AbstractBackgroundThe neural control of gastrointestinal muscle relies on circuit activity whose underlying motifs remain limited by small‐sample calcium imaging recordings confounded by motion artifact, paralytics, and muscle dissections. We present a sequence of resources to register images from moving preparations and identify out‐of‐focus events in widefield fluorescent microscopy.MethodsOur algorithm uses piecewise rigid registration with pathfinding to correct movements associated with smooth muscle contractions. We developed methods to identify loss‐of‐focus events and to simulate calcium activity to evaluate registration.Key ResultsBy combining our methods with principal component analysis, we found populations of neurons exhibit distinct activity patterns in response to distinct stimuli consistent with hypothesized roles. The image analysis pipeline makes deeper insights possible by capturing concurrently calcium dynamics from more neurons in larger fields of view. We provide access to the source code for our algorithms and make experimental and technical recommendations to increase data quality in calcium imaging experiments.ConclusionsThese methods make feasible large population, robust calcium imaging recordings and permit more sophisticated network analyses and insights into neural activity patterns in the gut.