Super-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures: cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery.

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关于样品，举例：单分子定位显微镜的优化程序

超分辨率显微镜深刻地改变了我们研究细胞结构的方式，揭示了未知的结构并完善了我们对细胞组装的看法。在各种技术中，单分子定位显微镜 (SMLM) 的分辨率可以达到大分子复合物的大小，并提供有关其纳米级原位排列的关键见解。因此，SMLM 是一项要求很高的技术，充分发挥其潜力需要专门优化的程序。在这里，我们描述了我们如何执行 SMLM 工作流程的连续步骤，重点是单色随机光学重建显微镜 (STORM) 以及用于固定样本纳米级地形 (DNA-PAINT) 成像的多色 DNA 点积累。我们提供了仔细的样品固定和典型细胞结构免疫染色的详细程序：细胞骨架、网格蛋白包被的小坑和细胞器。然后，我们为 SMLM 数据的最佳成像和处理提供指南，以优化重建质量并避免产生伪影。我们希望我们多年来发现的技巧和窍门以及这里的细节对希望制作最佳 SMLM 图像的研究人员有用，这是有意义的生物发现的先决条件。