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Atopic dermatitis (AD) is characterized by hyperkeratosis of epidermis and fibrosis within dermis in chronic skin lesions. Thus far, the histology of skin lesions has been evaluated only by examination of excised specimens. A noninvasive in vivo tool is essential to evaluate the histopathological changes during the clinical course of AD. We used Cr:forsterite laser-based multimodality nonlinear microscopy to analyze the endogenous molecular signals, including third-harmonic generation (THG), second-harmonic generation (SHG), and two-photon fluorescence (TPF) from skin lesions in AD. Significant differences in thickness of epidermis and stratum corneum (SC), and modified degrees of fibrosis in dermis (measured by THG signals and SHG signals, respectively), are clearly demonstrated in in vitro studies. Increased TPF levels are positively associated with the levels of the THG signals from the SC. Our in vitro observations of histological changes are replicated in the in vivo studies. These findings were reproducible in skin lesions from human AD. For the first time, we demonstrate the feasibility of preclinical applications of Cr:forsterite laser-based nonlinear microscopy. Our findings suggest that the optical signatures of THG, TPF, and SHG can be used as molecular markers to assess the pathophysiological process of AD and the effects of local treatment.

Noninvasive in vitro and in vivo assessment of epidermal hyperkeratosis and dermal fibrosis in atopic dermatitis