Visualizing laser-skin interaction in vivo by multiphoton microscopy

Recently, multiphoton microscopy has gained much popularity as a noninvasive imaging modality in biomedical research. We evaluate the potential of multiphoton microscopy for monitoring laser-skin reaction in vivo. Nude mouse skin is irradiated with an erbium:YAG laser at various fluences and immediately imaged by a multiphoton microscope. The alterations of cutaneous nonlinear optical properties including multiphoton autofluorescence and second-harmonic generation associated with laser irradiation are evaluated morphologically and quantitatively. Our results show that an erbium:YAG laser at a low fluence can selectively disrupt the stratum corneum, and this alteration may account for the penetration enhancing effect of laser-assisted transcutaneous drug delivery. At a higher fluence, the zone of tissue ablation as well as the disruption of the surrounding stratum corneum, keratinocytes, and dermal extracellular matrix can be better characterized by multiphoton microscopy as compared with conventional histology. Furthermore, the degree of collagen damage in the residual thermal zone can be quantified by second-harmonic generation signals, which have significant difference between control skin, skin irradiated with a 1.5-, 8-, and 16-J/cm ² erbium:YAG laser (P<0.05). We show that multiphoton microscopy can be a useful noninvasive imaging modality for monitoring laser-skin reaction in vivo.