In vivo two-photon calcium imaging provides detailed information about the activity and response properties of individual neurons. However, in vitro methods are often required to study the underlying neuronal connectivity and physiology at the cellular and synaptic levels at high resolution. This protocol provides a fast and reliable workflow for combining the two approaches by characterizing the response properties of individual neurons in mice in vivo using genetically encoded calcium indicators (GECIs), followed by retrieval of the same neurons in brain slices for further analysis in vitro (e.g., circuit mapping). In this approach, a reference frame is provided by fluorescent-bead tracks and sparsely transduced neurons expressing a structural marker in order to re-identify the same neurons. The use of GECIs provides a substantial advancement over previous approaches by allowing for repeated in vivo imaging. This opens the possibility of directly correlating experience-dependent changes in neuronal activity and feature selectivity with changes in neuronal connectivity and physiology. This protocol requires expertise both in in vivo two-photon calcium imaging and in vitro electrophysiology. It takes 3 weeks or more to complete, depending on the time allotted for repeated in vivo imaging of neuronal activity.