4Pi-SIMFLUX: 4Pi single-molecule localization microscopy with structured illumination

Single-molecule localization microscopy (SMLM) has transformed biological imaging by enabling nanoscale visualization of intricate subcellular structures. However, conventional three-dimensional SMLM techniques typically exhibit lower axial resolution than lateral resolution, hindering isotropic investigations. Interferometric approaches, such as 4Pi-SMLM, enhance axial resolution by approximately fivefold through dual-objective coherent fluorescence detection, surpassing lateral resolution. Here we present 4Pi-SIMFLUX, which integrates structured illumination into 4Pi-SMLM to double its lateral resolution, achieving near-isotropic three-dimensional localization precision of 2-3 nm. We demonstrate that 4Pi-SIMFLUX breaks the 10-nm resolution barrier in biological samples, resolving microtubule ultrastructure and nuclear pore complexes with exceptional detail and clarity, while accounting for label size and localization density. Furthermore, it enables simultaneous multicolor imaging for interrogating multiple cellular components and high-fidelity, whole-cell visualization that captures comprehensive spatial organization. 4Pi-SIMFLUX effectively bridges the axial-lateral resolution gap, establishing a robust tool for molecular-scale imaging in complex cellular environments.