BACKGROUND In ruminants, numerous antral follicles enter in final growth but only a dominant follicle will ovulate. Inside the follicle, follicular fluid (FF) and follicular cells have specific lipid contents, which change during follicular growth due to differential lipid uptake from blood and fatty acid metabolism of follicular cells. The objective was to develop a multimodal imaging approach to explore lipid distribution through the whole ovary. METHODS Ewe ovaries (Ovis aries) were fixed by 4% paraformaldehyde and analyzed using 3 Teslas Magnetic Resonance Imaging (MRI) instrument (Siemens). 3D images were acquired with an isotropic voxel size of 0.25 mm. 10 µm cryosections of the ovaries were analysed by Mass Spectrometry Imaging (MSI) using RapifleX MALDI-TOF spectrometer (Bruker). The MSI sequences were acquired with a spatial resolution of 30 µm/pixel, in positive ion mode to detect lipids in 100-1500 m/z range. Light microscopy 20x images of either hematoxylin, or Oil red stained sections were acquired using the AxioScanZ1 scanner (Zeiss). RESULTS MRI was performed on 28 whole ovaries. 15-71 antral follicles per ovary with inner diameter ranging from 0.25 mm to 10.5 mm were detected. Segmentation of 3D MRI images allowed measuring of FF volume in each follicle and determining their position. Five organs were sectioned, and either MSI of lipids, or histological staining were performed on ovary sections. Hierarchical clustering of lipid spectra, which discriminated antral follicles from blood vessels, luteal bodies and cortex, generated MSI segmentation 2D maps. Hematoxylin staining revealed the morphology of ovary sections. Lipid accumulation sites were detected by Oil red staining. Single 2D histological images were then aligned to MSI of adjacent sections. Both could be integrated within the 3D MRI ovary volume. By such 2.5D representations, the topology of lipids through ovarian compartments, or between the follicles of different sizes could be analyzed. CONCLUSIONS The combination of 3D MRI to optical microscopy and to 2D MSI has allowed access to new anatomical and structural information within the whole ovary, with especially a more precise mapping of lipids, enlightening their involvement in follicle differentiation through terminal folliculogenesis.