INTRODUCTION: In mammals, each reproduction cycle, numerous antral follicles enter in final growth but only dominant ones will ovulate while others go through atresia. During folliculogenesis, lipid metabolism is involved in the regulation of ovarian follicle growth, capacity to ovulate, corpus luteum (CL) formation, and luteolysis. 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 combining ex vivo 3D Magnetic Resonance Imaging (MRI), both 2D and 3D Mass Spectrometry Imaging (MSI) and 2D optic imaging (IO, immunohistology) to access new anatomical and structural information within the whole ovary in ewe (Ovis aries). We aimed to position the follicles and blood vessels within the ovary and precisely map the lipids to explore their distribution within the follicles at different stages. METHODS: Ovaries (N=28) were fixed with 4% paraformaldehyde and analyzed with a 3 Teslas MRI instrument (Siemens), acquiring 3D images with an isotropic voxel size of 0.25 mm. Two whole organs were cryosectioned to generate 10 µm-thick sections spaced with intervals around 100 µm. Slides were coated with DHB matrix using M5 sprayer (HTX Technologies) and analyzed by 2D MSI using a RapifleX TissueTyper MALDI-TOF mass spectrometer (Bruker). Lipids were detected in positive ion mode, in m/z 100-1200 mass range, at a lateral resolution of 30-40 µm. 2D/3D MSI data were treated by SCiLS Lab software. OI were performed by either histology using Oil red lipid staining or immunohistochemistry (Hemoglobin subunit ) on adjacent sections; 20x microscopy images were acquired using the AxioScanZ1 scanner (Zeiss). RESULTS: MRI performed on 28 whole ovaries allowed to detect 15 to 71 antral follicles per ovary with inner diameter ranging from 0.25 to 10.5 mm. Segmentation of 3D MRI images allowed measuring of FF volume in each follicle and determining their position. 72 and 123 MSI sequences from two ovaries were acquired and analyzed separately. From each section, 180-300 ion density maps were generated and revealed enrichment of different ions in different follicular compartments. Hierarchical clustering of specific lipid profiles resulted in segmentation maps, which clearly discriminated blood vessels, CL and interstitial tissue from antral follicles. From each ovary, all the images were aligned and merged into a 3D data set. 3D MSI representations (ion density maps or segmentation maps) allowed in situ cartography of the follicles by specific molecular signatures of follicular cells and fluids. In addition to lipids, blood vessels showed specific heme group (m/z 616.17) signature. Thus, multimodal analysis combining 2D MSI of lipids and immunodetection of Hemoglobin subunit  allowed mapping of blood vessels through the ovary. Single 2D histological or MSI images were then integrated within 3D MRI ovary volume. By such 2.5D representations, the topologies of ovarian structures could be more finely characterized. CONCLUSION: The combination of 3D MRI to optical microscopy and to 2D/3D-dimensional MSI of lipids allowed spatial mapping (Atlas) of the follicles relative to blood flow and discrimination of follicular compartments that may help to enlighten the involvement of lipids in follicle differentiation through terminal folliculogenesis.