Quantitative measurement of skin surface oiliness and shine using differential polarized images.

Excess amounts of skin surface oil can lead to adverse psychological consequences. Grease-spot photometry-based techniques measure sebum production rate. However, besides being tedious, these measurements are influenced by contact area, applied pressure, and time of application. Image analysis of polarized images has the potential to provide objective, quantitative information of skin oiliness. This study was designed to set up an imaging device for capturing and enhancing the changes in skin surface oiliness and to clinically and quantitatively, (via image analysis), evaluate varying levels of skin surface oiliness. Mineral oil was used to simulate skin surface oil. 40.5 µL of the mineral oil was applied within a two inch square area of interest on facial skin in twelve steps, from 1 to 40.5 µL, at 40% increments. The results indicate a strong correlation between the quantitative skin surface oiliness measurements and the clinical assessments. This sensitive technique has the potential to be utilized in future studies to evaluate product efficacies in reducing skin oiliness.

Long-wavelength Ultraviolet A1 and Visible Light Photoprotection: A Multimodality Assessment of Dose and Response.

Human skin is exposed to visible light (VL; 400-700 nm) and long-wavelength ultraviolet A1 (UVA1) radiation (370-400 nm) after the application of organic broad-spectrum sunscreens. The biologic effects of these wavelengths have been demonstrated; however, a dose-response has not been investigated. Ten subjects with Fitzpatrick skin phototype IV-VI were enrolled. Subjects were irradiated with 2 light sources (80-480 J cm-2 ): one comprising VL with less than 0.5% UVA1 (VL+UVA1) and the other pure VL. Skin responses were evaluated for 2 weeks using clinical and spectroscopic assessments. 4-mm punch biopsies were obtained from nonirradiated skin and sites irradiated with 480 J cm-2 of VL+UVA1 and pure VL 24 h after irradiation. Clinical and spectroscopic assessments demonstrated a robust response at VL+UVA1 sites compared with pure VL. Histology findings demonstrated a statistically significant increase in the marker of inflammation (P < 0.05) and proliferation (P < 0.05) at the irradiated sites compared with nonirradiated control. Threshold doses of VL+UVA1 resulting in biologic responses were calculated. Results indicate that approximately 2 h of sun exposure, which equates to VL+UVA1 dose (~400 J cm-2 ), is capable of inducing inflammation, immediate erythema and delayed tanning. These findings reinforce the need of photoprotection beyond the UV range.

Spectral characteristics of visible light-induced pigmentation and visible light protection factor.

Solar radiation is a major contributor to the development of skin cancer. Recent studies have shown that visible light (VL), a major portion of solar spectrum, induces biologic effects on the skin. Ultraviolet filters in currently available broad-spectrum sunscreens do not offer protection against VL. This study was designed to identify the spectral characteristics of the skin responses induced by VL, which can be utilized for time efficient in vivo VL testing. Thirty-one subjects were irradiated with a light source emitting visible light with less than 0.5% long wavelength UVA1 (VL + UVA1, 370-700 nm), and 41 subjects were irradiated with pure visible light (pure VL, 400-700 nm). Assessments including clinical photography, investigator's global assessment of pigmentation and erythema, and diffuse reflectance spectroscopy (DRS) performed immediately and seven days after irradiation. Clinical and spectroscopic data showed that VL + UVA1 spectral output induced significantly darker and persistent skin responses as compared to those induced by pure VL. Spectroscopic signatures of skin responses induced by both radiation sources were identified. The signatures were found to be specific to the radiation source and time of collection. A method to evaluate VL protection factor, using quantitative information from the spectral signatures obtained, was proposed.

The dynamics of pigment reactions of human skin to ultraviolet A radiation.

The pigment responses of human skin to broadband UVA radiation (320-400 nm) occur in three distinct phases. The first phase includes immediate pigment darkening (IPD), the pigment that appears immediately after irradiation. The second phase involves an intermediate step, termed persistent pigment darkening (PPD), which leads to the third phase of neomelanogenesis or delayed tanning (DT). Since DT results from synthesis of new melanin, it persists beyond 5-7 days. We conducted studies on human subjects to investigate the dynamic responses of the IPD and PPD reactions to broadband UVA radiation at threshold and superthreshold doses. The threshold doses for IPD, PPD, and DT were found to be approximately 1, 11, and 18 J/cm2 , respectively. The colorimetry ΔL* value corresponding to minimal clinically perceptible pigmentation was found to be 0.8 ± 0.1. IPD appeared immediately and had an associated decay constant of approximately 1.4 minutes. At doses greater than PPD threshold, IPD reaction decayed while PPD developed indicating toward IPD being used as a substrate in the formation of PPD.

Spectroscopic observations on human pigmentation.

BACKGROUND: The main chromophores of human skin are melanins and hemoglobins along with carotenoids, bilirubin, and other compounds. In an effort to study the spectral signatures of skin melanin, we measured absorption spectra in a variety of situations, including a method to show early signs of re-pigmentation in vitiligo. METHODS: To measure skin in vivo, the essential component was a "Bifurcated Optical Fiber" with one end connected to the light source and the second end connected to the spectrometer while the common end was placed on the skin. RESULTS: In a typical in situ "melanin in skin" spectrum, the absorbance values first rise gradually, from 750 to 600 nm, then rise moderately from 600 to 450 nm, and rise sharply from 450 nm to a broad peak at 335 nm, below which it gradually rolls down to much lower values. CONCLUSION: We successfully studied melanin spectroscopically in subjects with vitiligo lesions, obtaining the differential spectra. Higher melanin levels can be shown by steeper negative slopes of a straight line fitted between 620 and 720 nm. Also, absorption peak at 335 nm showed the presence of melanin.

Melanin quantification by in vitro and in vivo analysis of near-infrared fluorescence.

Objective measurements of melanin can provide important information for differentiating melanoma from benign pigmented lesions and in assessing pigmentary diseases. Herein, we evaluate near-infrared (NIR) fluorescence as a possible tool to quantify melanin. Various concentrations of in vitro Sepia melanin in tissue phantoms were measured with NIR fluorescence and diffuse reflectance spectroscopy. Similar optic measurements were conducted in vivo on 161 normal human skin sites. Diffuse reflectance spectroscopy was used to quantify the melanin content via Stamatas-Kollias algorithm. At physiologic concentrations, increasing in vitro melanin concentrations demonstrated higher fluorescence that was linearly correlated (R2  = 0.99, p < .001). At higher concentrations, the fluorescence signal plateaued. A linear relationship was also observed with melanin content in human skin (R2  = 0.59, p < .001). Comparing the fluorescence and reflectance signals with in vitro and in vivo samples, the estimated melanin concentration in human skin ranged between 0 and 1.25 mg/ml, consistent with previous quantitative studies involving invasive methods.

The impact of oral Polypodium leucotomos extract on ultraviolet B response: A human clinical study.

BACKGROUND: There is a rationale for adding systemic photoprotective agents to the current photoprotection regimen. OBJECTIVE: This study was designed to objectively evaluate the molecular and photobiologic effects of oral administration of Polypodium leucotomos extract (PLE). METHODS: In all, 22 subjects with Fitzpatrick skin phototype I to III were enrolled. On day 1, subjects were irradiated with visible light, ultraviolet (UV) A1, and UVB (using 308-nm excimer laser). Evaluation was done immediately and 24 hours after irradiation. On days 3 and 4, irradiation and evaluation process was repeated after ingestion of PLE. RESULTS: Clinical assessments and colorimetry data showed a decrease in UVB-induced changes in 17 of 22 subjects post-PLE administration; histology findings demonstrated such a decrease in all 22 subjects. LIMITATIONS: Only 2 doses of PLE were given. Furthermore, subjects with skin phototypes I to III only were studied. CONCLUSION: The results suggest that PLE can potentially be used as an adjunctive agent to lessen the negative photobiologic effects of UVB.

Water-Holding and Transport Properties of Skin Stratum Corneum of Infants and Toddlers Are Different from Those of Adults: Studies in Three Geographical Regions and Four Ethnic Groups.

BACKGROUND/OBJECTIVE: Epidermal structure, function, and composition are different in white infants and adults. We investigated whether ethnicity and location contribute to differences in functional and clinical measurements of skin barrier function during the first years of life and in adults. METHODS: Children (n = 397, ages 3-49 mos) and women (n = 117, mean age 31 yrs) were enrolled at independent centers in Beijing, China (ethnic Chinese), Skillman, New Jersey (white, African American), and Mumbai, India (ethnic South Asian). Water barrier properties of the stratum corneum were assessed using high-frequency conductance and transepidermal water loss (TEWL) on the dorsal forearm and upper inner arm. Digital imaging was used to evaluate facial erythema and scaling. RESULTS: Despite differences in local climate, TEWL was similar in adults. In children, conductance and TEWL decreased monotonically from age 3 months to 4 years. In children from Beijing, TEWL values were higher in both arm locations than in children in Mumbai and Skillman. No significant differences were observed in TEWL or conductance between the white and African American groups. CONCLUSION: In general, TEWL and conductance were greater on the upper inner arm than the dorsal forearm. Erythema and scaling were observed most often in subjects from Beijing and most infrequently in subjects from Mumbai. Stratum corneum water barrier properties were different in children and adults. Although there may be differences in these properties between ethnic groups in childhood, TEWL values were similar in adults across the three geographic locations and four ethnicities.

Visible Light Induces Melanogenesis in Human Skin through a Photoadaptive Response.

Visible light (400-700 nm) lies outside of the spectral range of what photobiologists define as deleterious radiation and as a result few studies have studied the effects of visible light range of wavelengths on skin. This oversight is important considering that during outdoors activities skin is exposed to the full solar spectrum, including visible light, and to multiple exposures at different times and doses. Although the contribution of the UV component of sunlight to skin damage has been established, few studies have examined the effects of non-UV solar radiation on skin physiology in terms of inflammation, and limited information is available regarding the role of visible light on pigmentation. The purpose of this study was to determine the effect of visible light on the pro-pigmentation pathways and melanin formation in skin. Exposure to visible light in ex-vivo and clinical studies demonstrated an induction of pigmentation in skin by visible light. Results showed that a single exposure to visible light induced very little pigmentation whereas multiple exposures with visible light resulted in darker and sustained pigmentation. These findings have potential implications on the management of photo-aggravated pigmentary disorders, the proper use of sunscreens, and the treatment of depigmented lesions.

Skin responses to micro scale field size of solar-simulated radiation--preliminary evaluation by reflectance confocal microscopy in vivo.

Erythema and pigment responses of human skin following an acute exposure to ultraviolet radiation (UVR) are frequently used to determine the photosensitivity of the skin. In this study we investigated the responses of the skin to a micro-scale area of UVR exposure (MiR) and compared the responses to a macro-scale area of exposure (MaR). Ten human volunteers were tested with solar-simulated radiation on their upper arm or back using a beam size of 8 mm and 0.2 mm in diameter. The fluence required to produce a minimally perceptible erythema (MED) using the MiR was found to be higher than that for the MaR. The erythema response extended beyond the exposed area and this became pronounced when the beam size was microscopic. Reflectance confocal microscopy in vivo revealed that MiR induced cellular alterations within a confined area of smaller dimensions than the area of exposure. Pigment responses were confined within the areas of cellular damage. The erythema expression of exposed skin recovered faster for the sites receiving MiR even when the applied fluence was higher than the MED for the MaR. Through the use of MiR we were able to visualize spatially dissimilar skin responses of erythema and pigmentation suggesting different cellular mechanisms.

New noninvasive approach assessing in vivo sun protection factor (SPF) using diffuse reflectance spectroscopy (DRS) and in vitro transmission.

BACKGROUND/PURPOSE: In the past 56 years, many different in vitro methodologies have been developed and published to assess the sun protection factor (SPF) of products, but there is no method that has 1:1 correlation with in vivo measurements. Spectroscopic techniques have been used to noninvasively assess the UVA protection factor with good correlation to in vivo UVA-PF methodologies. To assess the SPF of sunscreen product by diffuse reflectance spectroscopy (DRS) technique, it is necessary to also determine the absorbance spectrum of the test material in the UVB portion of the spectrum (290-320 nm). However, because of the high absorbance characteristics of the stratum corneum and epidermis, the human skin does not remit enough UVB radiation to be used to measure the absorption spectrum of the applied product on skin. In this work, we present a new method combining the evaluation of the absolute UVA absorption spectrum, as measured by DRS with the spectral absorbance 'shape' of the UVB absorbance of the test material as determined with current in vitro thin film spectroscopy. METHODS: The measurement of the in vivo UVA absorption spectrum involves the assessment of the remitted intensity of monochromatic UVA radiation (320-400 nm) before and after a sunscreen product was applied on skin using a spectrofluorimeter Fluorolog 3, FL3-22 (Yvon Horiba, Edison, NJ, USA). The probe geometry assures that light scattering products as well as colored products may be correctly assessed. This methodology has been extensively tested, validated, and reported in the literature. The in vitro absorption spectrum of the sunscreen samples and polyvinyl chloride (PVC) films 'surrogate' sunscreen standards were measured using Labsphere® UV-2000S (Labsphere, North Sutton, NH, USA). Sunscreens samples were tested using PMMA Helioplates (Helioscience, Marseille, France) as substrates. The UVB absorbance spectrum (Labsphere) is 'attached' to the UVA absorbance spectrum (diffuse reflectance) with the UVB absorbance matched to the UVA absorbance at 340 nm to complete the full spectral absorbance from which an estimate the SPF of the product can be calculated. RESULTS: Seventeen test materials with known in vivo SPF values were tested. Two of the tested products were PVC sunscreen thin films with 10-15 micrometers thickness and were used to investigate the absorption spectrum of these films when applied on different reflectance surfaces. Similar to the human in vivo SPF test, the developed methodology suggests limiting the use on Fitzpatrick skin phototypes I to III. The correlation of this new method with in vivo clinical SPF values was 0.98 (r2) with a slope of 1.007. CONCLUSION: This new methodology provides a new approach to determine SPF values without the extensive UV irradiation procedures (and biological responses) currently used to establish sunscreen efficacy. Further work will be conducted to establish methods for evaluation of products that are not photostable.

Non-invasive assessment of tryptophan fluorescence and confocal microscopy provide information on skin barrier repair dynamics beyond TEWL.

The stratum corneum (SC) serves a primary function of skin barrier and understanding the kinetics of SC formation may provide great insight for skin diagnosis and evaluation of therapies. Besides trans-epidermal water loss (TEWL), few methods have been characterized to assess skin barrier non-invasively in vivo, particularly for dynamic measurements on the same specimen over time. The objective of this study was to characterize alternative non-invasive methods to evaluate the dynamic processes involved in the recovery of normal human SC after total removal. TEWL, tryptophan fluorescence and reflectance confocal microscopy (RCM) were used to determine skin barrier function, cell turnover and epidermal morphology over a period of 10 days after total removal of the SC by tape stripping. The results show a biphasic recovery of TEWL over time, which contrasted with a linear increase of 2.3 µm/day in SC thickness. Tryptophan assessment of cell turnover also demonstrated a biphasic pattern attaining a maximum three to four times the levels of the control site 3 days after injury that slowly returned to baseline and displayed great correlation (R(2)  > 0.95) to viable epidermis thickness that also achieved a maximum about 3 days after injury with an approximate increase of 55%. When plotting the change of TEWL versus SC thickness, a single exponential function is observed [Δ-TEWL = 55 exp (-0.157×)] which contrasts with other proposed models. These methods were able to present rates for SC recovery processes beyond skin barrier (TEWL) that may provide new insights on kinetics of barrier formation for evaluation of skin conditions and treatments.

Irradiation of skin with visible light induces reactive oxygen species and matrix-degrading enzymes.

Daily skin exposure to solar radiation causes cells to produce reactive oxygen species (ROS), which are a primary factor in skin damage. Although the contribution of the UV component to skin damage has been established, few studies have examined the effects of non-UV solar radiation on skin physiology. Solar radiation comprises <10% of UV, and thus the purpose of this study was to examine the physiological response of skin to visible light (400-700 nm). Irradiation of human skin equivalents with visible light induced production of ROS, proinflammatory cytokines, and matrix metalloproteinase (MMP)-1 expression. Commercially available sunscreens were found to have minimal effects on reducing visible light-induced ROS, suggesting that UVA/UVB sunscreens do not protect the skin from visible light-induced responses. Using clinical models to assess the generation of free radicals from oxidative stress, higher levels of free radical activity were found after visible light exposure. Pretreatment with a photostable UVA/UVB sunscreen containing an antioxidant combination significantly reduced the production of ROS, cytokines, and MMP expression in vitro, and decreased oxidative stress in human subjects after visible light irradiation. Taken together, these findings suggest that other portions of the solar spectrum aside from UV, particularly visible light, may also contribute to signs of premature photoaging in skin.

The value of the ratio of UVA to UVB in sunlight.

The objective of this communication is to present the calculated ratio between UVA and UVB irradiance from sunrise to sunset and under a number of weather conditions. UVA plays an important role in the sun spectrum and a lot of attention has been paid lately regarding the protection of people from UVA. Solar spectra were collected in Kuwait City located at 29.3° North latitude (similar to that of Houston, TX) over a period of 8 months and under various weather conditions. Spectra were collected from 260 nm to 400 nm in 2 nm increments for solar elevation angles from 10° to 90° using a calibrated Optronics Laboratories OL-742 Spectroradiometer. The measurements reported in this study the ratio of UVA (320-400 nm) to UVB (280-320 nm) in solar terrestrial radiation remains essentially constant and equal to 20 for the part of the day when the solar elevation is greater than 60°. Consequently the value of the ratio of solar UVA/UVB should be considered as equal to 20 for studies in photobiology and photomedicine. When the wavelength limiting the range of UVA and UVB is 315 nm (i.e. UVB: 280-315 nm and UVA: 315-400 nm) the ratio of UVA to UVB becomes equal to 41.

Characterization and quantification of wound-induced hair follicle neogenesis using in vivo confocal scanning laser microscopy.

BACKGROUND: In vivo confocal scanning laser microscopy (CSLM) is a recently developed non-invasive technique for visualizing microscopic structures with the skin. CSLM has been used to characterize proliferative and inflammatory skin diseases, neoplastic skin lesions and pigmented lesions. OBJECTIVE: Here, we assessed the ability of CSLM to evaluate the formation of neogenic hair follicles after a full-thickness wound in mice. METHODS: Full-thickness wounds were made on the dorsal skin of 3-week-old mice. After scab detachment (SD), the number, width, length, space and volume of neogenic hair follicles were analyzed using CSLM. The results were compared with those from conventional methods, including staining for alkaline phosphatase (AP) and keratin 17 (K17) as well as histology. RESULTS: Quantification of neogenic hair follicles using CSLM compared favorably with the results from direct measurements on isolated epidermal tissue after immunostaining for K17, a marker for the epithelial portion of new hair follicles. CSLM detected 89% of K17-stained follicles. CSLM more accurately quantified the number of new follicles compared with AP staining, which detects the dermal portion of the new follicle. The width and length measurement from CSLM and histology were very close and correlated with each other. The minimum length of a neogenic hair follicle that could be detected by CSLM was 21 µm. The space between neogenic hair follicles was decreased in histological sections compared with CSLM. CONCLUSION: CSLM is an accurate and valuable method for counting and measuring neogenic hair follicles non-invasively. CSLM produces images similar to histology in mice. Measurements of microstructures using CSLM more accurately reflect actual sizes as this technique avoids fixation artifacts. In vivo visualization of developing follicles with CSLM allows the detection of serial changes in hair follicle formation, thus conserving the numbers of mice required for studies and improving the detection of temporal changes in developing hair follicles.

Impact of long-wavelength UVA and visible light on melanocompetent skin.

The purpose of this study was to determine the effect of visible light on the immediate pigmentation and delayed tanning of melanocompetent skin; the results were compared with those induced by long-wavelength UVA (UVA1). Two electromagnetic radiation sources were used to irradiate the lower back of 20 volunteers with skin types IV-VI: UVA1 (340-400 nm) and visible light (400-700 nm). Pigmentation was assessed by visual examination, digital photography with a cross-polarized filter, and diffused reflectance spectroscopy at 7 time points over a 2-week period. Confocal microscopy and skin biopsies for histopathological examination using different stains were carried out. Irradiation was also carried out on skin type II. Results showed that although both UVA1 and visible light can induce pigmentation in skin types IV-VI, pigmentation induced by visible light was darker and more sustained. No pigmentation was observed in skin type II. The quality and quantity of pigment induced by visible light and UVA1 were different. These findings have potential implications on the management of photoaggravated pigmentary disorders, the proper use of sunscreens, and the treatment of depigmented lesions.

Interpreting diffuse reflectance for in vivo skin reactions in terms of chromophores.

The measurement and quantification of skin reactions to insults involves certain assumptions about the relation between intensity of color appearance of the skin and the concentration of endogenous chromophores. The underlying assumption is that the Beer-Lambert law is obeyed, i.e., that a linear relation exists between the absorbance and the concentration of each chromophore and that the total absorbance is the linear superposition of the contributions of each chromophore. In this paper the authors compiled the results from a number of interventions on human skin that result in changes in its appearance and small deviations from the homeostatic state, where the results may be accounted for by a single or multiple chromophores. The validity of the assumptions is found to hold for a limited range of responses. The biological constraints need to be considered in certain cases because as we move away from the homeostatic state, complex biological processes are induced.

Simultaneous assessment of pulsating and total blood in inflammatory skin lesions using functional diffuse reflectance spectroscopy in the visible range.

We present a simple and cost-effective optical technique for the simultaneous assessment of pulsating and total blood noninvasively in an inflammatory skin lesion. Acquisitions of diffuse reflectance spectra in the visible range at 6 Hz are used to trace the oscillating components of reflectance. Measurements on erythematous lesions from a UV insult show slow changing signal at about 0.1 Hz and heart-driven regular oscillations at about 1 Hz simultaneously. The results demonstrate the potential of the technique in monitoring both pulsating and steady components of the blood in inflammatory lesions of the skin.