Membrane contact sites (MCSs) are hubs allowing various cell organelles to coordinate their activities. The dynamic nature of these sites and their small size hinder analysis by current imaging techniques. To overcome these limitations, we here design a series of reversible chemogenetic reporters incorporating improved, low-affinity variants of splitFAST, and study the dynamics of different MCSs at high spatiotemporal resolution, both in vitro and in vivo. We demonstrate that these versatile reporters suit different experimental setups well, allowing one to address challenging biological questions. Using these probes, we identify a pathway in which calcium (Ca²⁺) signalling dynamically regulates endoplasmic reticulum-mitochondria juxtaposition, characterizing the underlying mechanism. Finally, by integrating Ca²⁺-sensing capabilities into the splitFAST technology, we introduce PRINCESS (PRobe for INterorganelle Ca²⁺-Exchange Sites based on SplitFAST), a class of reporters to simultaneously detect MCSs and measure the associated Ca2+ dynamics using a single biosensor.