Supplementary Material for: In vivo characterization of colored tattoo ink distribution in human skin using non-invasive two-photon tomography combined with FLIM

In recent years, multicolored tattoos have gained popularity among young individuals. However, the behavior of tattoo inks in the skin, including their distribution, accumulation, and long-term effects, remains poorly understood, partly due to limited in vivo imaging techniques. This study employs picosecond-pulsed two-photon excited fluorescence lifetime imaging microscopy (TPE-FLIM) to non-invasively visualize the deposition of colored tattoo ink pigments (blue, red, green, yellow, orange, purple and black) in human skin in vivo at depths of up to ≈150 µm.
The study reveals that tattoo ink agglomerates are present across various skin layers, ranging from the stratum corneum to the reticular dermis. Tattoo ink pigments were predominantly observed within dermal cells, including mast cells, macrophages, and fibroblasts, suggesting that these cells act as long-term reservoirs for the pigments. Additionally, ink residues were detected in epidermal cells, including keratinocytes, dendritic cells, and basal cells, indicating a continuous low-level release of the ink even in old tattoos. Furthermore, the analysis showed that melanin can be distinguished from certain tattoo ink pigments using phasor approach combined with TPE autofluorescence imaging.
The TPE-FLIM technique enables in vivo visualization of epidermal dendritic cells and dermal fibroblasts, which are otherwise undetectable using conventional optical methods without staining. This method holds significant potential for clinical dermatology by offering insights into cellular uptake, accumulation, and long-term effects of tattoo inks, thereby supporting the management of tattoo-related complications.