目的·通过多尺度分辨率三维成像构建耳蜗及周围组织整体成像的标准化研究平台，实现生理状态下耳蜗的三维可视化分析。方法·8~12周龄胸腺细胞抗原1（Thy1）驱动黄色荧光转基因小鼠（Thy1-YFP-16小鼠）经心脏灌注后，获得单个耳蜗或耳蜗及全脑样品。使用PEGASOS技术进行组织透明化，再通过激光共聚焦显微镜4×和10×物镜对耳蜗及周围组织进行整体深度直接成像。对单个耳蜗样品，采用神经丝蛋白200（NF200）抗体及碘化丙啶（PI）荧光染料进行全组织免疫荧光染色，再结合PEGASOS和TESOS透明化及包埋技术进行处理；通过石蜡切片机序列切片及激光共聚焦显微镜63×油镜对整个耳蜗分层成像；使用ImageJ进行图像拼接获得三维图像后，通过Imaris软件处理图像、追踪单根神经纤维并测量长度，MATLAB计算路径曲率，GraphPad Prism进行统计分析。结果·通过组织透明化技术成功制备耳蜗及周围组织的透明化样品。共聚焦显微镜的中、低倍镜直接成像获得耳蜗及周围组织中等分辨率三维图像，重构了耳蜗的三维形态及其与前庭神经等周围结构相邻交错的空间位置；显微镜高倍镜下的连续切拍实现了整体耳蜗样本的单细胞分辨率三维成像，显示了耳蜗内THY1⁺纤维从毛细胞侧到蜗轴内的整体路径，并通过单根神经纤维追踪展示了螺旋神经节附近耳蜗传出神经系统与听神经纤维的不同走行特点。结论·利用PEGASOS、TESOS技术可实现对耳蜗及周围整体组织的多尺度分辨率三维成像；结合全组织免疫荧光染色，可用于获取耳蜗与毗邻组织、耳蜗内细胞与纤维的三维空间关系，实现对单神经纤维的路径追踪。

Objective ·To establish a standardized research platform for comprehensive imaging of the cochlea and its surrounding tissues through multi-scale resolution three-dimensional (3D) imaging, thereby enabling 3D visualization and analysis of the cochlea under physiological conditions. Methods ·The cochlea or combined cochlea and whole brain samples were obtained from 8‒12-week-old thymus cell antigen 1 (Thy1) -driven yellow fluorescent transgenic mice (Thy1-YFP-16 mice) after cardiac perfusion. Tissue clearing was performed using PEGASOS, followed by direct imaging of the cochlea and surrounding tissues through their entire depth using a laser confocal microscope with 4× and 10× objectives. For individual cochlear samples, whole tissue immunofluorescence staining was performed in combination with a neurofilament-200 (NF200) antibody and propidium iodide (PI) fluorescent dye, in the process of tissue clearing and embedding using PEGASOS and TESOS techniques. The entire cochlea was then subjected to serial sectioning with a paraffin microtome, and each section was imaged using a laser confocal microscope equipped with a 63× oil immersion objective. 3D reconstruction was performed using ImageJ for image stitching, followed by image processing, single-nerve-fiber tracking, and length measurement conducted in Imaris software. Fiber path curvature was calculated using MATLAB, and statistical analyses were performed with GraphPad Prism. Results ·Transparent cochlear and surrounding tissue samples were successfully prepared using tissue clearing techniques. The cochlea and its surrounding tissues were directly imaged by low- and medium-magnification objectives on the confocal microscope, which reconstructed the 3D morphology of the cochlea and delineated its spatial relationship with adjacent structures such as the vestibular nerve. Serial sectioning and imaging with a high-magnification objective enabled 3D imaging of the entire cochlear sample at single-cell resolution. This approach revealed the overall pathway of THY1⁺ fibers within the cochlea, extending from the hair cell region to the modiolus. Single nerve fiber tracing revealed distinct trajectory characteristics of the cochlear efferent nervous system and auditory nerve fibers near the spiral ganglion. Conclusion ·The PEGASOS and TESOS techniques enable multi-scale resolution 3D imaging of the cochlea and its surrounding tissues as an integrated whole. When combined with whole-tissue immunofluorescence staining, this methodology can be used to delineate the 3D spatial relationships between the cochlea and adjacent tissues, as well as between cells and nerve fibers within the cochlea itself. Furthermore, it allows for the tracing of individual nerve fiber pathways.