Second harmonic generation microscopy of inner ear otoconia

Abstract

Otoconia are microscopic biocrystals located within the vestibular system of the inner ear, that allow vertebrates to detect gravity and linear acceleration. When otoconia become degraded due to factors including aging, disease, and consumption of ototoxic medications, vestibular pathologies arise, and the body’s ability to maintain balance is significantly impaired. Vestibular pathologies such as benign-paroxysmal positional vertigo are a significant health concern across Canada. These pathologies lead to falls which cause debilitating conditions such as bone fractures, neurological damage, and negative social impacts. To develop methods to prevent degeneration of otoconia, and to induce and promote repair and regeneration, it is important to study their crystalline ultrastructure. In this study, we investigate the origin of second harmonic generation (SHG) signal in otoconia and their internal structural properties using SHG microscopy. SHG microscopy allows for optical sectioning of otoconia providing a 3D image of them, unlike previously used imaging techniques. To determine the origin of SHG signal of otoconia, calcite, the principal component of otoconia (>90 wt. -%) was imaged using SHG microscopy. Calcite gave ~41× less SHG signal compared to otoconia. Therefore, it was determined that calcite is unlikely the origin of SHG signal in otoconia. This finding indicates that proteins in otoconia are likely causing the SHG signal in otoconia as they make up the remaining portion of otoconia (<5 wt. -%). SHG images of otoconia revealed that they are polarization-dependent. This indicates that the proteins emitting SHG in otoconia are radially arranged, extending from a central region. Otoconia were also imaged after exposure to 0.5M ethylenediaminetetraacetic acid (EDTA), which degrades calcium carbonate. SHG signal increased over time, further supporting that proteins are causing the SHG signal in otoconia. It was concluded that SHG microscopy is a promising technique for investigating the internal structural properties of otoconia.