Label-free 4D holotomography with depth-adaptive segmentation for quantitative analysis of lipid droplet dynamics in hepatic organoids

Significance Quantifying lipid droplet (LD) remodeling in 3D hepatic organoids is often limited to endpoint staining or phototoxic live fluorescence imaging, thereby obscuring droplet-level kinetics. Aim We aimed to develop a label-free method to track LD dynamics in living hepatic organoids under different fatty-acid loads. Approach Time-lapse 3D refractive-index tomograms were acquired using holotomography and analyzed with a depth-adaptive, multi-threshold segmentation pipeline to quantify LD number, volume, sphericity, and refractive-index–derived concentration and dry mass at single-droplet resolution.

Oleic acid and linoleic acid induced LD accumulation while preserving organoid integrity, whereas palmitic acid triggered rapid structural collapse. Despite increases in total LD burden under both oleic acid and linoleic acid, droplet-level dynamics diverged: oleic acid produced volume-dominated accumulation via enlargement of fewer LDs and increased size heterogeneity, whereas linoleic acid produced number-dominated accumulation via sustained increases in LD number, yielding a more uniform population of small droplets.

Label-free holotomography with depth-adaptive analysis enables non-invasive, longitudinal, and multi-scale quantification of LD dynamics in intact organoids and reveals fatty-acid– dependent temporal modes of lipid storage. Statement of Discovery We developed a label-free, longitudinal 3D holotomography framework with depth-adaptive lipid droplet segmentation that quantifies single-droplet dynamics in living mouse hepatic organoids. Using this platform, we found that oleic acid and linoleic acid induce LD accumulation via distinct strategies—oleic acid via droplet enlargement and linoleic acid via sustained increases in droplet number—while palmitic acid rapidly compromises organoid integrity. Competing Interest Statement H.L., and Y.K.P. have financial interests in Tomocube, a company that commercializes HT instruments.