Cranial window implants in head-fixed rodents are becoming a preparation of choice for stable optical access to large areas of cortex over extended periods of time. Here, we provide a highly detailed and reliable surgical protocol for a cranial window implantation procedure for chronic widefield and cellular imaging in awake, head-fixed mice, which enables subsequent window removal and replacement in the weeks and months following the initial craniotomy. This protocol has facilitated awake, chronic imaging in adolescent as well as adult mice over several months from a large number of cortical brain regions; targeted virus and tracer injections from data obtained using prior awake functional mapping; and functionally-targeted two-photon imaging across all cortical layers in awake mice using a microprism attachment to the cranial window. Collectively, these procedures extend the reach of chronic imaging of cortical function and dysfunction in behaving animals.
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