Revelation of sound-induced motion patterns of fish auditory structures: a new experimental tomography-based 4D approach

现代硬骨鱼（Teleostei）在听觉能力和相应结构的形态上表现出高度的多样性，如内耳的耳石和鳔。尽管存在如此巨大的多样性，但提供听觉结构功能相互作用基本原理的实验证据很少，并且主要集中在金鱼或斑马鱼等少数模式物种上。这是因为在不通过手术暴露听觉结构的情况下，很难原位评估听觉结构的运动模式。在欧洲同步辐射设施之前的项目中，我们开发了一种装置，可以以一种非侵入性的方式将声音引起的鱼类听觉结构运动可视化。然而，移动的结构只被捕获在二维加上时间，从而忽略了它们的三维，阻碍了对它们的功能相互作用的解释。因此，在我们计划的项目中，我们设想了一种新的基于瑞士光源的4D（三维空间加时间）层析成像方法，以表征从鳔到韦伯听骨到耳石的声音诱导相互作用。为了解决听觉结构的变异性，我们将比较6种耳虫，即cypriniform物种Danio rerio、Pangio kuhlii和Sewellia lineolata，以及siluriform物种Kryptopterus vitreolus、Ancistrus dolichopterus和Corydoras aeneus。具体来说，我们的目标是开发一种具有高时空分辨率的原位运动捕获装置。这将首次允许根据声音成分（声压与声音引起的粒子运动）、频率、声级和种类，在3D方面量化听觉结构的运动模式。在Swiss Light Source之前的研究中，我们测试了一个20毫升的微型驻波管样装置的原型，用于时间分辨断层扫描（像素尺寸：2.75 μ m；帧率：2kHz），该装置可以在声压或声致粒子运动条件下运行。基于此，我们将通过在TOMCAT光束线上实现一种新的显微镜来提高空间（像素尺寸：< 1.0µm）和时间分辨率（帧率：20kHz）。运动模式将使用数据处理管道在旋转和平移方面进行量化。我们的研究将从根本上增强对硬骨鱼声音传播的认识，通过表征声音诱导运动从鳔到内耳的路径，同时测试几十年前制定的韦伯装置功能的假设。这些结果将为未来的鱼类运动模式建模研究提供重要的输入，这些研究无法用于濒危或化石物种等实验室实验。我们方法学的发展将开启新的生物力学应用，并有利于正在进行的研究，例如，阐明病理耳结构对人类中耳听小骨运动的影响。

Modern bony fishes (Teleostei) show a high diversity in auditory abilities and in the morphology of the corresponding structures such as the otoliths in the inner ears and the swimbladder. Despite this enormous diversity, experimental evidence providing insights into the basic principles of the functional interaction of auditory structures is rare and focuses on a few model species such as goldfish or zebrafish. This is due to the difficulty to evaluate the motion patterns of auditory structures in-situ without exposing them surgically. In previous projects at the European Synchrotron Radiation Facility, we developed a setup that enabled visualizing the sound-induced motion of fish auditory structures in a non-invasive way. The moving structures, however, were captured only in 2D plus time, thereby ignoring their three-dimensionality and hampering interpretations of their functional interplay.In our planned project, we therefore envisage a new 4D (three spatial dimensions plus time) tomography-based approach at the Swiss Light Source to characterize the sound-induced interaction from the swimbladder through the Weberian ossicles to the otoliths. To address the variability of the auditory structures, we will compare six otophysan species, namely the cypriniform species Danio rerio, Pangio kuhlii, and Sewellia lineolata, and the siluriform species Kryptopterus vitreolus, Ancistrus dolichopterus, and Corydoras aeneus. Specifically, we aim to develop a setup to capture the in-situ motion with high spatio-temporal resolutions. This will, for the first time, allow to quantify the motion patterns of the auditory structures in their 3D aspect depending on the sound component (sound pressure versus sound-induced particle motion), frequency, sound level, and species.In previous studies at the Swiss Light Source, we tested a prototype of a 20mL-miniature standing wave tube-like setup for time-resolved tomography (pixel size: 2.75µm; framerate: 2kHz) which can be run under sound pressure or sound-induced particle motion conditions. Based on this, we will increase the spatial (pixel size: < 1.0µm) and temporal resolutions (framerate: 20kHz) by implementing a new microscope at the TOMCAT beamline. Motion patterns will be quantified in terms of rotation and translation using a data processing pipeline.Our study will fundamentally enhance the knowledge of sound transmission in teleosts by characterizing the path of sound-induced motion from the swimbladder to the inner ears while testing hypotheses on the function of the Weberian apparatus formulated decades ago. The outcomes will yield vital input for future studies modelling motion patterns in fishes not available for lab experiments such as endangered or fossil species. Our methodological developments will unlock novel biomechanical applications and benefit ongoing studies which, for example, elucidate the effects of pathologic ear structures on the motion of the auditory ossicles in the human middle ear.