Toyonobu Yamashita

Observation of dermal collagen fiber in wrinkled skin using polarization-resolved second-harmonic-generation microscopy

Optical probe methods for in vivo assessments of cutaneous photoaging are necessary in fields such as anti-aging dermatology and skin cosmetic development. We investigated the relation between wrinkle direction and collagen orientation in ultraviolet-B-exposed (UVB-exposed) skin using polarization-resolved second-harmonic-generation (SHG) microscopy. A polarization anisotropic image of the SHG light indicated that wrinkle direction in UVB-exposed skin is predominantly parallel to the orientation of dermal collagen fibers. Furthermore, collagen orientation in post-UVB-exposed skin with few wrinkles changed from that of UVB-exposed wrinkled skin to that of no-UVB-exposed skin. The method proposed has the potential to become a powerful non-invasive tool for assessment of cutaneous photoaging.

In vivo observation of age-related structural changes of dermal collagen in human facial skin using collagen-sensitive second harmonic generation microscope equipped with 1250-nm mode-locked Cr:Forsterite laser

Abstract. In vivo visualization of human skin aging is demonstrated using a Cr:Forsterite (Cr:F) laser-based, collagen-sensitive second harmonic generation (SHG) microscope. The deep penetration into human skin, as well as the specific sensitivity to collagen molecules, achieved by this microscope enables us to clearly visualize age-related structural changes of collagen fiber in the reticular dermis. Here we investigated intrinsic aging and/or photoaging in the male facial skin. Young subjects show dense distributions of thin collagen fibers, whereas elderly subjects show coarse distributions of thick collagen fibers. Furthermore, a comparison of SHG images between young and elderly subjects with and without a recent life history of excessive sun exposure show that a combination of photoaging with intrinsic aging significantly accelerates skin aging. We also perform image analysis based on two-dimensional Fourier transformation of the SHG images and extracted an aging parameter for human skin. The in vivo collagen-sensitive SHG microscope will be a powerful tool in fields such as cosmeceutical sciences and anti-aging dermatology.

In vivo assessment of pigmentary and vascular compartments changes in UVA exposed skin by reflectance-mode confocal microscopy.

Abstract:  Exposure of the skin to ultraviolet A (UVA) results in various biological responses, skin‐colour changes being among the major ones. Although intense research has been performed on UVA‐induced pigmentation and vascular changes, the process of skin‐colour changes after UVA irradiation remains unclear. For a better understanding of the UVA tanning mechanism, we here performed a human study in 27 healthy volunteers with skin phototype (SPT) II to VI. After a single UVA exposure to inner forearm, the skin sites were imaged using reflectance‐mode confocal microscopy (RCM), for analysis of melanin and vascular changes. Punch biopsies were also taken from the UVA‐exposed or non‐exposed sites for histological examination. Skin sections were stained with Fontana‐Masson and evaluated by a sensitive tyrosinase assay for comparison with RCM images. Furthermore, the effect of blood flow on skin‐colour changes was evaluated visually after administration of an intradermal anesthesia of lidocaine with or without epinephrine. Our RCM analysis showed dendritic melanocytes and a different melanin distribution in the epidermal layer, clearly visible 1 week after the UVA exposure in subjects of SPT V which were supported by histological examination. However, no melanin distribution pattern changes were apparent immediately after the exposure, while RCM images showed accelerated capillary flow patterns. The restriction of this UVA induced‐accelerated blood flow by epinephrine inhibited partially or completely the immediate pigment darkening and delayed tanning. These in vivo studies confirmed that vascular change is an important factor for the development of the immediate pigment darkening and delayed tanning.

An innovative method to measure skin pigmentation

Background/purpose: It is crucial to establish an accurate method for measuring skin pigmentation in cosmetic science and clinical dermatology. Here, we report a non‐invasive precise method for measuring skin melanin content.

Motion-artifact-robust, polarization-resolved second-harmonic-generation microscopy based on rapid polarization switching with electro-optic Pockells cell and its application to in vivo visualization of collagen fiber orientation in human facial skin

Polarization-resolved second-harmonic-generation (PR-SHG) microscopy is a powerful tool for investigating collagen fiber orientation quantitatively with low invasiveness. However, the waiting time for the mechanical polarization rotation makes it too sensitive to motion artifacts and hence has hampered its use in various applications in vivo. In the work described in this article, we constructed a motion-artifact-robust, PR-SHG microscope based on rapid polarization switching at every pixel with an electro-optic Pockells cell (PC) in synchronization with step-wise raster scanning of the focus spot and alternate data acquisition of a vertical-polarization-resolved SHG signal and a horizontal-polarization-resolved one. The constructed PC-based PR-SHG microscope enabled us to visualize orientation mapping of dermal collagen fiber in human facial skin in vivo without the influence of motion artifacts. Furthermore, it implied the location and/or age dependence of the collagen fiber orientation in human facial skin. The robustness to motion artifacts in the collagen orientation measurement will expand the application scope of SHG microscopy in dermatology and collagen-related fields.

In vivo microscopic approaches for facial melanocytic lesions after quality-switched ruby laser therapy: time-sequential imaging of melanin and melanocytes of solar lentigo in Asian skin.

BACKGROUND The quality‐switched ruby laser (QSRL) has been widely used for the treatment of pigmented lesions, but clinical evaluations in most studies have been conducted on macroscopic skin color observation comparing the laser‐treated skin with its nontreated surrounding area. A few investigations examined skin changes after laser therapy at a cellular level, but almost none did so noninvasively. OBJECTIVE To elucidate the dynamic changes after QSRL irradiation of facial solar lentigo using noninvasive optical techniques. MATERIALS AND METHODS Time‐sequential imaging of Japanese female patients with a clinical diagnosis of solar lentigo was performed using ultraviolet photography, high‐magnification videomicroscopy, and reflectance‐mode confocal microscopy to examine pigmentary change after QSRL irradiation. RESULTS The present study showed that remaining melanocytes were visible in the solar lentigo of all subjects when crusts peeled off, despite hardly observable skin pigmentation to the naked eye. Moreover, noninvasive confocal imaging revealed that pigmented melanocytes varied in each solar lentigo after QSRL treatment, as indicated by melanin reflection level. CONCLUSIONS Optical techniques facilitate the evaluation of the in vivo dynamics of epidermal–melanocytic changes in solar lentigo after QSRL therapy and may be useful for monitoring outcomes after laser irradiation. The authors have indicated no significant interest with commercial supporters.

Image analysis of skin color heterogeneity focusing on skin chromophores and the age-related changes in facial skin

Heterogeneity with respect to skin color tone is one of the key factors in visual perception of facial attractiveness and age. However, there have been few studies on quantitative analyses of the color heterogeneity of facial skin. The purpose of this study was to develop image evaluation methods for skin color heterogeneity focusing on skin chromophores and then characterize ethnic differences and age‐related changes.

Extraction of Dermo-Epidermal Surface from 3D Volumetric Images of Human Skin

Analysis of the dermo-epidermal surface in three-dimensions has great value in evaluating cosmetics. One approach is based on the active contour model, which is used extensively in computer vision and image processing applications, particularly for local object boundaries with closed curve form. The dermo-epidermal surface, however, is a plane with open form. We have developed a method of automatically extracting the dermo-epidermal surface from volumetric confocal microscopic images, as well as constructing a 3D visual model of the surface by using the geometric information contained in the control points. Our method is a 3D extension of the active contour model, so we call it the active open surface model (AOSM). The initial surface for AOSM is an open curve plane, guided by a 3D internal force, a 3D external constraint force, and a 3D image force, which pull it towards the objective surface. The proposed tecnique has been applied to extract actual dermo-epidermal surface in the given volumetric confocal microscopic images.

A new quantitative evaluation method for age-related changes of individual pigmented spots in facial skin

Facial skin pigmentation is one of the most prominent visible features of skin aging and often affects perception of health and beauty. To date, facial pigmentation has been evaluated using various image analysis methods developed for the cosmetic and esthetic fields. However, existing methods cannot provide precise information on pigmented spots, such as variations in size, color shade, and distribution pattern. The purpose of this study is the development of image evaluation methods to analyze individual pigmented spots and acquire detailed information on their age‐related changes.

Observation of Collagen Fiber Structure in Dermis Tissue by a Second-Harmonic-Generation Microscope

Second-harmonic-generation (SHG) microscope is applied to observe collagen fiber structure in porcine dermis and mouse tendon. Difference of collagen fiber structure among different samples is clearly visualized as high contrast SHG images using a sample-scanning SHG microscope based on a transmission configuration. From comparison of the SHG image between a transmission detection mode and a reflection one, we confirm that the reflection-mode SHG imaging is also useful to observe the collagen fiber structure in the porcine dermis. Finally, we applied a laser-scanning confocal SHG microscope to optical-sectioning SHG imaging of the thick porcine dermis every 10-μm depth and confirmed that the collagen fiber structure in the dermis is spatially evolved along the depth direction. The proposed method will be a powerful tool for in vivo measurement of human dermis to monitor skin diseases and cosmetics.