Physiological studies in small animals can be complicated, but the complexity is increased dramatically when performing live‐animal synchrotron X‐ray imaging studies. Our group has extensive experience in high‐resolution live‐animal imaging at the Japanese SPring‐8 synchrotron, primarily examining airways in two‐dimensions. These experiments normally image an area of 1.8 mm×1.2 mm at a pixel resolution of 0.45 μm and are performed with live, intact, anaesthetized mice.There are unique challenges in this experimental setting. Importantly, experiments must be performed in an isolated imaging hutch not specifically designed for small‐animal imaging. This requires equipment adapted to remotely monitor animals, maintain their anesthesia, and deliver test substances while collecting images. The horizontal synchrotron X‐ray beam has a fixed location and orientation that limits experimental flexibility. The extremely high resolution makes locating anatomical regions‐of‐interest slow and can result in a high radia...