Advancing the monitoring of organelle contact sites in vitro and in vivo.

Organelle contact sites are highly dynamic and specialized regions where distinct organelles come into proximity, enabling direct inter-organelle communication. These structures play fundamental roles in cellular homeostasis by coordinating the exchange of lipids, metabolites, and ions, as well as regulating key processes such as organelle dynamics, mitochondrial fission, autophagy, and metabolic integration. Alterations in contact site architecture and function have been increasingly associated with a wide range of human diseases, including neurodegeneration, metabolic disorders, and cancer. Despite their biological relevance, the nanoscale nature and dynamic behaviour of contact sites have historically posed significant challenges for their accurate detection and functional characterization. Here, we provide a comprehensive overview of the methodologies currently available to study organelle contact sites, ranging from classical approaches such as electron microscopy and biochemical fractionation to advanced imaging techniques and genetically encoded reporters. We discuss recent developments in high-resolution and live-cell microscopy that have improved the spatial and temporal resolution of contact site analysis, as well as emerging tools designed to selectively label, quantify, and manipulate these interfaces. Attention is given to the next generation of engineered reporters capable of sensing molecular and ionic exchanges at contact sites, thereby moving beyond structural description toward functional interrogation. By critically evaluating the strengths and limitations of existing approaches, we aim to provide a framework for selecting appropriate tools and to highlight future directions in the field. Ultimately, advancing our ability to monitor and dissect organelle contact sites will be essential for understanding their contribution to cellular physiology and disease.