In Vivo Non-Invasive Observation of Dynamic Changes in Hair Shaft, Melanin, and Collagen During the Mice Hair Follicle Cycle.

Traditional evaluations of the hair follicle cycle rely on invasive histological methods, limiting continuous longitudinal observation. It was hypothesized that non-invasive optical imaging, particularly two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG), can accurately track and quantify in vivo dynamic changes in hair shafts, melanin, and collagen across multiple hair follicle cycles. The hair shafts of C57BL/6 mice were monitored in vivo during the anagen, catagen, and telogen phases using dermoscopy, photography, and electron microscopy. Specifically, TPEF under 800 nm excitation and imaging were used to quantify melanin dynamics, while SHG under 950 nm excitation was used to observe interfollicular collagen changes. To ensure accuracy, the in vivo optical measurements were cross-validated: melanin dynamics were confirmed via skin photography for overall tissue appearance and hematoxylin and eosin (HE) staining for hair bulb expression, while collagen changes were validated by assessing fiber density and orientation using a liquid crystal polarizing imaging system on Sirius Red-stained sections. The findings observations demonstrated that during the anagen phase, hair shaft diameter and melanin production progressively increased, accompanied by a significant reduction in interfollicular collagen density. During the catagen and telogen phases, melanin levels gradually decreased, and collagen density gradually recovered to baseline levels. This project establishes a validated, non-invasive in vivo methodology for evaluating the comprehensive dynamics of hair follicles, providing a robust experimental framework for the preclinical evaluation of active agents for hair loss treatments.