Approach to quantify human dermal skin aging using multiphoton laser scanning microscopy.

Extracellular skin structures in human skin are impaired during intrinsic and extrinsic aging. Assessment of these dermal changes is conducted by subjective clinical evaluation and histological and molecular analysis. We aimed to develop a new parameter for the noninvasive quantitative determination of dermal skin alterations utilizing the high-resolution three-dimensional multiphoton laser scanning microscopy (MPLSM) technique. To quantify structural differences between chronically sun-exposed and sun-protected human skin, the respective collagen-specific second harmonic generation and the elastin-specific autofluorescence signals were recorded in young and elderly volunteers using the MPLSM technique. After image processing, the elastin-to-collagen ratio (ELCOR) was calculated. Results show that the ELCOR parameter of volar forearm skin significantly increases with age. For elderly volunteers, the ELCOR value calculated for the chronically sun-exposed temple area is significantly augmented compared to the sun-protected upper arm area. Based on the MPLSM technology, we introduce the ELCOR parameter as a new means to quantify accurately age-associated alterations in the extracellular matrix.

Folic acid and creatine improve the firmness of human skin in vivo.

BACKGROUND: The decrease in firmness is a hallmark of skin aging. Accelerated by chronic sun exposure, fundamental changes occur within the dermal extracellular matrix over the years, mainly impairing the collagenous network. AIMS: Based on the qualitative and quantitative assessment of skin firmness, in vitro and in vivo studies were carried out to elucidate the effects of topical folic acid and creatine to counteract this age-dependent reduction in the amount of collagen. PATIENTS/METHODS: Topical application of a commercially available formulation containing folic acid and creatine was performed to study effects on skin firmness in vivo using cutometric analysis. Imaging and quantification of collagen density were carried out using multiphoton laser scanning microscopy (MPLSM). To investigate the effects of these compounds on collagen gene expression, procollagen synthesis, and collagen fibril organization, complementary in vitro studies on cultured fibroblast-populated collagen gels were carried out. RESULTS: The underlying structural changes in the collagen network of young and aged sun-exposed facial skin in vivo were visualized by MPLSM. Topical application of a folic acid- and creatine-containing formulation significantly improved firmness of mature skin in vivo. Treatment of fibroblast-populated dermal equivalents with folic acid and creatine increased collagen gene expression and procollagen levels and improved collagen fiber density, suggesting that the in vivo effects are based on the overall improvement of the collagen metabolism. CONCLUSIONS: Employing MPLSM, dermal changes occurring in photo-aged human skin were visualized in an unprecedented manner and correlated to a loss of firmness. Treatment of aged skin with a topical formulation containing folic acid and creatine counteracted this age-dependent decline by exerting sustained effects on collagen metabolism. Our results support previous findings on the efficacy of these actives.

Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm.

New imaging techniques using near-infrared (NIR) femtosecond lasers (fs-lasers) in multiphoton laser scanning microscopy (MPLSM) have great potential for in vivo applications, particularly in human skin. However, little is known about possible risks. In order to evaluate the risk, a "biological dosimeter" was used. We irradiated fresh human skin samples with both an fs-laser and a solar simulator UV source (SSU). DNA damage introduced in the epidermis was evaluated using fluorescent antibodies against cyclobutane-pyrimidin-dimers (CPDs) in combination with immunofluorescence image analysis. Four fs-irradiation regimes (at 800-nm wavelength) were evaluated differing in laser power and step width of horizontal scans. Fs-irradiation did not give CPDs at 15-mW or 30-mW irradiation power using 10 horizontal scans every 5 microns. CPDs could be seen at 60-mW laser power and 5-microm step size and at 35-mW using 1-micron step width. Quantitative comparison of SSU-induced CPDs showed that the 60-mW laser irradiation regime is comparable to UV-irradiation, giving 0.6 minimal erythemal dose (MED). The 1-micron irradiation regime was comparable to 0.45 MED. Under these experimental conditions, the risk of DNA damage due to fs-laser irradiation on skin is in the range of natural UV-exposure.

Assessing the risk of skin damage due to femtosecond laser irradiation.

We irradiated freshly excised skin biopsies with four irradiation regimes usually taken for multiphoton imaging. If there is any skin damaging, it is mainly an effect similar to the damaging effects of UV-irradiation. Using fluorescent antibodies against cyclobutane-pyrimidin-dimers (CPDs) in combination with immuno-fluorescence image analysis we quantitatively compared fs-irradiation effects with UV-irradiation (solar simulator). Based on these results we are giving a risk assessment. The results show that multi photon imaging using the parameters described here is in the ballpark of damaging occurring from every day sun exposure.