Elucidating subcellular architecture and dynamics at isotropic 100-nm resolution with 4Pi-SIM
Zheyuan Ouyang, Qian Wang, Xiaoyu Li, Qiuyang Dai, Muyuan Tang, Lin Shao, Wen Gou, Zijing Yu, Yanqin Chen, Bei Zheng, Linlin Chen, Chenqi Ping, Xiuli Bi, Bin Xiao, Xiaochun Yu, Changliang Liu, Liangyi Chen, Junchao Fan, Xiaoshuai Huang, Yongdeng Zhang, Xiaoshuai Huang, Yongdeng Zhang
Abstract
Three-dimensional structured illumination microscopy (3D-SIM) provides excellent optical sectioning and doubles the resolution in all dimensions compared with wide-field microscopy. However, its much lower axial resolution results in blurred fine details in that direction and overall image distortion. Here we present 4Pi-SIM, a substantial revamp of I5S that synergizes 3D-SIM with interferometric microscopy to achieve isotropic optical resolution through interference in both the illumination and detection wavefronts. We evaluate the performance of 4Pi-SIM by imaging various subcellular structures across different cell types with high fidelity. Furthermore, we demonstrate its capability by conducting time-lapse volumetric imaging over hundreds of time points, achieving a 3D resolution of approximately 100 nm. Additionally, we illustrate its ability to simultaneously image in two colors and capture the rapid interactions between closely positioned organelles in three dimensions. These results underscore the great potential of 4Pi-SIM for elucidating subcellular architecture and revealing dynamic behaviors at the nanoscale. The 4Pi-SIM microscope combines the benefits of structured illumination microscopy with 4Pi geometry for improved axial resolution, offering isotropic, multicolor super-resolution imaging of dynamic processes in living cells.