Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.
Group of Computational Biology and Applied Mathematics, Institute of Biology, Ecole Normale Supérieure, Paris, France.
Methods Mol Biol. 2022;2476:209-247. doi: 10.1007/978-1-0716-2221-6_16.
Single-molecule localization microscopy (SMLM) allows the super-resolved imaging of proteins within mammalian nuclei at spatial resolutions comparable to that of a nucleosome itself (~20 nm). The technique is therefore well suited to the study of chromatin structure. Fixed-cell SMLM has already allowed temporal "snapshots" of how proteins are arranged on chromatin within mammalian nuclei. In this chapter, we focus on how recent developments, for example in selective plane illumination, 3D SMLM, and protein labeling, have led to a range of live-cell SMLM studies. We describe how to carry out single-particle tracking (SPT) of single proteins and, by analyzing their diffusion parameters, how to determine whether proteins interact with chromatin, diffuse freely, or do both. We can study the numbers of proteins that interact with chromatin and also determine their residence time on chromatin. We can determine whether these proteins form functional clusters within the nucleus as well as whether they form specific nuclear structures.
单分子定位显微镜 (SMLM) 允许在哺乳动物核内以与核小体本身相当的空间分辨率 (~20nm) 对蛋白质进行超分辨率成像。因此，该技术非常适合研究染色质结构。固定细胞 SMLM 已经允许对哺乳动物核内染色质上蛋白质如何排列进行时间上的“快照”。在本章中，我们重点介绍了最近的发展，例如选择性平面照明、3D SMLM 和蛋白质标记，如何导致一系列活细胞 SMLM 研究。我们描述了如何进行单个蛋白质的单粒子追踪 (SPT)，并通过分析它们的扩散参数，确定蛋白质是否与染色质相互作用、自由扩散或两者兼而有之。我们可以研究与染色质相互作用的蛋白质数量，还可以确定它们在染色质上的停留时间。我们可以确定这些蛋白质是否在核内形成功能簇，以及它们是否形成特定的核结构。
