Preparation of cochlear scanning electron microscopy samples using Zero-OsO4 fixation

YUAN Shuolong; XU Liangwei; HUANG Wenjie; ZHOU Gehui; CHEN Wei

doi:10.3969/j.issn.1672-2922.2025.09.009

Abstract：Objective The aim of this study was to develop a method for cochlear scanning electron microscope(SEM)samples preparation without the use of highly toxic osmium tetroxide(OsO4)that imposes strict operational restrictions and safety risks.Methods The cochlea of normal hearing guinea pigs was fixed by 2.5%glutaraldehyde perfusion for 24 hours instead of the traditional OsO4 fixation.After decalcification with 10%EDTA for 3 days and microdissection to expose the basilar membrane,dehydration was completed by gradient ethanol and carbon dioxide critical point drying.Finally,by optimizing the multi-angle ion sputtering coating,a uniform 5-10nm conductive gold film was formed on the sample surface.Results There was no"osmium black"or structural deformation caused by OsO4 in the cochlea.The overall shape of the cochlea and the distribution of hair cells in each turn were clearly displayed at low power.The cilia bundles of inner and outer hair cells were arranged neatly and the microvilli of cuticular plate were visible at medium power.Conclusions The proposed method successfully overcame the defects in electrical conductivity and contrast ratio caused by OsO4 deficiency,with imaging resolutions comparable to that of conventional preparation with OsO4,especially clearly visible for sub-micron details of cilia(lateral links).By glutaraldehyde fixation combined with precise control of the sputtering process,this technology completely eliminates the safety and compliance barriers related to highly toxic reagents,significantly reduces experimental complexity and cost,and provides a safe,efficient and high-quality solution for the study of inner ear ultrastructure morphology that is generally suitable for most laboratories.