The sap extracted from sugar maple (Acer saccharum) trees contains high levels of sugar, and is collected for the production of maple syrup. The mechanisms driving sap exudation in maple trees are thought to be associated with: (1) the unique cellular structure of maple trees and existence of gas-filled fibres; (2) a freeze-thaw cycle where ambient temperatures fluctuate above and below 0 °C; and (3) the presence of sucrose within vessels of the tree’s xylem. Despite decades of research on the topic, the processes responsible for maple sap exudation have yet to be fully understood or experimentally validated. This research provides a comprehensive look at some of the processes that occur within maple trees, resulting in springtime sap exudation. The unique microstructure of a maple tree stem was characterised using light optical microscopy (LOM). X-ray micro-computed tomography (micro-CT) and synchrotron-based micro-CT were used to produce three-dimensional and in-situ visualizations of maple saplings in a natural state and throughout an induced freeze-thaw cycle. Using the combined results from different imaging experiments, we have quantified the microstructural conduits within maple xylem and determined the air-filled or water-filled status of vessels within maple xylem in response to freezing and thawing events. The number of embolisms increased as the tree reached minimum temperatures. However, upon re-thawing most vessels were able to recover their water-filled status. These findings have produced some of the first evidence of the microstructural changes within a maple tree during a freeze-thaw cycle, which will help resolve contrasting theories on the topic.