Investigation of intact mouse cochleae using two‐photon laser scanning microscopy

Abstract

Objectives The investigation of cochlear hair cells and lateral wall is a time‐consuming and labor‐intensive process. However, it is a mandatory experiment in audiology research. Here we suggest a novel method for investigating the inner ear microstructures from intact cochleae using two‐photon laser scanning microscopy (TPLSM). This technique guarantees fewer artifacts and technical simplicity. Methods Using TPLSM, we investigated the whole mount cochleae, decalcified cochleae, and cleared cochleae of wild type C57BL/6 mice. CX3CR1+/GFP mice were used to investigate the feasibility of visualizing cellular structures in the cochlear spiral ligament. All samples were investigated without staining. Results Endogenous fluorescence emission from the outer hair cells was strong enough to be distinguished from the other structures in all samples. From the single apical view, 50 and 90% of the whole hair cells of the decalcified cochleae and cleared cochleae, respectively, could be visualized without staining using TPLSM. Capillary structure of stria vascularis and spiral ligament could be visualized by endogenous fluorescence without staining. Conclusion We successfully investigated the hair cells and lateral wall of mouse cochleae using TPLSM without using staining or any destructive procedures. This method is easier, faster, and more reliable than conventional methods.

The Time Course of Monocytes Infiltration After Acoustic Overstimulation

“…We calculated the outer hair cell count as described previously ( Bae et al, 2020 ). Briefly, after fixation and decalcification as described above, the cochlea was separated into apical and basal portions under an optical microscope.…”

Two Photon Fluorescence Microscopy of the Unstained Human Cochlea Reveals Organ of Corti Cytoarchitecture

“…Recent advances in high resolution, deep penetration fluorescence microscopy techniques and the interfacing of these systems with miniature clinical endoscopes motivate investigation into whether these tools might be useful for intracochlear diagnostic applications; indeed, the abundance of endogenous fluorophores in the inner ear (e.g., flavin adenine dinucleotide (Sewell and Mroz, 1993) and nicotinamide adenine dinucleotide (Tiede et al, 2009) makes the cochlea a promising candidate for future fluorescence endoscopy. Following up on previous work in a mouse model by us (Yang et al, 2013;Romito et al, 2019) and others (Bae et al, 2020), here we demonstrate the ability of two-photon fluorescence microscopy (TPFM) to facilitate visualization of sensory cells and auditory nerve fibers in an unstained, non-decalcified cochlea from a former adult patient.…”

Toward Personalized Diagnosis and Therapy for Hearing Loss: Insights From Cochlear Implants

“…Advances in deep penetration fluorescence microscopy techniques have prompted the investigation into their application for intracochlear imaging. Previous work in mice models has demonstrated the utility of two-photon fluorescence microscopy (TPFM) for imaging of the inner ear by leveraging intrinsic optical properties of the tissue (5)(6)(7)(8). Given the thickness of the overlying otic capsule in which the cochlea is encased, optical scattering through bone re-mains a challenge.…”