Primary cilia are microtubule-based protrusions from the cell surface, which are approximately 0.3 µm in diameter and 3 µm in length. Because size approximates the optical diffraction limit, ciliary structures at the subdiffraction level can only be observed using a super-resolution microscope or electron microscope. Expansion microscopy (ExM) is an alternative super-resolution imaging technique, which uses a swellable hydrogel that enables the physical expansion of specimens. However, the efficacy of ExM has not been fully verified, and further improvements in the method are anticipated. In this study, we applied ExM for the observation of primary cilia and centrioles, and compared the acquired images with those obtained using conventional super-resolution microscopy. Furthermore, we developed a new tool, called the amplibody, for fluorescence-signal amplification, to compensate for the substantial decrease in fluorescence signal per unit volume inherent to physical expansion and for the partial proteolytic digestion of cellular proteins before expansion. We also demonstrate that the combinatorial use of the ExM protocol optimized for amplibodies and Airyscan super-resolution microscopy enables the practical observation of cilia and centrioles with high brightness and resolution.

原发纤毛是从细胞表面伸出的基于微管的突起，直径约为0.3 µm，长度约为3 µm。由于尺寸接近光学衍射极限，因此只能使用超分辨率显微镜或电子显微镜观察亚衍射级的睫状结构。扩展显微镜（ExM）是另一种超分辨率成像技术，它使用可膨胀的水凝胶来实现标本的物理扩展。但是，ExM的功效尚未得到充分验证，并且有望对该方法进行进一步的改进。在这项研究中，我们将ExM应用于原发纤毛和中心粒的观察，并将获得的图像与使用常规超分辨率显微镜获得的图像进行了比较。此外，我们开发了一种称为amplibody的新工具，用于荧光信号放大，以补偿物理扩展固有的每单位体积荧光信号的显着降低，并补偿扩展前细胞蛋白的部分蛋白水解消化。我们还证明，针对双抗体和Airyscan超分辨率显微镜优化使用的ExM协议的组合使用，可以实现具有高亮度和分辨率的纤毛和中心粒的实际观察。