The folded mitochondria inner membrane-cristae is the structural foundation for oxidative phosphorylation (OXPHOS) and energy production. By mechanically simulating mitochondria morphogenesis, we speculate that efficient sculpting of the cristae is organelle non-autonomous. It has long been inferred that folding requires buckling in living systems. However, the tethering force for cristae formation and regulation has not been identified. Combining electron tomography, proteomics strategies, super resolution live cell imaging and mathematical modeling, we reveal that the mitochondria localized actin motor-myosin 19 (Myo19) is critical for maintaining cristae structure, by associating with the SAM-MICOS super complex. We discover that depletion of Myo19 or disruption of its motor activity leads to altered mitochondria membrane potential and decreased OXPHOS. We propose that Myo19 may act as a mechanical tether for effective ridging of the mitochondria cristae, thus sustaining the energy homeostasis essential for various cellular functions.

Three-dimensional structured illumination microscopy (3D-SIM) provides excellent optical sectioning and doubles the resolution in all dimensions compared with wide-field microscopy. However, its much lower axial resolution results in blurred fine details in that direction and overall image distortion. Here we present 4Pi-SIM, a substantial revamp of I⁵S that synergizes 3D-SIM with interferometric microscopy to achieve isotropic optical resolution through interference in both the illumination and detection wavefronts. We evaluate the performance of 4Pi-SIM by imaging various subcellular structures across different cell types with high fidelity. Furthermore, we demonstrate its capability by conducting time-lapse volumetric imaging over hundreds of time points, achieving a 3D resolution of approximately 100 nm. Additionally, we illustrate its ability to simultaneously image in two colors and capture the rapid interactions between closely positioned organelles in three dimensions. These results underscore the great potential of 4Pi-SIM for elucidating subcellular architecture and revealing dynamic behaviors at the nanoscale.

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.

Superresolution (SR) fluorescence microscopy provides nanoscale resolution for studying subcellular structures and biological processes.^{1 – 7} However, the higher light dose required for SR imaging than conventional microscopy, phototoxicity, and photobleaching severely limit their applications in live-cell imaging.⁸ Structured illumination microscopy (SIM) demonstrates a higher photon efficiency than other SR microscopy.^{9 – 11} In particular, two-dimensinal (2D)-SIM can achieve a doubling resolution beyond the light diffraction limit using nine sequentially acquired images, making it useful for live-cell SR imaging. However, live-cell SR-SIM imaging still suffers from phototoxicity and photobleaching, and image restoration is an ill-posed inverse problem.^{9,10,12 – 15} Therefore, for raw images of low signal-to-noise ratio (SNR) caused by short exposure or excessive photobleaching, the conventional Wiener-based reconstruction method is prone to artifacts.^{16 – 19}