In vivo and real-time multicellular imaging enables the decoding of sensory circuits and the tracking of systemic drug uptake. However, in vivo imaging of the auditory periphery remains technically challenging owing to the deep location, mechanosensitivity and fluid-filled, bone-encased nature of the cochlear structure. Existing methods that expose the cochlea invariably cause irreversible damage to auditory function, severely limiting the experimental measurements possible in living animals. Here we present an in vivo surgical protocol that permits the imaging of cochlear cells in hearing mice. Our protocol describes a ventro-lateral approach for preserving external and middle ear structures while performing surgery, the correct mouse positioning for imaging cochlear cells with effective sound transmission into the ear, the chemo-mechanical cochleostomy for creating the imaging window in the otic capsule bone that prevents intracochlear fluid leakage by maintaining an intact endosteum, and the release of intracochlear pressure that separates the endosteum from the otic capsule bone while creating an imaging window. The procedure thus preserves hearing thresholds. Individual inner and outer hair cells, supporting cells and nerve fibers can be visualized in vivo while hearing function is preserved. This approach may enable future original investigations, such as the real-time tracking of ototoxic drug transport into the cochleae. The technique may be applied to the monitoring of sound-evoked functional activity in multiple cochlear cells, in combination with optogenetic tools, and may help to improve cochlear implantation in humans. The cochleostomy takes ~1 h and requires experience in surgery.
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