BACKGROUND-AIM Lipid metabolism is involved in the regulation of ovarian follicle growth, capacity to ovulate, corpus luteum (CL) formation, and luteolysis. Each cycle, from many antral follicles entering final growth, only dominant one can ovulate, others go through atresia. We aimed to map lipids through the whole ovary to explore their distribution according to follicle stage and the presence of blood vessels. METHODS Paraformaldehyde-fixed ovine ovaries were consecutively analyzed by ex vivo 3D Magnetic Resonance Imaging (MRI) and 2D/3D Mass Spectrometry Imaging (MSI). The whole organs were cut using cryostat to generate 10 μmthick sections spaced with intervals around 100 μm. Slides were coated with DHB matrix using M5 sprayer (HTX Technologies). MSI measurements were done on a RapifleX MALDI-TOF spectrometer (Bruker) in positive ion mode with m/z 100-1200 to detect lipids, with a lateral resolution of 30-40 μm. MSI data were treated by SCiLS Lab software. Immunohistochemistry was performed on adjacent sections, and 20x light microscopy images were acquired using the AxioScanZ1 scanner (Zeiss). RESULTS From two ovaries, 72 and 123 MSI sequences were acquired and analyzed separately. From each section, 180-300 ion density maps were generated, revealing different ions enriched in either follicular cells, or fluid, or blood vessels, or CL. After hierarchical clustering, segmentation maps clearly discriminated blood vessels, CL, and cortex/ interstitial tissue from antral follicles, due to specific lipid profiles. Then, for each ovary, all sections were aligned, merged into a 3D data set, in order to topography lipids between the follicles of different sizes and atretic stages. 3D representations allowed in situ cartography of blood vessels and reconstruction of dominant and subordinated follicles by specific molecular signatures of follicular cells and fluids. In addition, a multimodal analysis combining immunodetection of Hemoglobin subunit A and Proliferating Cell Nuclear Antigen confirmed lipid mapping to definite ovarian structures. CONCLUSIONS 2D and 3D-dimensional MSI of lipids allowed the spatial position of the follicles relative to blood flow and discrimination of follicular compartments that may help to enlighten the involvement of lipids in follicle differentiation.