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.

三维结构照明显微镜 (3D SIM) 的轴向分辨率限制为∼ 300 nm。在这里，我们提出了两种不同的、互补的方法来提高 3D SIM 中的轴向分辨率，而无需对光学系统进行最小修改或无需修改。我们表明，将镜子直接放置在样品对面，可以实现比 3D SIM 照明具有更高空间频率含量的四光束干涉，从而提供具有约120 nm 横向分辨率和 160 nm 轴向分辨率的近各向同性成像。我们还开发了一种深度学习方法，可实现约120 nm 各向同性分辨率。该方法可以与去噪相结合，以促进跨越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、描绘波形蛋白和微管丝的纳米级分布、观察小凹外壳蛋白和溶酶体标记物的相对位置以及可视化免疫突触早期阶段 T 细胞内的细胞骨架动态，展示了这些进展的潜力。形成。