This protocol describes the use of the genetically encoded troponin C–based calcium indicator TN-XXL to chronically monitor the functional properties of single neocortical neurons in the mouse visual cortex. A cranial window is implanted over the brain of a mouse expressing TN-XXL in pyramidal neurons of the cerebral cortex. Several days later, the visual cortex is mapped and photographed to facilitate repeated imaging of the same region using two-photon microscopy. Initial two-photon imaging may be done ~2 wk after the window is implanted. We show the application of this technique for long-term in vivo imaging of stimulus response properties. Beyond providing functional information, long-term imaging of TN-XXL–labeled neurons also enables the simultaneous monitoring of structural properties down to the level of single dendritic spines.