Here, we present a protocol for longitudinal two-photon calcium imaging and holographic optogenetic manipulation to investigate memory in mice. We describe chronic cranial window implantation, stereotaxic viral delivery, and head-fixed contextual fear conditioning synchronized with imaging acquisition and optical stimulation of identified neurons. We further detail procedures for image data processing pipelines for cross-session registration and longitudinal tracking of the same neuronal population. This protocol enables simultaneous neuronal activity recording, behavioral monitoring, and causal interrogation of neuronal ensembles. For complete details on the use and execution of this protocol, please refer to Wang et al. 1
Guo et al. (2026) studied this question.