Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Nat Biotechnol. 2021 Nov;39(11):1385-1393. doi: 10.1038/s41587-021-01042-y. Epub 2021 Oct 11.
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.
活细胞的高时空分辨率和高检测灵敏度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的 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较慢，需要高的峰值激发强度，或者受到高离焦背景的影响。在这里，我们提出了三维干涉晶格光片（3D-iLLS）成像技术，该技术通过将 4Pi 干涉仪与选择性平面照明相结合，在低激发光水平下提供高背景抑制和大大提高的体积分辨率。我们证明了 3D-iLLS 的轴向分辨率和单粒子定位精度分别为 100nm（FWHM）和<10nm（1σ）。我们在一系列系统中说明了 3D-iLLS 的性能：单个信使 RNA 分子、核内转录调控因子的纳米级组装、微管细胞骨架和线粒体细胞器。3D-iLLS 的增强的 4D 分辨率和增加的信噪比将有助于在亚细胞水平分析生物过程。
