Three-dimensional structured illumination microscopy with enhanced axial resolution
Xuesong Li, Yicong Wu, Yijun Su, Ivan Rey‐Suarez, Claudia Matthaeus, Taylor B. Updegrove, Zhuang Wei, Lixia Zhang, Hideki Sasaki, Yue Li, Min Guo, John Giannini, Harshad D. Vishwasrao, Jiji Chen, Shih-Jong J. Lee, Lin Shao, Huafeng Liu, Kumaran S. Ramamurthi, Justin W. Taraska, Arpita Upadhyaya, Patrick J. La Rivière, Hari Shroff
Abstract
The axial resolution of three-dimensional structured illumination microscopy (3D SIM) is limited to ∼300 nm. Here we present two distinct, complementary methods to improve axial resolution in 3D SIM with minimal or no modification to the optical system. We show that placing a mirror directly opposite the sample enables four-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with ∼120-nm lateral and 160-nm axial resolution. We also developed a deep learning method achieving ∼120-nm isotropic resolution. This method can be combined with denoising to facilitate volumetric imaging spanning dozens of timepoints. We demonstrate the potential of these advances by imaging a variety of cellular samples, delineating the nanoscale distribution of vimentin and microtubule filaments, observing the relative positions of caveolar coat proteins and lysosomal markers and visualizing cytoskeletal dynamics within T cells in the early stages of immune synapse formation.