Label-free measurements of lipid droplets in cells based on quantitative differential phase contrast imaging with high oblique illumination.

Lipid droplets (LDs) are dynamic organelles enclosed by a single layer of phospholipids and proteins, which play an important role in key physiological processes. As a critical parameter, LD size is a sensitive indicator of the effects of exogenous substances on adipogenesis. Accurate measurement of LD size in live cells is important for understanding disease mechanisms and developing targeted therapies. Fluorescence imaging offers high specificity, but it suffers from diffraction, making it difficult to distinguish smaller LDs and leading to inaccurate LD counting. Furthermore, fluorescent staining and labeling introduce phototoxicity and significant photobleaching, which disrupt the normal physiological state and impede long-term dynamic studies of LDs in live cells. Quantitative differential phase contrast (qDPC) imaging with high oblique illumination is proposed to measure LD size without any staining. According to simulations of illumination parameters, the annular LED array was designed with an illumination angle of 75 degrees. Using this qDPC configuration and L1-Retinex reconstruction, the resolution has approached 173 nm, as verified by the USAF-target. Furthermore, 200-nm microbeads produced detectable high-contrast phase signals. Then, an SEM-based calibration curve was established to obtain calibrated qDPC size estimates for microbeads ranging from 200 nm to 1 μm. In three cell lines, quantitative size, roundness, and refractive index of intracellular LDs are investigated, which can be applied to analyze their diverse dynamics. This method provides a high-resolution, label-free approach for size measurement and dynamic phase characterization of intracellular LDs.