Modern bony fishes (teleosts) cover a wide range of auditory capabilities (e.g. in terms of auditory sensitivity and the range of detectable frequencies) alongside a great diversity in their auditory structures (Braun and Grande, 2008; Ladich and Fay, 2013; Ladich and Schulz-Mirbach, 2016). Primarily, auditory structures refer to the inner ears including the mineralized otoliths, and can further include the swim bladder or any other gas-filled bladder, especially if these bladders approach or contact the inner ears (i.e. otophysic connection; Braun and Grande, 2008) as found in otophysan fishes. Otophysans such as zebrafish, Danio rerio (Cyprinidae), are important model organisms to study deafness and balance disorders in human medicine (e.g. Abbas and Whitfield, 2010). However, identifying the factors determining otolith motion and inner ear stimulation is still challenging in species such as zebrafish that have evolved specialized ancillary auditory structures (Weberian apparatus) for hearing enhancement (e.g. Schulz-Mirbach et al., 2019). Hence, knowledge of the interaction of fish auditory structures remains elusive (Popper et al., 2005; Ladich and Schulz-Mirbach, 2016).

The most straightforward and reliable way to study fish auditory structures ‘in action’ is the direct observation of the sound-induced in situ motion of the structures in the test subject. However, the technical challenge of gaining access to these internal structures, moving at amplitudes in the range of a few micrometers and at typical sound frequencies, without altering their response as a consequence of surgical procedures has hampered such research for many years. Only a limited number of experimental studies have investigated the sound-induced motion of the saccular otoliths (de Vries, 1950; Sand and Michelsen, 1978), the swim bladder walls (Popper, 1974; Clarke et al., 1975) or the whole set of auditory structures (Cox and Rogers, 1987). Nowadays, synchrotron radiation-based techniques provide powerful approaches to perform imaging of internal structures at high spatio-temporal resolution non-invasively (Mokso et al., 2015; Rack et al., 2010; Walker et al., 2014). Recent studies using hard X-ray phase contrast imaging at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) provided first insights into the sound-induced motion of fish auditory structures (Schulz-Mirbach et al., 2018, 2020). In these radiographic experiments using a standing wave tube-like tank, the motion of the swim bladder and the otoliths in the inner ears of goldfish (Carassius auratus, Otophysa) and the cichlid Etroplus canarensis was successfully visualized and motion patterns could be described qualitatively. However, the motion of the structures of interest could only be observed in 2D, i.e. in a certain orientation. In addition, ‘overlying’ structures such as gills or cranial bones, in part, hampered an undisturbed view of the moving auditory structures. Hence, any in-depth insights into the basic principles of the interaction of the auditory structures would require the visualization and characterization of the motion patterns during sound presentation covering the structures' full 3D aspect. A study on the flight motor motion of a blowfly performed at the TOMCAT beamline at the Swiss Light Source (SLS, Villigen, Switzerland) (Mokso et al., 2015; Walker et al., 2014) demonstrated that periodic motion patterns can be evaluated using tomography with high spatio-temporal resolution.

In mammals and birds, the inner ears show a rather clear division of labor consisting of a vestibular part (semicircular canals and in a wider sense the utricle and the saccule) and the portion serving the auditory sense, i.e. the lagena or cochlea (Manley and Clack, 2004; Witmer et al., 2008; Ladich, 2019). In these terrestrial vertebrates, sound pressure is the primary stimulus (Ladich, 2019; Fay and Popper, 2005). In contrast, in teleosts (and fishes in general), the otolith end organs, namely utricle, saccule and lagena, seem to serve both senses (‘mixed function hypothesis’; Platt and Popper (1981). Moreover, fish inner ears detect sound through particle motion (Rogers et al., 1988). The ability to additionally make use of the pressure component of sound is developed in several teleost groups (e.g. Otophysa, Clupeiformes, Mormyridae, Anabantiformes, several sciaenid species; Braun and Grande, 2008; Hawkins, 1993; Ramcharitar et al., 2006; Horodysky et al., 2008). Sound pressure detection in fishes is possible when the swim bladder or any gas-filled bladder acts as a pressure-to-displacement transducer (Rogers et al., 1988). This is most effective if the bladder is connected to the inner ears through anterior extensions or mechanically coupled by a chain of ossicles and ligaments as in otophysan fishes (Rogers et al., 1988; Schulz-Mirbach et al., 2019). Sound-induced pressure fluctuations provoke the compression and decompression of the gas in the bladder and result in the oscillation of the swim bladder walls. The oscillating walls then function as a secondary sound source, creating local particle motion, ultimately setting the otoliths in motion (Rogers et al., 1988; Schulz-Mirbach et al., 2019). As both sound components, acoustic particle motion and sound pressure, may play a role in stimulating fish inner ears, we set out to adapt the standing wave tube-like setup used at the ESRF for tomography at the SLS. The setup at the ESRF consisted of a horizontal 2L Plexiglas® tube equipped with a miniature inertial shaker at each end which could be driven under different phase conditions to create either a sound pressure or particle motion maximum in the tube center. The intended setup adaption implied, among others, a miniaturization from a horizontal 2 l tank to an upright 14.1 ml (one-shaker setup) or 40.8 ml (two-shaker setup) tube, respectively.

In our study, we aimed to develop a setup that enables non-invasive experiments characterizing the 3D motion of fish auditory structures during sound presentation, such that these experiments can be performed under sound-induced particle motion or sound pressure conditions. In the following, we characterize the approach, focusing on the acoustic setup.