abstract: The intestine has a complex and regular tissue organisation, composed of many different cell types. The maintenance of this tissue is one of the most intensive processes in the body, where many things could go wrong. However, exactly what processes regulate the precise arrangement of cells is still not fully understood. Intercellular signalling plays an important role in tissue maintenance. The main signalling pathways during intestinal homeostasis have been identified and studied extensively over the last two decades. In particular, Notch signalling is central to controlling key cell fate decisions, such as stem cell maintenance and differentiation towards either the secretory or absorptive lineage. Despite these advances in our understanding of intestinal homeostasis, critical questions have remained: How does Notch signalling regulate the formation of different cell types, i.e., progenitors and secretory cells? How is the balance between stem cell maintenance and the formation of the right number of differentiated cells regulated? In this study, we generated a transgenic mouse line expressing a fluorescent reporter for Notch pathway activity, from which we derived intestinal organoids for live-cell imaging. Single-cell tracking revealed, for the first time, that Notch signalling exhibits oscillatory behaviour in certain epithelial cell types. To investigate the functionality of these oscillations, we developed a microfluidics system to dynamically modulate the oscillation frequency, and observed their effects on tissue maintenance. With this, we demonstrated that specific frequencies of Notch signalling oscillations regulate differentiation of certain specialized cells in the small intestine. This study provides functional evidence for dynamic signal encoding in multicellular tissues, i.e., the transmission of biological information through parameters such as duration or oscillation period. This represents a milestone in the field of tissue biology, revealing a new mechanism of how signalling pathways regulate cell fate decisions in the intestine. It also establishes a new in vitro platform technology to control of signalling processes in multicellular systems.