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

Area of Science:

• Biophysics
• Microscopy
• Otolaryngology

Background:

• Otoconia and corpuscles are vital inner ear structures with poorly understood ultrastructure.
• Collagen and other biological tissues typically exhibit uniaxial symmetry in second harmonic generation (SHG) microscopy.
• Polarization-resolved SHG (PSHG) microscopy offers a method to probe ultrastructural parameters.

Purpose of the Study:

• To investigate the ultrastructural parameters of mouse inner ear otoconia and corpuscles using PSHG microscopy.
• To compare the SHG properties of otoconia and corpuscles with known biological emitters like collagen.
• To model the structural basis for observed SHG parameters in otoconia.

Main Methods:

• Dissection of otoconia and corpuscles from mouse inner ears.
• Application of polarization-resolved second harmonic generation (PSHG) microscopy.
• Calculation of the PSHG parameter 'ρ' (rho) for pixel-wise analysis.
• Simulation of otoconia structure using a biaxial emitter model.
• In situ SHG measurement during otoconia degradation.

Main Results:

• Average PSHG parameter 'ρ' values were 4.6 for corpuscles and -3.3 for otoconia.
• Otoconia displayed a negative 'ρ' value, indicating non-uniaxial SHG emitter symmetry, distinct from collagen.
• Corpuscle 'ρ' distribution was radial, similar to starch.
• Simulations predicted that increased disorder in a biaxial system leads to more negative 'ρ' values.
• Otoconia 'ρ' values significantly decreased during degradation, supporting the biaxial model.

Conclusions:

• Mouse inner ear otoconia possess a unique biaxial ultrastructure, differing from uniaxial biological tissues like collagen.
• PSHG microscopy effectively characterizes otoconial ultrastructure and its changes during degradation.
• The observed negative 'ρ' values in otoconia are consistent with a disordered biaxial SHG emitter system.