Laboratory of High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, USA.
Janelia Research Campus, Howard Hughes Medical Institute (HHMI), Ashburn, VA, USA.
Nat Biotechnol. 2023 Sep;41(9):1307-1319. doi: 10.1038/s41587-022-01651-1. Epub 2023 Jan 26.
The axial resolution of three-dimensional structured illumination microscopy (3D SIM) is limited to ∼300 nm. Here we present two distinct, complementary methods to improve axial resolution in 3D SIM with minimal or no modification to the optical system. We show that placing a mirror directly opposite the sample enables four-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with ∼120-nm lateral and 160-nm axial resolution. We also developed a deep learning method achieving ∼120-nm isotropic resolution. This method can be combined with denoising to facilitate volumetric imaging spanning dozens of timepoints. We demonstrate the potential of these advances by imaging a variety of cellular samples, delineating the nanoscale distribution of vimentin and microtubule filaments, observing the relative positions of caveolar coat proteins and lysosomal markers and visualizing cytoskeletal dynamics within T cells in the early stages of immune synapse formation.
三维结构光照明显微镜（3D SIM）的轴向分辨率限制在约 300nm。在这里，我们提出了两种不同的、互补的方法，通过对光学系统进行最小或不修改来提高 3D SIM 中的轴向分辨率。我们表明，在样品的正对面放置一面镜子，可以实现具有比 3D SIM 照明更高空间频率内容的四束干涉，从而提供具有约 120nm 侧向和 160nm 轴向分辨率的各向同性成像。我们还开发了一种深度学习方法，实现了约 120nm 的各向同性分辨率。该方法可以与去噪相结合，以促进数十个时间点的体积成像。我们通过对各种细胞样本进行成像来证明这些进展的潜力，描绘出中间丝和微管丝的纳米尺度分布，观察网格蛋白包被蛋白和溶酶体标记物的相对位置，并观察免疫突触形成早期 T 细胞内细胞骨架动力学。
