An optimized tissue processing pipeline for multiscale imaging of the intact peripheral auditory pathway

Area of Science:

• Neuroscience
• Bioimaging
• Auditory Science

Background:

• The peripheral auditory pathway, including the cochlea and cochlear nucleus (CN), is crucial for hearing.
• Analyzing this pathway often involves sectioning, leading to data loss and difficulty in studying neuronal connections.
• Multiscale imaging is needed to understand the complex structure and function of the auditory system.

Purpose of the Study:

• To develop a comprehensive pipeline for multiscale analysis of the intact peripheral auditory pathway.
• To enable holistic assessment of the cochlea, cochlear root, and CN within a single specimen.
• To provide researchers with a versatile framework for studying auditory function and pathology.

Main Methods:

• Integration of micro-computed tomography (µCT), light-sheet fluorescence microscopy, confocal microscopy, and STED microscopy.
• A novel dissection technique preserving the cochlea-CN connection for volumetric imaging.
• Adapted ethyl cinnamate-based clearing, novel slice preparation, and a validated antibody library for robust labeling.

Main Results:

• Successful volumetric imaging of neuronal populations and quantification of spiral and vestibular ganglion neurons without sectioning loss.
• Enabled vascular network segmentation and nanoscale protein distribution analyses.
• Demonstrated robust labeling from whole-organ to synaptic scales.

Conclusions:

• The developed pipeline offers a unified framework for investigating auditory function and pathology.
• Maximizes information extraction from individual specimens, aligning with 3R principles.
• Advances the study of hearing impairment and related genetic models by providing an accessible, comprehensive analysis tool.