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 $5microns$. CPDs could be seen at $60-mW$ laser power and $5-μm$ 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.

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.