Si cantilevers

To further investigate possible internal nanogranular structure of otoconia, and to correlate this with the nanoscale distribution of organic matrix in the same sample, atomic force microscopy (AFM) was conducted on 1-µm-thick, ultramicrotome-cut sections from wild-type mice. AFM height and amplitude images were taken using a Multimode Nanoscope IIIa atomic force microscope (Veeco, Santa Barbara, CA, USA) operating in the tapping mode in air at room temperature using a nonvertical engage E-scanner and NanoScope version 5.30 software (Veeco/Bruker-AXS Inc., Madison, WI, USA). In the AFM experiments, V-shaped tapping mode probes (typical tip apex radius of approximately 7 nm) with Si cantilevers having a spring constant k = 42 N/m (Bruker-AXS Inc.) were used. The tip force exerted on the surface was optimized by the amplitude set-point being as high as possible to reduce imaging artefacts. The Feret diameters of the units comprising the observed nanostructure were calculated using ImageJ software (1.x version) (Schneider et al., 2012) . At least 200 Feret diameters of the otoconial nanostructure domains were calculated from the AFM images (obtained in amplitude mode) after performing the software's high-pass processing to enhance domain boundaries.

Electrical measurements were performed with conductive probes (Pt-Ir covered Si cantilevers with a low spring constant, k = 0.2 Nm -1 , SCM-PIC by Bruker) in contact mode by measuring simultaneously both topography and electrical current images. In these measurements, the conducting probe makes contact with the sample, acting like a nanoelectrode, and maps a current image at a fixed bias of -5V. The current was measured by a preamplifier.