Fast, three-dimensional, live-cell super-resolution imaging with multiplane structured illumination microscopy

Three-dimensional structured illumination microscopy (3D-SIM) doubles the spatial resolution along all dimensions and is used widely in cellular imaging. However, its temporal resolution is constrained by the need for sequential plane-by-plane movement of the sample using a piezo stage for imaging, which often increases the acquisition time to several seconds per volume. To address this limitation, we develop 3D multiplane SIM (3D-MP-SIM), which simultaneously detects multiplane images and reconstructs them using synergistically evolved reconstruction algorithms. Compared with conventional 3D-SIM imaging, 3D-MP-SIM achieves an approximately eightfold increase in the temporal resolution of volumetric super-resolution imaging, with lateral and axial spatial resolutions of about 120 and 300 nm, respectively. The rapid acquisition substantially reduces motion artefacts during the imaging of dynamic structures, such as late endosomes, in live cells. Moreover, we demonstrate the capabilities of 3D-MP-SIM via high-speed time-lapse volumetric imaging of the endoplasmic reticulum at rates of up to 11 volumes per second. We also show the feasibility of dual-colour imaging by observing rapid and close interactions among intra- and intercellular organelles in 3D space. These results highlight the potential of 3D-MP-SIM for explaining dynamic behaviours and interactions at the subcellular level and in three dimensions. Three-dimensional multiplane structured illumination microscopy, combining three-beam interference, multiplane detection and a synergistically evolved reconstruction algorithm, enables 3D imaging at rates of up to 11 volumes per second in live cells with lateral and axial spatial resolutions of 120 and 300 nm, respectively.