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Nat Protoc. 2014 Nov;9(11):2515-2538. doi: 10.1038/nprot.2014.165. Epub 2014 Oct 2.

Removable cranial windows for long-term imaging in awake mice.

Author information

Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS701, Boston, MA 02115, USA.
Center for Integrative Neuroscience and Department of Physiology, University of California, San Francisco, 675 Nelson Rising Lane, San Francisco, CA 94158, USA.
Department of Neurobiology, Duke University Medical School, Durham, NC 27710, USA.
Janelia Farm Research Campus, 19700 Helix Drive, Ashburn, VA 20147, USA.
Allen Institute for Brain Science, 551 N 34th Street, Seattle, Washington 98103, USA.
Neuro-Electronics Research Flanders, a joint research initiative of imec, VIB and KU Leuven, Kapeldreef 75, 3001 Leuven, Belgium.
Department of Neurology, F.M. Kirby Neurobiology Center, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Contributed equally


Cranial window implants in head-fixed rodents are becoming a preparation of choice for stable optical access to large areas of the cortex over extended periods of time. Here we provide a highly detailed and reliable surgical protocol for a cranial window implantation procedure for chronic wide-field and cellular imaging in awake, head-fixed mice, which enables subsequent window removal and replacement in the weeks and months after the initial craniotomy. This protocol has facilitated awake, chronic imaging in adolescent and 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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