Imaging of excised cochleae by micro-CT: staining, liquid embedding, and image modalities

Assessing the complex three-dimensional (3D) structure of the cochlea is crucial to understanding the fundamental aspects of signal transduction in the inner ear and is a prerequisite for the development of novel cochlear implants. X-ray phase-contrast computed tomography offers destruction-free 3D imaging with little sample preparation, thus preserving the delicate structure of the cochlea. The use of heavy metal stains enables higher contrast and resolution and facilitates segmentation of the cochlea. For μ-CT of small animal and human cochlea, we explore the heavy metal osmium tetroxide (OTO) as a radiocontrast agent and delineate laboratory from synchrotron CT. We investigate how phase retrieval can be used to improve the image quality of the reconstructions, both for stained and unstained specimens. Image contrast for soft tissue in an aqueous solution is insufficient under the in-house conditions, whereas the OTO stain increases contrast for lipid-rich tissue components, such as the myelin sheaths in nervous tissue, enabling contrast-based rendering of the different components of the auditory nervous system. The overall morphology of the cochlea with the three scalae and membranes is very well represented. Further, the image quality of the reconstructions improves significantly when a phase retrieval scheme is used, which is also suitable for non-ideal laboratory settings. With highly brilliant synchrotron radiation (SR), we achieve high contrast for unstained whole cochleae at the cellular level. The OTO stain is suitable for 3D imaging of small animal and human cochlea with laboratory , and relevant pathologies, such as a loss of sensory cells and neurons, can be visualized. With SR and optimized phase retrieval, the cellular level can be reached even for unstained samples in aqueous solution, as demonstrated by the high visibility of single hair cells and spiral ganglion neurons.