Abstract The anatomy of the auditory system in humans and animals is highly specialized and adapted to their environment and hearing abilities, but its structure and response can be significantly altered by hearing impairing conditions. In modern bony fishes, hearing structures show a high diversity, involving the swim bladder or specialized derivatives of the vertebrae as found in otophysans (e.g.: zebrafish). Intriguingly, these derivatives have mechanically very similar functions as the constituents of the human middle ear. The swim bladder (if present) acts as a sound pressure-sensitive membrane analogous to the tympanic membrane, while the Weberian apparatus, akin to the human middle ear ossicles, mechanically transmits the vibrations to the ear stones and sensory cells, with a similar function as the human inner ear. Investigating the motion patterns of these hearing structures non-destructively under near in-vivo conditions (no fixation, tissue degradation, minimal surgical exposure, etc.) over the full scale of the hearing organs is a challenge due to their location within the body, the micron-level motion amplitudes, and the high relevant vibration frequencies. We use retrospectively gated time-resolved tomographic microscopy [1] at the TOMCAT beamline, SLS, PSI, to image the dynamics of hearing structures in fresh-frozen ex-vivo human temporal bones at 128 Hz [2] and fresh post-mortem small fish specimen from 350 to 1500 Hz [3] when subjected to sound wave stimuli. Phase contrast reconstructions feature voxel sizes of 1.1 or 2.75 μm with exposure times per frame down to 50 μs. Large area reconstructions are stitched from multiple high-resolution 4D scans. 3D motion trajectories of specific points of interest on the hearing structures are extracted from the 4D data via sub-pixel accuracy volume correlation (see Fig. 1). Using this technique, the 3D motion pattern of the full human middle ear ossicular chain has been successfully visualized and quantified [2]. Measurements carried out on several different fish species enable us to quantify and compare auditory capabilities across taxa and to test longstanding hypotheses on the function of fish hearing systems. Our research demonstrates the ability to image fast periodic processes in the kHz regime at an effective temporal resolution > 10 kHz and sub-micron spatial sensitivity. The technique is ideally suited for stimulated biomechanical processes but can be readily adapted to other realms such as materials science or oscillatory rheology – provided that the sample changes are (quasi-) periodic in nature. References: [1] R. Mokso et al., Sci. Rep. 5, 8727 (2015).[2] M. Schmeltz et al., Commun. Biol. 7, 157 (2024)[3] I.P. Maiditsch et al., J. Exp. Biol. 225, jeb243614 (2022)