Practical method for superresolution imaging of primary cilia and centrioles by expansion microscopy using an amplibody for fluorescence signal amplification.

Primary cilia are microtubule-based protrusions from the cell surface that 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 be observed only by using a superresolution microscope or electron microscope. Expansion microscopy (ExM) is an alternative superresolution imaging technique that 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 to the observation of primary cilia and centrioles and compared the acquired images with those obtained using conventional superresolution 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 superresolution microscopy enables the practical observation of cilia and centrioles with high brightness and resolution.