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Treatment and management of alopecia are highly determined by an accurate diagnosis, which can be challenging due to the lack of methods to properly visualize hair follicles. Current standard diagnosis is based on dermoscopy for non-scarring alopecia and scalp biopsy for scarring types of alopecia. Dermoscopy can be inconclusive, while biopsy is a painful procedure. In this study, we used a clinical tomograph based on multiphoton microscopy (MPM) to non-invasively image the scalp of 5 healthy subjects and of 12 patients affected by non-scarring alopecia (androgenetic and areata) and scarring alopecia (frontal fibrosing). MPM is capable of non-invasive in vivo imaging of follicular structures in human scalp including hair shafts, hair follicles and sebaceous glands via two-photon excited fluorescence (TPEF) from keratin and NADH/FAD and of the papillary dermis surrounding the hair follicles through second harmonic generation (SHG) from collagen and TPEF from elastin fibers. In normal and non-scarring alopecia patients, MPM often identified presence of sebaceous glands associated with hair follicles, while MPM images of scarring alopecia were characterized by miniaturization of hair follicles as well as by presence of macrophages and lymphocytes surrounding hair follicles. A quantitative analysis involving measurement of hair follicle diameter sizes showed they were significantly smaller in scarring comparing to non-scarring alopecia patients and to normal scalp subjects (p < 0.043). This study shows, in a limited number of patients, that MPM imaging can non-invasively identify morphological features that distinguish scarring from non-scarring alopecia.
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