Background: Micro-lipid droplets (mLDs) appear in adipocytes upon lipolytic stimulation. LDs may grow by spontaneous, homotypic fusion.Results: Scavenging of fatty acids prevents mLD formation. LDs grow by a slow transfer of lipids between LDs.Conclusion: mLDs form due to fatty acid overflow. LD growth is a controlled process.Significance: Novel mechanistic insights into LD remodeling are provided.

Lipid droplets (LDs) are the main lipid storage depots for neutral lipids (‘fat’) mainly triacylglycerols (TAGs) in all eukaryotic cells. Synthesis, storage, and mobilization of TAGs are critical cellular processes to maintain cellular lipid and energy homeostasis. Although many molecular and biochemical details of neutral lipid metabolism have been revealed basic cell biological aspects of LDs such as the mode of LD breakdown and of LD growth are still elusive. We have applied label‐free coherent anti‐Stokes Raman scattering (CARS) microscopy, long‐term four‐dimensional live cell imaging and three‐dimensional electron microscopy to study lipid droplet remodelling in intact murine and human adipocytes. We show that under selected nutritional conditions neutral lipid breakdown and synthesis can occur in parallel and suggest an underlying mechanism to prevent cellular lipid overflow and lipotoxicity. Furthermore, we demonstrate that LD of adipocytes grow by lipid transfer between closely associated organelles without physical interaction over large LD surface areas. In addition, we demonstrate the specific detection of isotope labeled fatty acids and their incorporation into LD using CARS microscopy. Advantages and limitations of chemical imaging versus fluorescence labeling for monitoring lipids in intact cells are discussed. FWF, LIPOTOX F3005‐B12; DK‐ME W901‐B05, GOLD (GEN‐AU); KoRS‐CB; BWS.