Background

Lipid droplets (LD) are dynamic cytoplasmic organelles that serve as intracellular energy storage, mainly in the form of triglycerides and cholesterol esters (van Dierendonck et al., 2022). In addition to their role in lipid storage and transport, LD are increasingly recognized to be involved in the regulation of other cellular processes, including inflammation, cell activation, and metabolism (Xu et al., 2018; Agudelo et al., 2020). Accumulation of LD is also related to several pathologies as obesity, diabetes, atherosclerosis, or lung diseases (Xu et al., 2018; Agudelo et al., 2020). Alveolar macrophages (AM) are the first line of defense against respiratory pathogens and play key roles in lung lipid metabolism (Agudelo et al., 2020). Excessive amounts of intracellular lipids in AM have been described in lung pathologies such as chronic obstructive pulmonary disease, acute lung injury, idiopathic pulmonary fibrosis, or pulmonary alveolar proteinosis, among others (Agudelo et al., 2020). Nevertheless, the mechanisms responsible for LD accumulation are not fully elucidated. Fluorescence-based live-cell imaging is the most widely used technique for LD studies, even though the phototoxicity of fluorescent dyes may disturb LD dynamics. Here, we present a protocol for live imaging of the LD accumulation without the addition of fluorescent labels, using 3D holotomographic microscopy (Nanolive). This label-free microscopy method reports the changes of the refractive indices (RIs) in three dimensions at high spatial and temporal resolution, allowing label-free analysis of organelle biology and kinetics studies with a low level of phototoxicity (Sandoz et al., 2019). We have performed this protocol in the MH-S alveolar macrophage cell line, grown in the presence of oleic acid to induce LD formation. Using this live-cell approach, foam cell formation can be analyzed under a physiological context, excluding the artefacts induced by the addition of different chemicals.