Amira aviso 6

To produce 3D data for modelling, 17 ears of C. gorgonensis were scanned using a SkyScan 1172 X-ray micro-computed tomography scanner (Bruker Corporation, Billerica, MA, USA) with a resolution between 1.3 and 2.9 µm (55 kV source voltage, 180 µA source current, 300ms exposure and 0.1° rotation steps). As experimental procedures required removal of the cuticular pinnae, eight additional specimens with intact pinnae were scanned. The micro-computed tomography projection images were reconstructed with NRecon (v.1.6.9.18, Bruker Corporation, Billerica, MA, USA) to produce a series of orthogonal slices. The 3D segmentation of the ear, measurements of the ear cross section and width, and volumetric measurements of the pinnal cavities were performed with the software Amira-Aviso 6.7 (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Micro-computed tomography stereolithography files (STL) were generated for numerical modelling using established protocols (Jonsson et al., 2016 (link); Veitch et al., 2021 (link)) and to 3D print ear models.
For 2D measurements of the cavity slit area, pinnal protrusion, and the distance between the pinnal cavities, an Alicona InfiniteFocus microscope (G5, Bruker Alicona Imaging, Graz, Austria) at 5× objective magnification was used to capture images of collection specimens with intact pinnae, with a resolution of about 100 nm (n = 8 ears).

Most specimens were maintained in the colony until natural death by senescence for use in other studies, but for micro-CT imaging, one male and one female were euthanized by placement in 90% ethanol. These specimens were scanned using a SkyScan 1172 μ-CT scanner (Bruker Corporation, Billerica, MA, USA). The forelegs were removed and mounted in custom built holders before scanning (voxel size 2 µm, 55 kV, 180 µA, 800 ms exposure, 0.1° rotation step). μ-CT projection images were reconstructed to produce orthogonal slices with NRecon (v. 1.6.9.18, Bruker Corporation, Billerica, MA, USA). For three-dimensional segmentation of the trachea, the slice data were imported into Amira-Aviso 6.7 (Thermo Fisher Scientific, Waltham, MA, USA) and the trachea manually selected using the magic wand tool every five slices throughout the whole foreleg, followed by interpolation to connect the selected geometries and generate a three-dimensional surface.

To produce an idealised geometry of the C. gorgonensis inner-ear, a female specimen was scanned using (i) a SkyScan 1172 X-ray μ -CT scanner (Bruker Corporation, Billerica, MA, USA) with a resolution of 1.6 μ m (40 kV source voltage, 165 μA source current, 2200 ms exposure and 0.1° rotation steps) and (ii) synchrotron X-ray CT imaging at the Diamond Manchester Imaging Branchline (I13-2, Diamond Light Source, Oxford). We used monochromatic light and a 4× objective with a pco.edge 5.5 detector, providing a voxel size of 0.8125 μm. The obtained images were then reconstructed with NRecon (v.1.6.9.18, Bruker Corporation, Billerica, MA, USA) for a series of orthogonal slices.
The 3D segmentation of the inner-ear was performed with the software Amira-Aviso 6.7 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and were used for obtaining the dorsal wall thickness and the dimensions of the auditory vesicle, through the Center Line Tree module in AMIRA. For the 2D measurement of cap cell surface area, scolopale cell radius and dendrite length, an Alicona InfiniteFocus microscope (G5, Bruker Alicona Imaging, Graz, Austria) at ×5 objective magnification was used, with a resolution of about 100 nm.