中华耳科学杂志2025，Vol.23Issue(9)：1053-1057,5.DOI:10.3969/j.issn.1672-2922.2025.09.009
无四氧化锇固定制备耳蜗扫描电子显微镜样本的研究
Preparation of cochlear scanning electron microscopy samples using Zero-OsO4 fixation
摘要
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.关键词
四氧化锇/耳蜗/扫描电子显微镜/纤毛Key words
osmium tetroxide/cochlea/scanning electron microscope/cilia引用本文复制引用
袁硕龙,徐良慰,黄文捷,周舸晖,陈伟..无四氧化锇固定制备耳蜗扫描电子显微镜样本的研究[J].中华耳科学杂志,2025,23(9):1053-1057,5.基金项目
科技部重点研发计划(2022YFC2402701) （2022YFC2402701）
