Three-dimensional structured illumination microscopy with enhanced axial resolution

Nat Biotechnol. 2023 Sep;41(9):1307-1319. doi: 10.1038/s41587-022-01651-1. Epub 2023 Jan 26.


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.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cytoskeleton
  • Imaging, Three-Dimensional* / methods
  • Lighting*
  • Lysosomes
  • Microscopy, Fluorescence / methods