Abstract
Cortical computations arise from patterns of neuronal activity that span across all cortical layers and cell types. Three-photon excitation has extended the depth limit of in vivo imaging within the mouse brain to encompass all cortical layers. However, simultaneous three-photon imaging throughout cortical layers has yet to be demonstrated. Here, we combine non-unity magnification remote focusing with adaptive optics to achieve single-cell resolution imaging from two temporally multiplexed planes separated by up to 600 µm. This approach enables the simultaneous acquisition of neuronal activity from genetically defined cell types in any pair of cortical layers across the mouse neocortical column.