Supplemental Material for: Retinoid delivery and effects in human skin cells using fluorescent two-photon imaging techniques

In this study, fluorescence lifetime imaging microscopy (FLIM) was used to visualize the spatiotemporal uptake and intracellular behaviour of several natural and synthetic retinoids in immortalized human keratinocytes (HaCaT cells) and dermal fibroblasts (BJ-1 cells). </p><p dir="ltr">Results: Taking advantage of the intrinsic fluorescence properties of retinoids, FLIM enabled the discrimination of different retinoid species and their metabolites within cells based on distinct fluorescence lifetime signatures.

Treatment with retinoids produced measurable changes in fluorescence lifetime, signal intensity and cellular morphology compared with untreated controls, indicating differences in intracellular accumulation and metabolic processing. These changes were most pronounced for retinol, which demonstrated marked increases in fluorescence intensity and lifetime over time (average 3.7-fold increase in intensity and 1.9-fold increase in mean time-weighted lifetime). </p><p dir="ltr">Conclusion: Collectively, these findings demonstrate that FLIM provides a powerful, label-free approach for visualizing the intracellular disposition of retinoids and may offer new insights into their cellular pharmacokinetics and metabolism within skin cells.</p>