Three-dimensional structured illumination microscopy with enhanced axial resolution

• Published in: Nature biotechnology Vol. 41; no. 9; pp. 1307 - 1319
• Main Authors: Li, Xuesong, Wu, Yicong, Su, Yijun, Rey-Suarez, Ivan, Matthaeus, Claudia, Updegrove, Taylor B, Wei, Zhuang, Zhang, Lixia, Sasaki, Hideki, Li, Yue, Guo, Min, Giannini, John P, Vishwasrao, Harshad D, Chen, Jiji, Lee, Shih-Jong J, Shao, Lin, Liu, Huafeng, Ramamurthi, Kumaran S, Taraska, Justin W, Upadhyaya, Arpita, La Riviere, Patrick, Shroff, Hari
• Format: Journal Article
• Language: English
• Published: United States Nature Publishing Group 01.09.2023; Nature Publishing Group US
• Subjects: Cytoskeleton; Deep learning; Filaments; Frequency dependence; Illumination; Imaging; Interference; Lymphocytes; Lymphocytes T; Microscopy; Vimentin
• ISSN: 1087-0156; 1546-1696; 1546-1696
• DOI: 10.1038/s41587-022-01651-1

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.