Supplementary Material for: Human skin responses to UV (predominantly UVC at 254 nm) irradiation – in vivo kinetics of morphological changes and recovery using non-invasive two-photon tomography

Introduction: The effects of ultraviolet (UV) radiation on the skin are manifold. On the one hand, it aids in the production of vitamin D and is used in phototherapy. On the other hand, it can cause cell damage leading to premature skin aging and the development of various skin pathologies, including skin cancer. Despite the importance of this issue, the development and recovery of morphological changes induced by UV irradiation are still almost unexplored. Methods: In this in vivo study, two-photon tomography combined with fluorescence lifetime imaging was used to investigate structural changes in the skin of healthy volunteers over 30 days after a single UV irradiation (the lamp spectrum includes UVC ≈39.8%, UVB ≈7.5%, UVA ≈8.3%, and visible ≈44.4%) applied for 120 s at a total dose of 43.1 mJ/cm². Results: The results show that UV (predominantly UVC at 254 nm) irradiation leads to an increase in the nucleus/cell ratio, appearance of a bright perinuclear rim, disruption of the membrane, expanded intercellular space, and the appearance of sunburn cells in the epidermis, and a decrease in collagen type I in the dermis, which are recovered 30 days post-UV irradiation. The phasor plot approach enabled the separation of dermal cells into four clusters, allowing fluorescence lifetime analysis of resting and activated mast cells as well as M1 and M2 macrophages. We observed that UV (predominantly UVC at 254 nm) radiation can activate mast cells, leading to a decrease their fluorescence lifetime, whereas no effect was observed in macrophages. Conclusion: The results obtained in this in vivo study confirm that UV (predominantly UVC at 254 nm) has damaging effect on all skin layers. Two-photon tomography is an effective non-invasive method for in vivo imaging morphological changes in the skin following UV irradiation and for studying severe photodamage.