Nature Methods, 2 December, 2025, DOI：https://doi.org/10.1038/s41592-025-02911-z

Molecular-scale isotropic 3D super-resolution microscopy via interference localization

Shihang Luo, Xian’ao Zhao, Yuanyuan Li, Chunyan Fan, Ruina Liu, Ran Gong, Weixing Li, Nana Ma, Zhenghong Yang, Tao Xu, Wei Ji & Lusheng Gu

Abstract

Three-dimensional (3D) nanoscale imaging reveals the detailed morphology of subcellular structures; however, conventional single-molecule localization microscopy is constrained by limited axial resolution. Here we introduce ROSE-3D, an interferometric localization approach that enables isotropic 3D super-resolution imaging with uniform performance across the entire depth of field. Compared with conventional astigmatism-based methods, ROSE-3D improves lateral localization precision by 2–6 times and axial precision by 3.5–8 times over a depth of field of approximately 1.2?μm. Leveraging its multicolor and whole-cell imaging capabilities, ROSE-3D resolves, in situ, the nanoscale organization of nuclear lamins and the assemblies of mitochondrial fission-related protein DRP1. These results establish ROSE-3D as a powerful tool for interrogating nanoscale cellular architecture.

Article link：https://www.nature.com/articles/s41592-025-02911-z