Noninvasive in vivo imaging reveals differences between tectorial membrane and basilar membrane traveling waves in the mouse cochlea

Significance The membranes within the cochlea vibrate in response to sound. However, measuring these vibrations to study the sense of hearing has been a technological challenge because invasive techniques have been required. Herein, we describe a new technique capable of depth-resolved displacement measurements in 3D space with picometer sensitivity within the unopened mouse cochlea. We used this technique to make, to our knowledge, the first measurements of the tectorial membrane, the structure that overlies the sensory hair cell stereociliary bundles, within a healthy cochlea. We found that the tectorial membrane sustains traveling wave propagation differently than the more commonly measured basilar membrane. This finding provides a clearer understanding of the mechanical stimulus at the level of the inner hair cell responsible for non-linear sound encoding. Sound is encoded within the auditory portion of the inner ear, the cochlea, after propagating down its length as a traveling wave. For over half a century, vibratory measurements to study cochlear traveling waves have been made using invasive approaches such as laser Doppler vibrometry. Although these studies have provided critical information regarding the nonlinear processes within the living cochlea that increase the amplitude of vibration and sharpen frequency tuning, the data have typically been limited to point measurements of basilar membrane vibration. In addition, opening the cochlea may alter its function and affect the findings. Here we describe volumetric optical coherence tomography vibrometry, a technique that overcomes these limitations by providing depth-resolved displacement measurements at 200 kHz inside a 3D volume of tissue with picometer sensitivity. We studied the mouse cochlea by imaging noninvasively through the surrounding bone to measure sound-induced vibrations of the sensory structures in vivo, and report, to our knowledge, the first measures of tectorial membrane vibration within the unopened cochlea. We found that the tectorial membrane sustains traveling wave propagation. Compared with basilar membrane traveling waves, tectorial membrane traveling waves have larger dynamic ranges, sharper frequency tuning, and apically shifted positions of peak vibration. These findings explain discrepancies between previously published basilar membrane vibration and auditory nerve single unit data. Because the tectorial membrane directly overlies the inner hair cell stereociliary bundl