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

We present a comprehensive tissue processing and imaging pipeline tailored for multiscale analyses of the intact peripheral auditory pathway, encompassing the cochlea, cochlear root, and cochlear nucleus (CN) within a single specimen. This workflow integrates advanced imaging modalities - including micro-computed tomography (µCT), light-sheet fluorescence microscopy, confocal microscopy, and super-resolution STED microscopy - to enable a holistic assessment of fixed temporal bones. This methodological pipeline incorporates a unique and novel dissection technique that maintains the delicate connection between the cochlea and CN, facilitating volumetric imaging of neuronal populations and precise quantification of spiral and vestibular ganglion neurons without sectioning-related loss. The presented workflow further supports vascular network segmentation and nanoscale protein distribution analyses, thus offering versatility for diverse cochlear research applications. To promote widespread adoption, an adapted ethyl cinnamate-based clearing protocol, a novel slice preparation procedure as well as a validated antibody library are provided that enable robust labeling of cochlear structures ranging from whole-organ visualization to synaptic-scale analyses. By maximizing information extraction from individual specimens, this accessible pipeline aligns with 3R principles and provides a unified framework for investigating auditory function and pathology, advancing the study of hearing impairment and related genetic models.