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Summary: 2-photon all-optical physiology combines in vivo 2-photon calcium imaging and optogenetics, which enables both the read out and manipulation of neuronal microcircuits with single-cell resolution. Here, we describe a protocol for achieving optimized co-expression of calcium indicator and opsin. To enable longitudinal designs, we introduce a template for virus injection and chronic window implantation. We also highlight key aspects of performing 2-photon imaging and suggest an analysis algorithm for the binarization of putatively action-potential (AP)-related calcium transients.For complete details on the use and execution of this protocol, please refer to Fu et al. (2021).