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Transcranial two-photon microscopy allows long-term imaging of neurons, glia, and vasculature in the intact cortex of living animals. So far, this technique has been primarily used to acquire images in anesthetized animals. Here, we describe a detailed protocol for high-resolution two-photon imaging of neuronal structures in the cortex of awake head-restrained mice. Surgery is done within 1 h in anesthetized mice. After animals recover from anesthesia, two-photon imaging can be performed multiple times over minutes to days, allowing longitudinal studies of synaptic plasticity and pathology without the complication induced by anesthesia reagents.

Transcranial Two-Photon Imaging of Synaptic Structures in the Cortex of Awake Head-Restrained Mice