Mitochondria-ER contact sites (MERCS) are playing critical roles in various homeostatic functions, such as calcium/redox homeostasis, lipid transfer, mitophagy/autophagy and regulation of organelle dynamics. Hundreds of proteins have been shown to either have a structural or functional role in these contacts. ER-mitochondria tethering is a tightly regulated process which is modulated in conditions such as starvation, ER stress and mitochondrial dynamics modifications. Despite diverse proteins have been reported to participate in MERCS, their role in several cellular pathways and pathologies remain to be fully elucidated. Biochemical and imaging approaches have been used to address these questions. However, MERCS are dynamics and their abundance can vary rapidly, which make their characterization a real challenge. Currently, not only one experimental approach has to be used to fully characterize an organelle contact site, but a combination of different methods. Old methods such as, cell fractionation can be used to isolate ER-mitochondria interaction in combination with electron microscopy methods to assess morphological MERCS structure. Fluorescence-based techniques are also widely used, which allow a more dynamic assessment of MERCS, despite a lower resolution. In this workshop, we will present the combination of three fluorescent-based techniques: in situ proximity ligation assay (PLA), a contact site sensor based on split-GFP (SPLICS) and a novel FRET-based proximity biosensor (MERLIN-FRET) to study MERCS in cells and tissues sections. We will analyze ER-mitochondria interactions in a model of type 2 diabetes. Indeed, disruption of MERCS is an early causal trigger of hepatic insulin resistance and steatosis observed in obesity and type 2 diabetes.