A novel epifluorescence microscope design and software package to record naturalistic behaviour and cell activity in freely moving Caenorhabditis elegans

Understanding how neural circuits drive behavior requires imaging methods that capture cellular dynamics in freely moving animals. Here, we introduce Wormspy, a compact, flexible, and cost-effective microscope system paired with an open-source software package, specifically designed for high-magnification epifluorescence imaging in Caenorhabditis elegans . By integrating dual-channel fluorescence optics, off-the-shelf components, and a motorized stage, Wormspy enables simultaneous recording of neuronal activity and behavioral dynamics without restraining the animal. Our system incorporates both manual and automated tracking—including DeepLabCut-based feature extraction—to maintain precise centering of the subject, even during complex locomotor behaviors. We demonstrate the utility of Wormspy across multiple paradigms: quantifying body wall muscle calcium transients during locomotion, capturing rapid sensory-evoked responses in the polymodal ASH neuron during aversive stimuli, and resolving food-related activity in the AWC ^ON neuron. Notably, Wormspy further distinguishes subcellular calcium events in the RIA axonal compartments that correlate with head bending kinematics. This versatile platform not only reproduces known phenotypes, such as altered gait in gar-3 mutants, but also uncovers nuanced sensorimotor correlations previously inaccessible with conventional methods. Wormspy’s modular design and open-source framework lower the technical and financial barriers to high-resolution, behaviorally relevant neural imaging. Our findings establish Wormspy as a robust tool for dissecting the neural underpinnings of behavior in freely moving organisms, with potential applications extending to other small model systems.