Imaging hair cells through laser-ablated cochlear bone

Abstract

We report an innovative technique for the visualization of cells through an overlying scattering medium by combining femtosecond laser bone ablation and two-photon excitation fluorescence (TPEF) microscopy. We demonstrate the technique by imaging hair cells in an intact mouse cochlea ex vivo. Intracochlear imaging is important for the assessment of hearing disorders. However, the small size of the cochlea and its encasement in the densest bone in the body present challenging obstacles, preventing the visualization of the intracochlear microanatomy using standard clinical imaging modalities. The controlled laser ablation reduces the optical scattering of the cochlear bone while the TPEF allows visualization of individual cells behind the bone. We implemented optical coherence tomography (OCT) simultaneously with the laser ablation to enhance the precision of the ablation and prevent inadvertent damage to the cells behind the bone.

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

“…Given the thickness of the overlying otic capsule in which the cochlea is encased, optical scattering through bone re-mains a challenge. Laser ablation to thin the otic capsule has been shown to limit optical scattering and allow for TPFM imaging directly through bone (9). In preparation for the eventual clinical application of TPFM, an unstained, nondecalcified human cadaveric temporal bone was imaged through a cochleostomy using TPFM, revealing cellular and subcellular architecture (10).…”