A protocol for cortical and deep-brain calcium imaging in freely moving mice with the Mini2P system.

Two-photon calcium imaging has transformed our understanding of neuronal circuit function, yet conventional approaches require head fixation, limiting the study of neural activity during natural behavior. Miniaturized two-photon microscopy (Mini2P) overcomes this constraint by enabling calcium imaging in freely moving animals while preserving the optical single-cell resolution of conventional two-photon microscopy. However, its implementation requires the careful integration of multiple technically demanding procedures, presenting substantial challenges for laboratories adopting this approach. Here, we present a comprehensive, step-by-step protocol for implementing Mini2P calcium imaging in freely moving mice, emphasizing procedures that are unique to or particularly critical for successful implementation of this technique. This protocol covers system preparation and performance checks, optical alignment, field-of-view selection, baseplate installation, and strategies for maintaining imaging stability during microscope attachment and reattachment. We also address behavioral habituation, integration with behavioral tasks, imaging acquisition, quality control, and troubleshooting as a part of an integrated experimental workflow. Representative imaging data acquired through cranial windows in the primary visual and somatosensory cortices, and through a gradient-index lens in the medial prefrontal cortex, illustrate the applicability of this workflow across different brain regions and optical preparations. Designed for adaptation across brain regions and behavioral paradigms, the protocol provides practical guidance for identifying technical challenges and verifying successful implementation. By establishing clear decision points, validation criteria, and troubleshooting strategies at each stage, this workflow aims to improve reproducibility and facilitate the adoption of the Mini2P for investigating neuronal circuit dynamics during freely moving behavior.