2‐D mapping of skin chromophores in the spectral range 500 – 700 nm

The multi‐spectral imaging technique has been used for distant mapping of in‐vivo skin chromophores by analyzing spectral data at each reflected image pixel and constructing 2‐D maps of the relative concentrations of oxy‐/deoxy‐haemoglobin and melanin. Instead of using a broad visible‐NIR spectral range, this study focuses on narrowed spectral band 500–700 nm, speeding‐up the signal processing procedure. Regression analysis confirmed that superposition of three Gaussians is optimal analytic approximation for the oxy‐haemoglobin absorption tabular spectrum in this spectral band, while superposition of two Gaussians fits well for deoxy‐haemoglobin absorption and exponential function – for melanin absorption. The proposed approach was clinically tested for three types of in‐vivo skin provocations: ultraviolet irradiance, chemical reaction with vinegar essence and finger arterial occlusion. Spectral range 500–700 nm provided better sensitivity to oxy‐haemoglobin changes and higher response stability to melanin than two reduced ranges 500–600 nm and 530–620 nm. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Retrieving skin properties from in vivo spectral reflectance measurements

A previously developed inverse method was applied to in vivo normal‐hemispherical spectral reflectance measurements taken on the inner and outer forearm as well as the forehead of healthy white Caucasian and black African subjects. The inverse method was used to determine the thickness and melanin concentration in the epidermis, dermal blood volume fraction and oxygen saturation, and skin's spectral scattering coefficient. It was established that changes in melanin concentration due to racial difference and tanning, and differences in epidermal thickness and blood volume with anatomical location were detectable. The retrieved values were also consistent with independent measurements reported in the literature. The same method could be used for optical diagnosis of pathologies affecting the structure and pigmentation of human skin. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

In vivo multiphoton microscopy of melasma

Melasma is a skin disorder characterized by hyperpigmented patches due to increased melanin production and deposition. In this pilot study, we evaluate the potential of multiphoton microscopy (MPM) to characterize non‐invasively the melanin content, location, and distribution in melasma and assess the elastosis severity. We employed a clinical MPM tomograph to image in vivo morphological features in melasma lesions and adjacent normal skin in 12 patients. We imaged dermal melanophages in most dermal melasma lesions and occasionally in epidermal melasma. The melanin volume fraction values measured in epidermal melasma (14% ± 4%) were significantly higher (p < 0.05) than the values measured in perilesional skin (11% ± 3%). The basal keratinocytes of melasma and perilesions showed different melanin distribution. Elastosis was predominantly more severe in lesions than in perilesions and was associated with changes in melanin distribution of the basal keratinocytes. These results demonstrate that MPM may be a non‐invasive imaging tool for characterizing melasma.     

Enhanced spatial resolution for snapshot hyperspectral imaging of blood perfusion and melanin information within human tissue

We report a reconstruction method to achieve high spatial resolution for hyperspectral imaging of chromophore features in skin in vivo. The method utilizes an established structure‐adaptive normalized convolution algorithm to reconstruct high spatial resolution of hyperspectral images from snapshot low‐resolution hyperspectral image sequences captured by a snapshot spectral camera. The reconstructed images at chromophore‐sensitive wavebands are used to map the skin features of interest. We demonstrate the method experimentally by mapping the blood perfusion and melanin features (moles) on the facial skin. The method relaxes the constrains of the relatively low spatial resolution in the snapshot hyperspectral camera, making it more usable in imaging applications.     

Smartphone‐based spectral imaging otoscope: System development and preliminary study for evaluation of its potential as a mobile diagnostic tool

We develop a novel smartphone‐based spectral imaging otoscope for telemedicine and examine its capability for the mobile diagnosis of middle ear diseases. The device was applied to perform spectral imaging and analysis of an ear‐mimicking phantom and a normal and abnormal tympanic membrane for evaluation of its potential for the mobile diagnosis. Spectral classified images were obtained via online spectral analysis in a remote server. The phantom experimental results showed that it allowed us to distinguish four different fluids located behind a semitransparent membrane. Also, in the spectral classified images of normal ears (n = 3) and an ear with chronic otitis media (n = 1), the normal and abnormal regions in each ear could be quantitatively distinguished with high contrast. These preliminary results thus suggested that it might have the potentials for providing quantitative information for the mobile diagnosis of various middle ear diseases.     

Role of light in human skin color viariation.

The major source of color in human skin derives from the presence within the epidermis of specialized melanin-bearing organelles, the melanosomes. Tanning of human skin on exposure to ultraviolet light results from increased amounts of melanin within the epidermis. Melanosomes synthesized by melanocytes are acquired by keratinocytes and transported within them to the epidermal surface. In some cases, the melanosomes are catobolized en route. New information indicates that the multicellular epidermal melanin unit (melanocyte and associated pool of keratinocytes) rather than the melanocyte alone is the focal point for the control of melanin metabolism within mammalian epidermis. Gross human skin color derives from the visual impact of the summed melanin pigmentation of the many epidermal melanin units. In theory, constitutive skin color in man designates the genetically-determined levels of melanin pigmentation developed in the absence of exposure to solar radiation or other environmental influences; facultative skin color or "tan" characterizes the increases in melanin pigmentation above the constitutive level induced by ultraviolet light. The details of genetic regulation of pigment metabolism within the epidermal melanin units are being clarified. In some mammals at least, the function of epidermal melanin units is significantly influenced by hormones which may be regulated by radiations received through the eyes. Based on an evolutionary history of the human family which exceeds ten million years, it is proposed that melanin pigmentation may have played a number of roles in human adaptions to changing biologic and physical environments.     

ELECTRON MICROSCOPE STUDIES OF EPIDERMAL MELANOCYTES, AND THE FINE STRUCTURE OF MELANIN GRANULES

Melanocytes and melanin granules have been studied by electron microscopy in normal human and cat skin, and in hyperplastic human skin lesions. The melanocytes have always been found as free cells within the epidermis,i.e., on the epidermal side of the dermal membrane. Melanocytes frequently rest on the dermal membrane or bulge towards the dermis. In such cases the uninterrupted dermal membrane is uniformly thin and smooth in appearance, in contrast with the regions alongside Malpighian cells, where it appears appreciably thicker and seemingly anchored to the basal cell layer. Two types of melanin granules have been distinguished according to their location in the melanocytes and to morphological characteristics which may only express different stages in the maturation of the granules: (a) light melanin granules in which a structure resembling a fine network is apparent; (b) dense melanin granules which, in osmium-fixed preparations, appear as uniformly dense masses surrounded by a coarsely granular, intensely osmiophilic shell. Treatment of sections of osmium-fixed tissues with potassium permanganate has revealed within the dense granules the existence of an organized framework in the form of a regular, crystalline-like lattice. It is suggested that this basic structure is protein in nature and may include the enzymatic system capable of producing melanin. The existence is reported of fine filaments located in the cytoplasm of melanocytes and morphologically distinct from the tonofilaments found in Malpighian cells.     

Syndecan‐2 regulates melanin synthesis via protein kinase C βII‐mediated tyrosinase activation

Syndecan‐2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan‐2 in melanogenesis. Syndecan‐2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA‐mediated knockdown of syndecan‐2 was associated with reduced melanin synthesis, whereas overexpression of syndecan‐2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan‐2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan‐2 expression, and this up‐regulation of syndecan‐2 was required for UVB‐induced melanin synthesis. Taken together, these data suggest that syndecan‐2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin‐associated diseases.     

大鲵皮肤色素提取工艺及抗氧化研究

为优化大鲵皮肤黑色素的提取工艺条件,探讨大鲵皮肤黑色素组成成分及体外抗氧化活性,采用酶法和碱溶酸沉法提取大鲵皮肤黑色素,以氢氧化钠浓度、液料比、提取温度为影响色素提取率因素,优化黑色素提取工艺条件,用紫外-可见光谱仪、红外光谱仪和超高效液相质谱仪测定黑色素的光谱特性,测定其抗氧化性。结果表明:大鲵皮肤黑色素最佳提取工艺条件为氢氧化钠浓度1.5 mol/L、液料比1∶15、提取温度45℃,黑色素提取率达0.65%。大鲵皮肤黑色素的紫外最大吸收波长为214 nm,由真黑色素和脱黑色素两种色素组成,其对超氧阴离子自由基的清除率为30.51%,对羟基自由基的清除率为54.17%。大鲵皮肤黑色素具有一定的体外抗氧化能力。     

Comparison of Long‐Term Survival of Pigmented Epidermal Reconstructs Cultured In Vitro vs. Xenografted on Nude Mice

Epidermal reconstructs incorporating pigment cells have been used in vitro over the last decade to study the physiology of the epidermal melanin unit. However, the major limitation of this technology is the duration of the assays, which need to be completed within 2–3 weeks to obviate the problem of epidermal senescence and excessive terminal differentiation. This becomes a major problem for studying long‐term biological phenomena in photoprotection and epidermal skin cancers. We report here a simplified surgical technique in immunotolerant mice allowing long‐term studies. The creation of a vascularized mouse skin flap is the key point of the surgical procedure. Long‐term pigmentation of the xenografts seemed macroscopically successful, but surprisingly microscopy at 11 and 16 weeks postgrafting showed mostly dermal pigment aggregates and rare Melan‐A positive dermal and epidermal pigment cells. In the same reconstructs maintained in vitro, dermal pigment and dermal pigment cells were never noted. It could be speculated that in our model, the colonization of the xenografted dead human dermis by murine cells influences melanocyte survival.     

Comparison of long-term survival of pigmented epidermal reconstructs cultured in vitro vs. xenografted on nude mice

Epidermal reconstructs incorporating pigment cells have been used in vitro over the last decade to study the physiology of the epidermal melanin unit. However, the major limitation of this technology is the duration of the assays, which need to be completed within 2-3 weeks to obviate the problem of epidermal senescence and excessive terminal differentiation. This becomes a major problem for studying long-term biological phenomena in photoprotection and epidermal skin cancers. We report here a simplified surgical technique in immunotolerant mice allowing long-term studies. The creation of a vascularized mouse skin flap is the key point of the surgical procedure. Long-term pigmentation of the xenografts seemed macroscopically successful, but surprisingly microscopy at 11 and 16 weeks postgrafting showed mostly dermal pigment aggregates and rare Melan-A positive dermal and epidermal pigment cells. In the same reconstructs maintained in vitro, dermal pigment and dermal pigment cells were never noted. It could be speculated that in our model, the colonization of the xenografted dead human dermis by murine cells influences melanocyte survival.     

Variation in melanin content and composition in type V and VI photoexposed and photoprotected human skin: the dominant role of DHI

A combination of techniques, including high-performance liquid chromatography (HPLC), spectrophotometric measurements, and a novel method for quantifying melanosome morphology, were applied to the analysis of melanin content and composition in highly pigmented (Fitzpatrick type V and VI) human skin. We found that total epidermal melanin content is significantly elevated in photoexposed type V and VI skin (approximately 1.6 x), while analysis of individual melanin components suggests that pheomelanin content increases only slightly, whereas 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-eumelanin and to a greater extent 5,6-dihydroxyindole (DHI)-eumelanin content are both markedly elevated. Analysis of the relative composition of epidermal melanin in these subjects revealed that DHI-eumelanin is the largest single component (approximately 60-70%), followed by DHICA-eumelanin (25-35%), with pheomelanin being a relatively minor component (2-8%). Moreover, there was a comparative enrichment of DHI-eumelanin at photoexposed sites, with a corresponding decline in the relative contributions from DHICA-eumelanin and pheomelanin. There was also a good correlation and close agreement between the concentration of spheroidal melanosomes determined by morphological image analysis and the concentration of pheomelanin determined by a combination of HPLC and spectrophotometric analysis (r = 0.89, P < 0.02). This study demonstrates the usefulness of melanosome morphology analysis as a sensitive new method for the quantification of melanin composition in human skin. The data also suggest that DHI-eumelanin formation is the dominant pathway for melanin synthesis in heavily pigmented (Fitzpatrick V and VI) skin types in vivo, and is the favoured pathway when melanin production is increased in chronically photoexposed skin.     

Three‐dimensional multispectral optoacoustic mesoscopy reveals melanin and blood oxygenation in human skin in vivo

Optical imaging plays a major role in disease detection in dermatology. However, current optical methods are limited by lack of three‐dimensional detection of pathophysiological parameters within skin. It was recently shown that single‐wavelength optoacoustic (photoacoustic) mesoscopy resolves skin morphology, i.e. melanin and blood vessels within epidermis and dermis. In this work we employed illumination at multiple wavelengths for enabling three‐dimensional multispectral optoacoustic mesoscopy (MSOM) of natural chromophores in human skin in vivo operating at 15–125 MHz. We employ a per‐pulse tunable laser to inherently co‐register spectral datasets, and reveal previously undisclosed insights of melanin, and blood oxygenation in human skin. We further reveal broadband absorption spectra of specific skin compartments. We discuss the potential of MSOM for label‐free visualization of physiological biomarkers in skin in vivo.

Cross‐sectional optoacoustic image of human skin in vivo. The epidermal layer is characterized by melanin absorption. A vascular network runs through the dermal layer, exhibiting blood oxygenation values of 50–90%. All scale bars: 250 µm     

Histological and histochemical examination of American Indian scalps, mummies, and a shrunken head

The histological and histochemical detail remaining in Jivaro shrunken heads, North American Indian scalps, and mummified remains from Peru, Chile, and the American southwest, is remarkable when these specimens are rehydrated and fixed by Sandison's methods. Special techniques and stains used frequently in the histochemistry of the skin brought out details which were less evident or not apparent in the routine hematoxylin and eosin preparations. The polarizing microscope was particularly useful in demonstrating keratin and collagen fibers. Use of the silver stain for melanin not only aided in demonstrating hairs, but also outlined the pattern of epidermal detail by supranuclear accumulation and melanin dust in the stratum corneum. The hematoxylin-phloxine-alcian blue orange G stain for prekeratin, keratin, and mucin showed long persistence of the prekeratin and keratin staining properties. The stain identified strands as epidermis where they would otherwise have been overlooked. The Luxol Fast Blue stain, birefringence, and appearance with phase contrast microscopy are promising means for evaluating the degradation of skin collagen under conditions of dry preservation. The first loss of histological information occurs in extravascular cells of the dermis; mast cells, eosinophils, and other perivascular cells could not be identified in any of the specimens. Fine elastic fibers near the epidermis disappear before the larger, mid-dermal elastic fibers. Some of the disease processes which could be diagnosed in very old mummified skin are discussed in terms of the remaining structures.     

Ethnic variation in melanin content and composition in photoexposed and photoprotected human skin

We have examined the quantity and composition of melanin in both photoprotected (volar upper arm) and chronically photoexposed (dorsal forearm) skin from a range of different ethnic skin types including African, Indian, Mexican, Chinese and European. The most lightly pigmented (European, Chinese and Mexican) skin types have approximately half as much epidermal melanin as the most darkly pigmented (African and Indian) skin types. However, the composition of melanin in these lighter skin types is comparatively more enriched with lightly coloured, alkali-soluble melanin components (up to three-fold). Regardless of ethnicity, epidermal melanin content is significantly greater in chronically photoexposed skin than it is in corresponding photoprotected skin (up to two-fold). However, by comparison there is only a modest enrichment of lightly coloured, alkali soluble melanin components in photoprotected skin (up to 1.3-fold). Analysis of melanosomes extracted from the epidermis in these subjects indicates that the proportion of spheroidal melanosomes is low in all skin types examined (<10%). This suggests that in human skin, pheomelanin is a very minor component of epidermal melanin, even in the lightest (European) skin types. Analysis of melanosome size revealed a significant and progressive variation in size with ethnicity: African skin having the largest melanosomes followed in turn by Indian, Mexican, Chinese and European. On the basis of these findings, we propose that variation in skin pigmentation is strongly influenced by both the amount and the composition (or colour) of the melanin in the epidermis. Variation in melanosome size may also play a significant role. However, the data also suggest that in human skin there are subtle differences in the mechanisms associated with the maintenance of constitutive pigmentation and facultative hyperpigmentation, respectively.     

Use of mathematically enhanced spectral analysis and spectral contrast techniques for the liquid chromatographic and capillary electrophoretic detection and identification of pharmaceutical compounds

The use of mathematically enhanced ultraviolet/visible (UV/VIS) absorbance spectral analysis and spectral contrast software techniques in high performance liquid chromatography (HPLC) and micellar electrokinetic capillary electrophoresis (MECC) as an aid for the determination of peak homogeneity, identification, and tracking during method development was investigated. Various structurally similar pharmaceutical compounds, and compounds present as either cis/trans isomers, diastereomers, or enantiomers were used as test compounds to probe the limits of this technique. Two tricyclic antidepressants, nortriptyline and imipramine, were employed to study the effects of HPLC mobile phase composition and pH on the ability to identify and track peaks during method development. It was found that method changes altered the spectral matches used for identification, but not enough to cause incorrect peak identification. It was also shown using HPLC that the cis/trans isomers of doxepin and the diastereomers ephedrine and pseudoephedrine could be distinguished. The mathematically enhanced spectral analysis and spectral contrast software techniques were also employed with MECC. Peaks tracking during method development as pH and the concentration of surfactant changes is shown for a separation of various penicillin type antibiotics. It was shown that during chiral MECC (CMECC) analyses ephedrine/pseudoephedrine diastereomers as well as ephedrine enantiomers could be distinguished. The determination of enantiomers is possible in CMECC since enantiomers are eluted as diastereomeric complexes, as opposed to HPLC where they are eluted in their native state. © 1996 Wiley-Liss, Inc.     

Effect of keratinocytes on regulation of melanogenesis in culture of melanocytes

Melanocytes are the melanin-producing cells by melanogenesis, and the pigment melanin is primarily responsible for the color of skin. These cells contain dendrites that are in close contact with neighboring keratinocytes. Keratinocytes produce and secrete factors that regulate the proliferation and melanogenesis of melanocytes in vitro. Therefore, adopting only melanocyte pure culture may not clearly reflect the skin physiology in vivo. In this study, we applied a two-culture model using melanocytes and keratinocytes from human skin, such as melanocyte pure culture and melanocyte co-culture with keratinocyte. And then, there was compared the responses of melanocytes under different culture conditions (treatment with arbutin, MSH-α and UV-B irradiation). The results show that there was no significant difference in melanocyte proliferation and melanogenesis between arbutin and MSH-α treatment. However, the co-culture model was more stable than the pure culture model in terms of melanocyte proliferation and melanogenesis upon UV-B irradiation. Therefore, the co-culture model was superior to the pure culture as a useful method for the study of melanocytes and epidermal melanin unit.     

The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes

Malignant transformation of melanocytes is associated with changes in melanogenesis. Therefore, fluorescence of melanin may be an informative indicator of this process. But the conventionally excited autofluorescence of melanin in skin tissue is ultra-weak and its main part in the visible spectral region is hidden by the much stronger fluorescence from other endogenous fluorophores. Here, using a new mode of stepwise two-photon excitation, melanin-dominated fluorescence spectra of pigmented skin lesions are reported. From these, pure melanin fluorescence spectra of normal pigmented skin, melanocytic nevi and malignant pigmented melanoma were analyzed. They show distinctly different spectral shapes: melanoma gave a characteristic fingerprint with a fluorescence band peaking at 640 nm, independent of the melanoma subtype. The melanin fluorescence spectra peaked at 590 nm for all types of common melanocytic nevi. These differences in the fluorescence spectra are probably based on different contents of eumelanin and pheomelanin. In a series of 167 cases with melanocytic nevi and melanomas, the sensitivity of this new method to diagnose melanoma was 93.5%, the specificity 80.0% and the diagnostic accuracy 82.6%. The two-photon excitation fluorescence method is a new diagnostic tool which may in future supplement conventional dermatohistopathology.     

A fluorescent imaging method for analyzing the biodistribution of therapeutic monoclonal antibodies that can distinguish intact antibodies from their breakdown products

Many monoclonal antibodies have been developed for therapy over the last 2 decades. In the development of therapeutic antibodies, the preclinical assessment of an antibody's biodistribution is important for the prediction of the antibody's efficacy and safety. For imaging analyses of such biodistributions, radioisotope (RI) labeling and fluorescence labeling methods are typically used, but the resulting data are limited because these methods cannot distinguish breakdown products from intact antibodies. To resolve this problem, we developed a novel method using fluorescent resonance energy transfer (FRET)-type labeling and a spectral unmixing tool. With FRET-type labeling (labeling with 2 species of fluorophore), different fluorescence properties of labeled intact antibodies and their breakdown products (the hydrolyzed/digested type of breakdown products) are made visible. With the spectral unmixing tool, the fluorescence of a solution containing the intact antibody and its breakdown products could be unmixed in proportion to their contents. Moreover, when labeled antibodies that targeted either human epidermal growth factor receptor-2 or epidermal growth factor receptor were injected into nude mice implanted subcutaneously with tumor cells, the accumulation of the injected labeled antibodies and their breakdown products in the tumor could be separately analyzed by both whole-mouse imaging and a tumor homogenate analysis. These results suggest that our method using FRET-type labeling and a spectral unmixing tool could be useful in distinguishing breakdown products from intact antibodies.