Live cell imaging with high spatiotemporal resolution and high detection sensitivity facilitates the study of the dynamics of cellular structure and function. However, extracting high-resolution 4D (3D space plus time) information from live cells remains challenging, because current methods are slow, require high peak excitation intensities or suffer from high out-of-focus background. Here we present 3D interferometric lattice light-sheet (3D-iLLS) imaging, a technique that requires low excitation light levels and provides high background suppression and substantially improved volumetric resolution by combining 4Pi interferometry with selective plane illumination. We demonstrate that 3D-iLLS has an axial resolution and single-particle localization precision of 100 nm (FWHM) and <10 nm (1σ), respectively. We illustrate the performance of 3D-iLLS in a range of systems: single messenger RNA molecules, nanoscale assemblies of transcription regulators in the nucleus, the microtubule cytoskeleton and mitochondria organelles. The enhanced 4D resolution and increased signal-to-noise ratio of 3D-iLLS will facilitate the analysis of biological processes at the sub-cellular level.

具有高时空分辨率和高检测灵敏度的活细胞成像有助于研究细胞结构和功能的动力学。然而，从活细胞中提取高分辨率 4D（3D 空间加时间）信息仍然具有挑战性，因为目前的方法速度慢、需要高峰值激发强度或存在高失焦背景。在这里，我们展示了 3D 干涉式晶格光片 (3D-iLLS) 成像，该技术需要低激发光水平并通过将 4Pi 干涉法与选择性平面照明相结合来提供高背景抑制和显着提高的体积分辨率。我们证明 3D-iLLS 的轴向分辨率和单粒子定位精度分别为 100 nm (FWHM) 和 <10 nm (1σ)。我们说明了 3D-iLLS 在一系列系统中的性能：单信使 RNA 分子、细胞核中转录调节因子的纳米级组装、微管细胞骨架和线粒体细胞器。3D-iLLS 增强的 4D 分辨率和增加的信噪比将有助于在亚细胞水平上分析生物过程。