Three-dimensional super-resolution imaging of whole cells using a high numerical aperture oblique-plane microscope

Single-molecule localization microscopy (SMLM) significantly advanced the investigation of protein complexes and organelle interactions. However, whole-cell SMLM imaging remains constrained by the field of view and penetration depth of imaging. We present a high numerical aperture oblique-plane microscope (HOPE-STORM) for whole-cell super-resolution imaging. This system achieves an effective numerical aperture of up to 1.40 and is compatible with DNA points accumulation for imaging in nanoscale topography (DNA-PAINT), allowing for the resolution of the three-dimensional structure of nuclear pore complexes (NPCs) in cells at 7.5 nm. We demonstrated the system's capability for dual-color super-resolution imaging, visualizing nanoscale structures within intact cells. Utilizing HOPE-STORM, we investigated the interaction between dynamin-related protein 1 (DRP1) complexes and the mitochondrial outer membrane at mitochondrial fission sites. For the first time, we measured the 3D structure of DRP1 complexes in situ, and classified the mitochondrial fission sites based on the quantification analysis of DRP1 complex. These findings contribute to our understanding of mitochondrial fission mechanisms and the structural dynamics of DRP1.