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00925njm 22002777a 4500 dc Pérez-Montero, Andrea author Zaragoza, Oscar author Luque, Alfonso author Hortelano, Sonsoles author Acebo, Paloma author 2023-03-05 Lipid droplets (LD), triglycerides and sterol esters among them, are well known for their capacity as lipid storage organelles. Recently, they have emerged as critical cytoplasmic structures involved in numerous biological functions. LD storage is generated de novo by the cell and provides an energy reserve, lipid precursors, and cell protection. Moreover, LD accumulation can be observed in some pathologies as obesity, atherosclerosis, or lung diseases. Fluorescence imaging techniques are the most widely used techniques to visualize cellular compartments in live cells, including LD. Nevertheless, presence of fluorophores can damage subcellular components and induce cytotoxicity, or even alter the dynamics of the organelles. As an alternative to fluorescence microscopy, label-free techniques such as stimulated Raman scattering and coherent anti-stokes Raman scattering microscopy offer a solution to avoid the undesirable effects caused by dyes and fluorescent proteins, but are expensive and complex. Here, we describe a label-free method using live-cell imaging by 3D holotomographic microscopy (Nanolive) to visualize LD accumulation in the MH-S alveolar macrophage cell line after treatment with oleic acid, a monounsaturated fatty acid that promotes lipid accumulation. Bio Protoc. 2023 Mar 5;13(5):e4629. 10.21769/BioProtoc.4629 2331-8325 Bio-protocol 36908642 http://hdl.handle.net/20.500.12105/16116 3D holotomographic microscopy Alveolar macrophages Lipid droplet Lung pathologies Metabolism Oleic acid Visualization of Lipid Droplets in the Alveolar Macrophage Cell Line MH-S with Live-cell Imaging by 3D Holotomographic Microscopy (Nanolive)