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

Luo, Shihang; Zhao, Xian'ao; Li, Yuanyuan; Fan, Chunyan; Liu, Ruina; Gong, Ran; Li, Weixing; Ma, Nana et al. · Nat Methods · 2026

basic_science · Level V

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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.

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