Characterization of inner ear granular bodies by polarization-resolved second harmonic generation microscopy

Otoconia and corpuscles dissected from the inner ear of mice were investigated using polarization-resolved second harmonic generation microscopy (PSHG) to extract ultrastructural parameters. The PSHG parameter, ρ, typically related to helical tilt in collagen, was calculated for each image pixel, and the average ρ values were found to be 4.6 for corpuscles and -3.3 for otoconia. The negative ρ from otoconia was measured unambiguously, indicating that the symmetry of SHG emitters is not predominantly uniaxial, in contrast to typical biological SHG emitters such as collagen, myosin, or starch. The internal distribution of ρ values of corpuscles was radial and resembled the average ρ values of starch. Simulating otoconia structure as consisting of a biaxial system with two similar emitters > 90° apart predicts that disorder alters the measured ρ values, making them increasingly negative. An SHG measurement of otoconia during degradation was performed, revealing that the ρ values of otoconia significantly decrease with degradation in agreement with this model.