Imaging and quantifying organelle contact sites using a reversible splitFAST complementation system

Organelle contact sites are crucial hubs for inter-organelle logistics; yet, visualizing these dynamic foci of sub-micro scale in living cells is challenging. In this chapter, we describe how to use the FABCON (Fluorogen-Activated Bimolecular complementation at CONtact sites) toolkit to detect and quantify contact sites. FABCON labels contact sites via a reversible, fluorogen-dependent complementation of the splitFAST system. This protocol first describes the engineering principle of FABCON, allowing customization to model systems of interest. Next, we provide detailed instructions for using FABCON to monitor mitochondria-lipid droplet interactions in mammalian cells and how to quantify levels of contact sites via intensity-based measurement and line scanning. FABCON can be broadly applied to visualize and quantify other contact sites. With proper validation and optimization, FABCON provides a robust platform to study the dynamic regulation of organelle contact sites.