Region-Specific Lens Insertion Strategies For In vivo Calcium Imaging In Mice Across Cortical And Hippocampal...

In vivo calcium imaging of neuronal populations using surgically implanted optical lenses is widely used to monitor neuronal population activity during behavior. However, surgical success and imaging quality depend critically on anatomical constraints that vary across brain regions. This protocol provides region-specific surgical guidance for the selection and implantation of imaging lenses in commonly targeted cortical and hippocampal areas. Specifically, it describes a surgical workflow encompassing target localization and skull leveling, region-specific craniotomy, and, when required, dura removal or aspiration of overlying tissue, as well as the selection, insertion, and fixation of appropriate lenses and holders. In particular, we describe three implantation strategies: conventional lens implantation for the ventral hippocampus, lens implantation with tissue aspiration for the dorsal hippocampus, and prism lens implantation for cortical imaging. We further provide practical guidance on how differences in tissue depth, vascular architecture, and circuit organization inform key surgical parameters, including lens diameter, craniotomy size, the extent of tissue removal, insertion speed, hemostatic procedures, and fixation methods. By explicitly documenting surgical considerations that are often underreported in written methods, this protocol aims to reduce trial-and-error during lens implantation, and improve experimental reproducibility. Overall, this protocol provides a practical guide for selecting and reliably implementing region-appropriate lens implantation strategies across cortical and hippocampal circuits.