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.引言：紫外线（ultraviolet, UV）辐射对皮肤的影响具有多重性。一方面，其可辅助维生素D的合成，并应用于光疗领域；另一方面，紫外线可造成细胞损伤，引发皮肤过早老化以及包括皮肤癌在内的多种皮肤病变。尽管该问题意义重大，但紫外线辐照诱导的皮肤形态学变化的发生与恢复过程，目前仍几乎未被探索。方法：本体内研究采用双光子断层成像（two-photon tomography）联合荧光寿命成像（fluorescence lifetime imaging）技术，对单次紫外线辐照（光源光谱包含UVC≈39.8%、UVB≈7.5%、UVA≈8.3%及可见光≈44.4%）后30天内的健康志愿者皮肤结构变化进行探究。该辐照时长为120秒，总剂量为43.1 mJ/cm²。结果：研究结果显示，以254nm短波紫外线（UVC）为主的紫外线辐照会导致表皮中核质比升高、出现明亮的核周亮环、细胞膜受损、细胞间隙扩大以及晒伤细胞生成，同时真皮内I型胶原含量降低；上述变化均在紫外线辐照后30天得以恢复。相量图（phasor plot）分析方法可将真皮细胞分为四个聚类簇，从而实现对静息肥大细胞（mast cells）、活化肥大细胞以及M1型和M2型巨噬细胞的荧光寿命分析。我们观察到，以254nm UVC为主的紫外线辐射可激活肥大细胞，使其荧光寿命缩短，而巨噬细胞未出现此类变化。结论：本体内研究所得结果证实，以254nm UVC为主的紫外线辐射对皮肤各层均具有损伤作用。双光子断层成像是一种有效的非侵入性方法，可用于紫外线辐照后皮肤形态学变化的体内成像，以及严重光损伤的相关研究。