicals can damage the DNA, proteins and lipids of the living cells. The consequences can be skin aging, immune suppression and even skin cancer. Humans have developed a protective mechanism against the action of free radicals in the form of antioxidant substances. Several of these antioxidants cannot be produced by humans and have to be acquired via food, such as carotenoids. Optical, non-invasive methods, like resonance Raman spectroscopy, allow a qualitative and quantitative online detection of the kinetics of antioxidants such as carotenoids in the skin. By employing this method it has been shown that the uptake of carotenoids in food can lead to an accumulation in the skin. On the other hand, stress, illness and UV-radiation can reduce the concentration of antioxidant substances in the skin. A high concentration of antioxidant substances is protective and associated with a reduction in skin wrinkling.     

Functional Food und Bioverfügbarkeit im Zielorgan Haut

Reactive free radicals can be produced in the skin by the action of environmental factors, such as sun radiation and toxins. These radicals can damage the DNA, proteins and lipids of the living cells. The consequences can be skin aging, immune suppression and even skin cancer. Humans have developed a protective mechanism against the action of free radicals in the form of antioxidant substances. Several of these antioxidants cannot be produced by humans and have to be acquired via food, such as carotenoids. Optical, non-invasive methods, like resonance Raman spectroscopy, allow a qualitative and quantitative online detection of the kinetics of antioxidants such as carotenoids in the skin. By employing this method it has been shown that the uptake of carotenoids in food can lead to an accumulation in the skin. On the other hand, stress, illness and UV-radiation can reduce the concentration of antioxidant substances in the skin. A high concentration of antioxidant substances is protective and associated with a reduction in skin wrinkling.     

Stratum corneum adhesive tape stripping: influence of anatomical site, application pressure, duration and removal

BACKGROUND: Tape stripping is a common method for investigating stratum corneum (SC) physiology as well as bioavailability and bioequivalence of topical drugs. OBJECTIVES: To investigate the influence of procedures (anatomical site, pressure, pressure duration, tape removal rate) inherent in each stripping protocol on changes in skin physiology. METHODS: Tape stripping was performed using tapes on the forearm, forehead and back. On the forearm different pressures (165 and 330 g cm(-2)), durations of pressure (2 and 10 s), and removal rate (slow and rapid removal) were used. Changes in skin physiology were evaluated by measurement of transepidermal water loss (TEWL) and hydration. RESULTS: A significant influence of all parameters on the TEWL increase as a function of tape strip number was observed. The fastest increase was demonstrated on the forehead, followed by the back and, lastly, the forearm. Rapid removal produced a protracted increase in comparison with slow removal. Pressure for 10 s induced a faster increase in TEWL than 2 s pressure. Likewise, pressure at 330 g cm(-2) induced an earlier increase than pressure at 165 g cm(-2). Skin hydration was not influenced by the variables tested. CONCLUSIONS: Tape stripping results are influenced dramatically by all investigated parameters. A dynamic SC stress test to investigate SC cohesion more closely is proposed based on the present observations.     

我国正常人皮肤表面皮脂和水分含量的研究

目的：了解我国不同年龄、不同性别正常人皮肤角质层含水量、皮肤表面皮脂含量及皮肤皮脂的分泌速度。方法：利用皮肤表面皮脂测量仪和皮肤水分含量测量仪对我国北方325名正常人的前额及前臂屈侧皮肤皮脂量和皮肤含水量进行测量。结果：12岁以前,男女前额皮脂量无差异;13岁以后,男性前额的皮脂量明显高于女性。除36～50岁年龄组外,女性前额的皮脂分泌快于男性。男女前臂的皮脂量无差异。除男性13～35岁组的前额皮肤含水量高于女性外,其他各年龄组各部位男、女皮肤含水量无显著差别。13～35岁组男、女性前额的含水量均高于前臂。其他各年龄组前额和前臂皮肤含水量无明显部位差异。13-35岁组男性前额皮肤含水量最高。结论：正常人皮肤表面含水量和皮脂含量因性别、年龄和部位的不同而有差异。     

Kinetics of blood flow after topical application of benzyl nicotinate on different anatomic sites

Cutaneous characteristics, e.g., thickness of the SC and density of follicles, affect the penetration of topically applied substances. In the present study, the penetration of benzyl nicotinate, causing a vasodilation, was studied on three anatomic sites (forearm, forehead and calf) differing in their skin characteristics. Therefore, the blood flow of the superficial dermal plexus and that of the larger capillaries in the deeper skin layers was simultaneously measured using a laser Doppler flowmeter. In addition, the cutaneous temperature and redness were determined as a function of time. These four biological reactions were measured in turn on a skin area treated with a gel containing benzyl nicotinate and on an untreated control area. The highest basal levels were observed on the forehead. Topical application of benzyl nicotinate resulted in an increase in each biological response. Compared to the other sites, the maximal values were reached earliest on the forehead, which also showed the fastest decrease. No significant differences were obtained comparing the kinetic data of the calf with that of the forearm. The results indicate an additional contribution by the numerous vellus hair follicles of the forehead to the penetration and exposure of the drug.     

Number and distribution of interstitial cells lining the epidermis of normal human skin from different anatomical locations

A study was conducted to determine the number and distribution of interstitial cells lining the epidermis of normal human skin from various anatomical locations. Five to seven normal human skin specimens per each anatomical site were collected from surgical specimens, mostly with pigmented nevi. Ten anatomical locations were classified; and a total of 65 normal human skin samples were evaluated. The number of interstitial cells lining the epidermis was quantified under light microscopy using a computer-assisted image analyzer. Two types of interstitial cells were recognized beneath the dermo-epidermal junction of normal human skin: cells containing oval nuclei and spindle-shaped cells containing elongated nuclei. As for the number of oval cells, no significant difference was found among the anatomical locations. In contrast, significantly greater numbers of spindle-shaped cells were found in the palm (4.11 + 1.24; p < 0.01), sole (3.52 + 0.83; p < 0.001) and buttock (2.52 + 0.49; p < 0.01), compared with those in the anterior trunk (0.60 + 0.22). In normal skin of the palm and sole, the number of spindle-shaped cells located beneath the apices of rete ridges (7.35 + 1.56) was significantly greater than along the dermal papillae (1.39 + 0.39, p < 0.01). However, cells containing oval nuclei also predominated beneath the apices of rete ridges, but the difference was not significant. In summary, the present study demonstrated that the number of spindle-shaped cells, quantified by H & E staining, was significantly greater beneath the apices of rete ridges than in dermal papillae. The number was greater in palm and sole skin compared with other samples of normal human skin. This data may relate to the glabrous nature of palm and sole skin.     

Frictional properties of human skin: relation to age, sex and anatomical region, stratum corneum hydration and transepidermal water loss

Differences in the dynamic skin friction coefficients (mu) were investigated with respect to age, sex, and anatomical region. A total of 29 volunteers consisting of seven young females, seven old females, seven young males, and eight old males participated in the study. Measurements were obtained from II anatomical regions, namely, the forehead, upper arm, volar and dorsal forearm, postauricular, palm, abdomen, upper and lower back, thigh, and ankle. The friction data were compared with stratum corneum hydration and transepidermal water loss (TEWL). The dynamic friction coefficient did not vary significantly between age and sex groups but varied considerably among the anatomical regions of the body. The forehead and postauricular had the highest mu (0.34 +/- 0.02) while the abdomen had the lowest (0.12 +/- 0.01); the remaining regions had an average mu value of 0.21 +/- 0.01. Similarly, no sex differences were observed for TEWL and stratum corneum hydration. Capacitance was only significantly lower on the palms of the elderly. Regional differences showed a higher state of hydration on the forehead and postauricular as well as the upper arm, upper and lower back when compared with the volar forearm. TEWL was generally lower in the elderly on all anatomical regions except the postauricular and palm. A significant correlation was established between mu and capacitance for most regions. Between mu and TEWL significant correlation was observed only on the palm and thigh. These findings suggest that frictional properties of skin are dependent on more than water content or non-apparent sweating and the role of sebum secretion is suggested as one possible factor.     

In vivo measurement of the water content in the dermis by confocal Raman spectroscopy

Background/purpose: Dermal water plays an important role in the physical properties of the skin. Recently, researchers have attempted to directly measure the dermal water content in vivo using magnetic resonance imaging, near infrared spectroscopy, and Raman spectroscopy. However, these methods have limitations. Although confocal Raman spectroscopy has been developed to measure the water content in the skin, no reports have suggested that this instrument can measure the dermal water content. This report describes a method for measuring the dermal water content in vivo using confocal Raman spectroscopy. Methods: We used a confocal Raman spectrometer and adjusted the laser exposure time and depth increments according to the skin depth. Age‐related changes in the dermal water content of the forearm were examined in 30 young and 30 elderly male subjects. Diurnal changes in the dermal water content of the forearm were examined in 12 elderly male subjects. Results: Adjusting the exposure time and depth increment dramatically improved the signal‐to‐noise ratios of the Raman spectra. Elderly dermis had significantly higher water content than young dermis. Moreover, the dermal water content displayed a diurnal change. Conclusion: This study demonstrates that the dermal water content can be measured in vivo using confocal Raman spectroscopy.     

Integrated real‐time Raman system for clinical in vivo skin analysis

Background: Raman spectroscopy is a non‐invasive optical technique that can probe the molecular structure and conformation of biochemical constituents. The probability of Raman scattering is exceedingly low (∼10⁻¹⁰), and consequently up to now the practical application of Raman spectroscopy to clinical medicine has been limited by either the weak spectral signal or by the long data acquisition times. Recent advances in Raman hardware and probe design have reduced spectral acquisition times, paving the way for clinical applications. Methods: We present an integrated real‐time Raman spectroscopy system for skin evaluation and characterization, which combines customized hardware features and software implementation. Real‐time data acquisition and processing includes CCD dark‐noise subtraction, wavelength calibration, spectral response calibration, intensity calibration, signal saturation detection, cosmic ray rejection, fluorescence background removal, and composition modeling. Real‐time in vivo Raman measurement of volar forearm skin is presented to illustrate the methods and modeling. Results: The system design implemented full‐chip vertical hardware binning to improve the signal‐to‐noise ratio by 16‐fold. The total time for a single in vivo measurement with analysis can be reduced to 100 ms with this implementation. Human skin was well modeled using the base Raman spectra. Conclusion: In vivo real‐time Raman can be a very promising research and practical technique for skin evaluation.