Protocol for the in vivo quantification of mitochondria-associated ER membranes in Caenorhabditis elegans

Summary Mitochondria-associated membranes (MAMs) are specialized contact sites between the endoplasmic reticulum and mitochondria, with multiple functional aspects. Here, we present a confocal microscopy-based protocol for quantifying the area of MAM-like domains in intestinal cells of the nematode Caenorhabditis elegans using organelle-specific fluorescent reporters. We describe steps for worm synchronization, microscope setup, sample preparation, and image acquisition. We then detail procedures for manual single-image analysis and scalable automated batch processing, enabling robust quantification of ER-mitochondria contacts across experimental conditions. For complete details on the use and execution of this protocol, please refer to Roussos et al.¹