Unveiling Lipid Droplet Transport Dynamics as Biomarkers of Senescence Using Label-Free, Time-Lapse Holotomography.

Accumulation and density of lipid droplets (LDs) in cells have been identified as a potential biomarker to detect senescent cells. However, their intracellular dynamic transport and alterations during senescence remain largely unclear. To address this knowledge gap, senescence was induced in human microglia cells using hydrogen peroxide (H₂O₂) to investigate both short-term and long-term effects of H₂O₂ treatment on LD dynamics. We captured time-lapse holotomograms of LDs using label-free refractive index (RI)-based holotomography and quantified 11 dynamic parameters of LDs through single-particle tracking. These quantified parameters were then compared across healthy cells, short-term H₂O₂-treated pre-senescent cells (H₂O₂-treated cells), and long-term H₂O₂-induced senescent cells (senescent cells). The results revealed that LD dynamics are significantly altered in both H₂O₂-treated pre-senescent cells and H₂O₂-induced senescent cells, though with differing trends. Healthy cells exhibited higher values in all LD dynamic parameters compared to senescent cells, with the exception of the mean directional change rate, which is lower. In addition, H₂O₂-treated cells showed higher values in dynamic parameters such as total displacement, mean straight-line velocity, and confinement ratio compared to healthy and senescent cells, due to the observed linear migration of LDs during H₂O₂ treatment. We found that the altered movement of LDs is closely related to H₂O₂-induced damage to microtubule networks. These findings suggest that altered LD dynamics, along with associated molecules and pathways, may serve as potential biomarkers for identifying senescent cells, thereby aiding in the development of novel therapeutic targets and senolytics.