Superresolution microscopy is becoming increasingly widespread in biological labs. While it holds enormous potential for biological discovery, it is a complex imaging technique that requires thorough optimization of various experimental parameters to yield data of the highest quality. Unfortunately, it remains challenging even for seasoned users to judge from the acquired images alone whether their superresolution microscopy pipeline is performing at its optimum, or if the image quality could be improved. Here, we describe how superresolution microscopists can objectively characterize their imaging pipeline using suitable reference standards, which are stereotypic so that the same structure can be imaged everywhere, every time, on every microscope. Quantitative analysis of reference standard images helps characterizing the performance of one’s own microscopes over time, allows objective benchmarking of newly developed microscopy and labeling techniques, and finally increases comparability of superresolution microscopy data between labs.

超分辨率显微镜在生物实验室中变得越来越普遍。尽管它具有巨大的生物学发现潜力，但它是一种复杂的成像技术，需要对各种实验参数进行全面优化以产生最高质量的数据。不幸的是，即使是经验丰富的用户也只能从获得的图像中判断他们的超分辨率显微镜管线是否在其最佳性能下运行，或者是否可以提高图像质量，这仍然是一个挑战。在这里，我们描述了超分辨率显微学家如何使用合适的参考标准客观地表征他们的成像管道，这些参考标准是定型的，以便可以在任何地方，每次使用每台显微镜对同一结构进行成像。