Synergistic effects of long-wavelength ultraviolet A1 and visible light on pigmentation and erythema.

BACKGROUND: Visible light (VL) induces multiple cutaneous effects. Sunscreen testing protocols recommended by regulatory bodies throughout the world require the use of solar simulators with spectral output in the ultraviolet (UV) domain only. However, sunlight contains VL and infrared radiation also. OBJECTIVES: This study aimed to evaluate the contributions of VL and UVA on pigmentation and erythema, and optimize parameters for in vivo testing. METHODS: Ten subjects with Fitzpatrick skin phototype IV-VI were enrolled. Subjects were irradiated on their back with VL using two light sources: one containing pure VL and one containing VL with less than 0·5% UVA1 (VL+UVA1). Four different irradiances were administered to investigate reciprocity behaviour. Assessments, including photography, Investigator's Global Assessment, colorimetry and spectroscopy, were performed immediately, 24 h, 7 days and 14 days post-irradiation. RESULTS: Pigmentation was observed with both light sources; however, pigment intensity was greater with VL+UVA1 than with pure VL. Reciprocity was observed in pure VL sites, but not VL+UVA1. Variation in spectral output had greater impact on pigment intensity than irradiance. Clinical erythema was observed on the VL+UVA1 side, but not on the pure VL side. A protocol for testing photoprotection product efficacy against VL-induced effects has been proposed. CONCLUSIONS: The findings suggest a synergistic relationship between VL and UVA1 and emphasize the need for developing means of photoprotection against VL.

Review of applications of fluorescence excitation spectroscopy to dermatology.

Endogenous molecules that exhibit fluorescence hold the potential to serve as reporters of tissue structure, activity and physiology. Fluorescence excitation spectroscopy is one means to measure and express tissue's innate fluorescence. This review focuses on the application of endogenous fluorescence ultraviolet excitation spectroscopy to dermatology.

Assessing facial wrinkles: automatic detection and quantification.

BACKGROUND: As people mature, their skin gradually presents lines, wrinkles, and folds that become more pronounced with time. Skin wrinkles are perceived as important cues in communicating information about the age of the person. Nowadays, documenting the facial appearance through imaging is prevalent in skin research, therefore detection and quantitative assessment of the degree of facial wrinkling can be a useful tool for establishing an objective baseline and for assessing benefits to facial appearance due to various dermatological treatments. However, few image-based algorithms for computationally assessing facial wrinkles are present in the literature, and those that exist have limited reliability. METHODS: In this work, an algorithm for automatic detection of facial wrinkles is developed, based on estimating the orientation and the frequency of elongated spatial features, captured via digital image filtering. RESULTS: The algorithm is tested against one set of clinically validated 11-point wrinkle scales present on the face. The algorithm is employed for assessing the presence of forehead furrows on a set of 100 clinically graded facial images. The proposed computational assessment correlates well with the corresponding clinical scores. CONCLUSION: We find that the results are in better agreement with clinical scoring when the wrinkle depth information, approximated via filter responses, is combined with the wrinkle length information as opposed to the case when the two measures are considered separately.

Documentation of normal stratum corneum scaling in an average population: features of differences among age, ethnicity and body site.

BACKGROUND: Scaling skin involves an imbalance between cell proliferation and desquamation, resulting in partially detached corneocytes at the stratum corneum (SC) surface that become visible as they scatter light. OBJECTIVES: The purpose of this study was to document scaling skin with no associated pathology, to estimate the range of normal corneocyte detachment in the average population, and to determine if age, pigmentation and/or body sites of different exposures contribute to differences observed in the SC. METHODS: Healthy African-American and Caucasian female subjects (n = 151) from a typical central New Jersey population, aged between 14 and 75 years, were evaluated on the dorsal forearm and upper inner arm. Dermatoscopy and adhesive tape were used to evaluate the appearance and adhesion of surface corneocytes. Transepidermal water loss and conductivity were measured to assess water-handling properties of the SC. Measurements were conducted during the winter. RESULTS: Corneocyte detachment observed with dermatoscopy became more prevalent with age and was more severe on the dorsal forearm and in Caucasian subjects. The distribution of the amount of corneocyte removal with adhesive tape increased with age. The range of values was larger in the dorsal forearm than the upper inner arm and was greater in Caucasian subjects than African-American subjects. Minimal changes were observed for water-handling properties. CONCLUSIONS: The architecture of the outer SC appears different between ages, body sites of different exposures, and individuals of different pigmentation groups, but minimal differences in water-handling properties are observed.

Caucasian facial L* shifts may communicate anti-ageing efficacy.

An ageing study was conducted to capture skin colour parameters in the CIELab system from Caucasians of both genders and all available adult ages. This study produced a linear correlation between L* and age for a Caucasian population between 20 and 59 years of age as follows: (L* value) = -0.13 × (Age in years) + 63.01. Previous studies have addressed age-related changes in skin colour. This work presents a novel consumer correlated quantitative linear model of skin brightness by which to communicate age-related changes. Two product assessment studies are also presented here, demonstrating the ability of anti-ageing products to deliver on objective and subjective improvements in skin brightness. It was determined to be possible to use the fundamental Caucasian L*-age correlation to describe product benefits in a novel quantitative and consumer-relevant fashion, through the depiction of a 'years back' calculation.

Infant skin physiology and development during the first years of life: a review of recent findings based on in vivo studies.

Infant skin is often presented as the cosmetic ideal for adults. However, compared to adult skin it seems to be more prone to develop certain pathological conditions, such as atopic dermatitis and irritant contact dermatitis. Therefore, understanding the physiology of healthy infant skin as a point of reference is of interest both from the cosmetic as well as from the clinical point of view. Clinical research on healthy infants is, however, limited because of ethical considerations of using invasive methods and therefore until recently data has been scarce. Technical innovations and the availability of non-invasive in vivo techniques, such as evaporimetry, electrical impedance measurement, in vivo video and confocal microscopy, and in vivo fibre-optic based spectroscopy, opened up the field of in vivo infant skin physiology research. Studies incorporating such methods have demonstrated that compared to adult, infant skin continues to develop during the first years of life. Specifically, infant skin appears to have thinner epidermis and stratum corneum (SC) as well as smaller corneocytes at least until the second year of life. The water-handling properties are not fully developed before the end of the first year and infant SC contains more water and less amounts of natural moisturizing factors. Such findings re-evaluate the old notions that skin is fully matured at birth. Armed with this knowledge, we are in a position not only to better understand infant dermatological conditions but also to design better skin care products respecting the distinct qualities of infant skin.

Measurement of skin texture through polarization imaging.

BACKGROUND: Determination of skin surface texture is of particular importance in the field of dermatology as such measurements can be used for skin diagnostics and evaluation of therapeutic or cosmetic treatments. Profilometry of skin replicas, three-dimensional imaging and computer vision have been successfully used to measure and document skin texture. Nevertheless, the development of a simpler and faster technique may prove to be advantageous in a clinical setting. OBJECTIVES: We propose the use of polarization imaging with high angles of incidence as a simple alternative to measure/document skin texture/roughness. METHODS: A system based on digital photography and polarization optics was developed to acquire and compute texture images. Optimization of the system configuration was conducted to enhance the contrast for measuring skin roughness. The method was validated against roughness standards and tested in clinical studies. Measurements were made in subjects aged from 9 to 70 years and image analysis was used to evaluate texture. RESULTS: The developed texture scale was shown to correlate closely to the results from clinical assessment and from roughness standards. Frequency domain analysis showed a significantly different power spectrum for the texture images of young subjects when compared with older subjects. The evaluation of texture as a function of age showed that facial skin roughness increased linearly from teenage to 40 years followed by a plateau thereafter. CONCLUSIONS: The system proved to be a useful clinical tool for assessing skin texture. The age-related results may indicate that some skin texture features are formed before the age of 40 years.

In vivo measurement of skin erythema and pigmentation: new means of implementation of diffuse reflectance spectroscopy with a commercial instrument.

BACKGROUND: Various physical, chemical and biological insults, including exposure to ultraviolet (UV) radiation, cause erythema and change in pigmentation in human skin. These reactions provide an important measure of the cutaneous response to the insult. OBJECTIVES: To present a new implementation of a method for objective in vivo measurement of erythema and pigmentation. METHODS: The method is based on acquisition of reflectance spectra in the visible range using a commercially available spectrophotometer. The probe of this instrument incorporates an integrating sphere that captures the light remitted from the skin in a wide range of angles. We corrected the acquired reflectance spectra for the contribution of specular reflections by an amount given by the Fresnel equation and verified this correction experimentally. This correction is particularly important when measurements are performed on heavily pigmented skin. The corrected reflectance spectra are then transformed into absorbance spectra. To analyse these spectra, we developed an algorithm which can be used to calculate apparent concentrations of oxyhaemoglobin, deoxyhaemoglobin and melanin. This method was tested in clinical studies of skin reactions induced by exposure to UV radiation. These experiments involved three groups of subjects with progressively darker complexion (constitutive pigmentation). Each group consisted of 10 subjects. Erythema was measured 1 day after UV exposure, and pigmentation (melanin content) 1 week later. Results Distinct apparent absorbance spectra were obtained for dark, intermediate and fair skin. There was good agreement between reconstructed spectra and experimental data at relevant wavelengths. Difference absorption spectra were able to show the dose dependence of UV-induced responses, and erythema and pigmentation values obtained by the spectroscopic method showed good correlation with those derived by subjective visual grading. CONCLUSIONS: The results demonstrate that the presented methodology provides an objective noninvasive way of measuring UV-induced reactions independently of the level of constitutive pigmentation.

Skin viscoelasticity displays site- and age-dependent angular anisotropy.

One of the dominant characteristics of skin aging is loss of elasticity. Although the changes in the mechanical properties of the skin over several decades of life are substantial, objective measurements have failed to capture their magnitude thus far. Moreover, the mechanical properties of the skin are not uniform in all directions, and there is a need to understand this angular anisotropy. In this work we present a methodology of documenting the angular anisotropy of skin elasticity with high sensitivity and dynamic range using the Reviscometer RVM 600 (Courage & Khazaka Electronic GmbH, Cologne, Germany). The method is based on determining the directional dependence of the speed of an acoustic shear wave on the skin surface at intervals of 3 degrees . Based on the angular distribution of the resonance running time, we define two parameters: the anisotropy and the angular dispersion width. We find that with increasing age the anisotropy increases, while the angular dispersion width decreases. The ratio of these values provides a sensitive parameter for the assessment of the directional behavior of the skin mechanical properties. This parameter provides a large effective dynamic range capable of demonstrating close to an order of magnitude differences in skin viscoelasticity from infants up to adults 75 years of age. Furthermore, we show that the direction of the angular anisotropy relates to the direction of the dermal cleavage lines as defined by Langer, indicating that the anisotropy of the mechanical properties of skin stems from structural parameters. Based on these results, we conclude that the proposed methodology is able to capture accurately the age-dependent changes of the mechanical properties of the skin and to demonstrate a structure-function relationship.

Spectroscopic characteristics of human melanin in vivo.

In this paper we present the absorption characteristics of human melanin in the visible range of wavelengths and specifically in the range 620-720 nm. The spectroscopy of melanin is studied by measuring remittance spectra of normal skin and vitiligo-involved skin of volunteers-patients. It is assumed that the spectral differences between adjacent areas of normally pigmented skin, and to some degree amelanotic skin, can only be due to the variations of the melanin filter. The ratio of the remittance spectrum of the vitiligo-involved skin with the spectrum of the normal skin in the range 620-720 nm can be fitted with a straight line for all the volunteers. A very strong correlation is obtained between the intercept and the slope for all the volunteers, which leads us to conclude that it is indeed melanin that we are measuring in all the volunteers and that it is the same substance spectroscopically for all the volunteers.

Absorption mechanisms of human melanin in the visible, 400-720 nm.

In this paper we propose that human melanin absorbs visible radiation through two distinct mechanisms: one that is in effect over the entire visible range and is linear in wavelength, and a second one that is evident at wavelengths in the range 400-500 nm and is exponential in frequency. These mechanisms are apparent in all human diffuse reflectance spectra that we have collected. We show that the absorber is the same in all human volunteer skin samples. By studying the diffuse reflection spectra of DOPA-melanin in solution and DOPA-melanin in powder form, we find that we can correlate the absorption mechanisms, one with melanin in solution (a low molecular weight form) and the other with melanin in powder (a high molecular weight form). Therefore, we propose that melanin exists in two distinct states. This model is of biologic significance, as it provides a reasonable interpretation for the diffuse reflection spectra obtained from delayed pigment (UVB-induced) and immediate pigment (UVA-induced). Delayed pigment appears as an increase of both forms of melanin (neomelanogenesis), whereas immediate pigment appears as an increase in the higher molecular weight form with a commensurate decrease in the lower molecular weight form: the two mechanisms change independently of each other. Finally, we show that we can distinguish spectroscopically between the delayed pigment and the immediate pigment.

The in vivo fluorescence of tryptophan moieties in human skin increases with UV exposure and is a marker for epidermal proliferation.

We have investigated the in vivo fluorescence of human skin with UV excitation and the effect of UV irradiation on the UV fluorescence. A particular chromophore was found to be very sensitive to suberythemogenic UV radiation. This chromophore has the spectral characteristics of tryptophan residues in proteins and is characterized by a fluorescence excitation maximum at 295 nm. The fluorescence of this chromophore in human epidermis has an excitation maximum that is coincident with the maximum of the action spectrum of most UV-induced photobiologic responses to human skin. The fluorescence of the chromophore was found to increase with UV exposure. The action spectrum was determined by following the increase of the emission at 345 nm with excitation at 295 nm as a function of skin exposure to a number of wavelengths in the UV region of the spectrum. The results show that irradiation in the UVB (290-320 nm) is more effective in producing the change in the fluorescence of tryptophan. Irradiation in the UVA (320-380 nm) was found to be capable of producing the increase but to a smaller extent. Whereas tryptophan fluorescence is found in both the epidermis and the dermis, it is only the epidermal component that increases with UV exposure. The change in 295 nm fluorescence with UV exposure was determined to be oxygen dependent. The fluorescence of tryptophan moieties measured in situ was found to increase when epidermal proliferation increases. This was verified by inducing epidermal repair after mechanical insult (tape stripping). The results suggest two possible scenarios for the UV-induced increase of the fluorescence: a prompt photooxidation of tryptophan moieties or a fast proliferation response to the insult created by UV irradiation.

Skin melanin, hemoglobin, and light scattering properties can be quantitatively assessed in vivo using diffuse reflectance spectroscopy.

Noninvasive and real-time analysis of skin properties is useful in a wide variety of applications. In particular, the quantitative assessment of skin in terms of hemoglobin and melanin content, as well as in terms of its light scattering properties, is a challenging problem in dermatology. We present here a technique for examining human skin, based on the in vivo measurement of diffuse reflectance spectra in the visible and near-infrared ranges of the electromagnetic spectrum. Spectra were measured by means of a fiber optic probe, and they were analyzed using an analytical model of light diffusion in the skin. The results of the analysis indicate that it is possible to obtain quantitative information about hemoglobin and melanin content, as well as basic information regarding the scattering properties of the skin.

Fluorescence excitation spectroscopy provides information about human skin in vivo.

Fluorescence spectroscopy of human skin has the potential to provide useful morphologic and biochemical information. The endogenous fluorescence of human skin has been investigated in vivo on normal human volunteers as well as on patients with psoriasis and it was found that characteristic bands can be identified in the fluorescence spectra that are associated with specific skin fluorophores. One epidermal band (295 nm excitation, attributed to tryptophan) and two dermal bands (335 and 370 nm excitation, attributed to collagen cross-links) were consistently present in all fluorescence spectra. In addition, the fluorescence spectra obtained from lesions and nonlesional sites of psoriatic patients differed from those obtained from healthy volunteers and the hyperproliferative state of the lesions was characterized by a significantly larger signal at 295 nm excitation. These results indicate that fluorescence spectroscopy is a promising technique for the investigation of human skin in vivo.

Endogenous skin fluorescence is a good marker for objective evaluation of comedolysis.

OBJECTIVE: evaluation of comedone lesions, especially in vivo, remains a challenge. We have used the rhino mouse model in combination with topical application of all-trans retinoic acid as a comedolytic agent, to investigate the potential of fluorescence spectroscopy as a noninvasive technique in the assessment of noninflammatory acne. The results indicate that there is a strong correlation between the fluorescence excitation spectral features assessed in vivo, and the histologic changes identified, particularly the size of the utriculi as well as the dermal and epidermal thickness. We conclude that fluorescence excitation spectroscopy represents a promising novel and useful tool in the quantitative evaluation of the pseudocomedones and could also be used for the rapid and noninvasive assessment of comedolysis induced by the application of pharmacologic agents such as retinoids.

Iontophoretic delivery of ALA provides a quantitative model for ALA pharmacokinetics and PpIX phototoxicity in human skin.

Photodynamic therapy (PDT) with topical 5-aminolevulinic acid (ALA) is increasingly employed for skin cancer, yet ALA dosing is crude. Using iontophoresis, we developed a rapid and quantifiable system for topical ALA delivery, with measurement of subsequent PpIX fluorescence and phototoxicity. ALA was iontophoresed from a 2% solution into upper inner arm skin of 13 healthy volunteers. Six doses of ALA were delivered with a series of charges varying from 3-120 milliCoulombs (mC); four additional doses were given with a charge of 60 mC. Five hours post-iontophoresis, sites were irradiated with broad-band yellow-red light, the series of six ALA doses receiving 100 J/cm2, while the four identical doses received 6.25, 12.5, 25, and 50 J/cm2, respectively. Resultant erythema was measured by reflectance spectroscopy. The time course of PpIX fluorescence was ALA-dose-dependent. With charge < or = 24 mC, PpIX fluorescence peaked at 3 h and returned to zero at 9-10 h, whereas charges > 24 mC had a sustained peak at 5-10 h, falling to zero by 24 h. Pre-irradiation, PpIX fluorescence correlated with ALA dose (r = 1.0). PpIX fluorescence fell immediately post-irradiation (p < 0.0001); recovery levels at 3 h correlated with ALA dose (p < 0.0001). Delayed erythema correlated with ALA dose and irradiation dose (p < 0.0001, p < 0.01, respectively). Both PpIX fluorescence intensity pre-irradiation and fall in PpIX fluorescence post-irradiation correlated with erythema (r = 0.98). Hence, PpIX synthesis is ALA-dose-dependent, and phototoxicity can be predicted from ALA dose, irradiation dose, and photobleaching of PpIX. This reproducible system allows accurate dosimetry in topical PDT and facilitates study of ALA metabolism.

Attenuated total reflection-Fourier transform infrared spectroscopy as a possible method to investigate biophysical parameters of stratum corneum in vivo.

We investigated the use of attenuated total reflection-Fourier transform infrared spectroscopy as a method to study differences in the molecular components of human stratum corneum in vivo. These variations as a function of the anatomic site and of the depth into its layered structure are important to understand the biology and physiology of the tissue. In this preliminary study we have investigated spectroscopic changes in 18 healthy individuals. Total reflection-Fourier transform infrared spectroscopy represents a potentially powerful tool to study biophysical properties of surfaces. We observed that, in vivo, biophysical parameters of the stratum corneum (such as hydration, lipid composition, and conformation of the aliphatic chains) are indeed dependent on the anatomic site. As in total reflection-Fourier transform infrared spectroscopy experiments the penetration depth of the evanescent field into the stratum corneum is comparable with the thickness of a layer of corneocytes, this technique can be used to follow the distribution of lipids, water, and proteins as a function of depth into the tissue. We found that, in vivo, these molecular components are non-uniformly distributed, in agreement with the presence of water and lipid reservoirs as observed with ex vivo ultrastructural investigations. Composition and conformational order of lipids are also a function of depth into the stratum corneum. Finally we compared the in vivo superficial hydration measured using the infrared absorption of the OH stretch of water, with the hydration measured using the Skicon hygrometer. Our results indicate that total reflection-Fourier transform infrared spectroscopy might be useful to measure important chemical and biophysical parameters of stratum corneum in vivo.

Aging and effects of ultraviolet A exposure may be quantified by fluorescence excitation spectroscopy in vivo.

The fluorescence properties of skin chromophores such as tryptophan and collagen cross-links might be useful markers of aging and photoaging. As the fluorescence of pepsin-digestible collagen cross-links was found to increase with aging and decrease with photoaging we investigated the characteristics of this dependence. In vivo fluorescence excitation spectra (emission at 380 nm) of SKH hairless mouse model skin are characterized by two bands centered near 295 nm and 335 nm due, respectively, to epidermal tryptophan moieties and pepsin-digestible collagen cross-links. Several groups of hairless mice were followed over a period of 18 mo to document changes in skin fluorescence with aging. Other groups of animals were exposed to either broad band or narrowband ultraviolet A radiation to determine the effects of ultraviolet A exposure on the fluorescence of the dermal collagen cross-links and to determine an action spectrum for the induced changes. We also found that the intensity of pepsin-digestible collagen cross-links in vivo increases linearly with age and that the fluorescence of epidermal tryptophan decreases linearly with age. We found that the fluorescence of pepsin-digestible collagen cross-links decreases immediately following exposure to ultraviolet A whereas epidermal tryptophan fluorescence increases. Both changes were dose dependent but the increase in tryptophan fluorescence occurred exclusively in young animals (2--6 mo old). We found that the ultraviolet-induced fluorescence decrease of pepsin-digestible collagen cross-links is wavelength specific. The action spectrum for the ultraviolet A effect on the in vivo fluorescence of pepsin-digestible collagen cross-links shows a distinct maximum at 335 nm that corresponds to the maximum in the fluorescence excitation spectrum due to pepsin-digestible collagen cross-links. Our results seem to indicate that in vivo fluorescence of epidermal tryptophan moieties and collagen cross-links in the dermal matrix may serve as markers for skin aging, for photoaging, and for immediate assessment of exposure to ultraviolet A radiation.

A single parameter, oxygenated hemoglobin, can be used to quantify experimental irritant-induced inflammation.

To quantify the dose-response relation of irritant-induced erythema, we examined inflammation in human skin after application of an irritant, using perpendicular polarized photography and diffuse reflectance spectroscopy as compared to clinical visual scoring. The ventral forearms of 11 healthy subjects were patch-tested for 24 h under occlusion in finn chambers with five concentrations of the irritant sodium lauryl sulfate. The tested sites and three control sites were evaluated clinically for erythema at 24, 48, and 72 h after occlusion, photographed using standard and perpendicular polarized photography, and measured by diffuse reflectance spectroscopy. All photographs were evaluated for erythema by three investigators. Diffuse reflectance spectra were analyzed, and changes in apparent oxyhemoglobin and deoxyhemoglobin concentrations were estimated. Clinical and photographic assessments of erythema yielded similar linear dose-response relations. A linear dose-response relation, with no minimum threshold, also was obtained for changes in the apparent oxyhemoglobin concentration with increasing irritant dose, whereas the apparent deoxyhemoglobin concentrations were unchanged with increasing dose. These results show that diffuse reflectance spectroscopy permits the characterization of irritant-induced inflammation in terms of a single parameter, the apparent concentration of oxyhemoglobin, and that irritant-induced inflammation primarily involves the capillaries and the superficial arterial plexus.