Super-resolution microscopies have become an established tool in biological research. However, imaging throughput remains a main bottleneck in acquiring large datasets required for quantitative biology. Here we describe multifocal flat illumination for field-independent imaging (mfFIFI). By integrating mfFIFI into an instant structured illumination microscope (iSIM), we extend the field of view (FOV) to >100 × 100 µm² while maintaining high-speed, multicolor, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching adjacent images for fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM with ultrastructure expansion microscopy to collect three-dimensional (3D) images of hundreds of centrioles in human cells, or thousands of purified Chlamydomonas reinhardtii centrioles, per hour at an effective resolution of ~35 nm. Classification and particle averaging of these large datasets enables 3D mapping of posttranslational modifications of centriolar microtubules, revealing differences in their coverage and positioning.

超分辨率显微镜已经成为生物学研究中的既定工具。但是，成像通量仍然是获取定量生物学所需的大型数据集的主要瓶颈。在这里，我们描述了用于场无关成像（mfFIFI）的多焦点平面照明。通过将mfFIFI集成到即时结构照明显微镜（iSIM）中，我们将视场（FOV）扩展到> 100×100 µm ²同时以两倍于衍射极限的分辨率保持高速，多色，体积成像。我们通过拼接相邻图像进一步扩展有效FOV，以对数十个细胞进行快速活细胞超分辨率成像。最后，我们将平场iSIM与超微结构扩展显微镜相结合，以〜35 nm的有效分辨率每小时收集人类细胞中数百个百日圆或成千上万个纯化的莱茵衣藻的百年式的三维（3D）图像。这些大型数据集的分类和粒子平均可以对中心微管的翻译后修饰进行3D映射，从而揭示其覆盖范围和位置的差异。