Recently, biologists have gained access to several far-field fluorescence nanoscopy (FN) technologies that allow the observation of cellular components with ~20 nm resolution. FN is revolutionizing cell biology by enabling the visualization of previously inaccessible subcellular details. While technological advances in microscopy are critical to the field, optimal sample preparation and labeling are equally important and often overlooked in FN experiments. In this review, we provide an overview of the methodological and experimental factors that must be considered when performing FN. We present key concepts related to the selection of affinity-based labels, dyes, multiplexing, live cell imaging approaches, and quantitative microscopy. Consideration of these factors greatly enhances the effectiveness of FN, making it an exquisite tool for numerous biological applications.

最近，生物学家已经获得了几种远场荧光纳米镜 (FN) 技术，可以以约 20 nm 的分辨率观察细胞成分。FN 通过实现以前无法访问的亚细胞细节的可视化正在彻底改变细胞生物学。虽然显微镜技术的进步对该领域至关重要，但最佳的样品制备和标记同样重要，但在 FN 实验中经常被忽视。在这篇综述中，我们概述了进行 FN 时必须考虑的方法和实验因素。我们提出了与基于亲和力的标签、染料、多重分析、活细胞成像方法和定量显微镜的选择相关的关键概念。考虑到这些因素极大地提高了 FN 的有效性，使其成为众多生物应用的绝佳工具。