3D imaging and quantitative analysis of adipocytes in situ and ex situ

Different adipose tissues (AT) have been described, including subcutaneous and visceral tissues (SCAT and VAT). They display different morphological structures, physiological and metabolic functions. Imaging adipocytes in the whole AT was not feasible because of the large adipocyte sizes and the lipid-full content of the droplets that increased the refractive index. Tissue clearing is then required mainly through a delipidation step, which induces also a tissue shrinkage. Our aim was to image in 3D freshly extracted adipocytes and compare them to those within their tissues. Trout ATs were stained with 5DTAF (extracellular matrix) and Nile Red (lipids). After clearing with Histodenz, 3D images were obtained using a confocal microscope, and adipocytes were segmented and measured. In situ, major differences in adipocyte size and shape were observed between the VAT and SCAT. Ex situ, only the size mattered because all cells were round outside their tissues. This method can be applied to other species, such as mice. In situ, adipocyte sphericity was even higher in the SCAT from a Swiss and a C57Bl6. This approach demonstrates that 3D adipocyte imaging with lipid labeling enables accurate morphological characterization, provides insights into depot-specific structural features, and supports optimization of cell isolation protocols