Beijing National Research Center for Molecular Sciences, CAS Key Laboratory of Molecule Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China; email:
Department of Chemistry, University of the Chinese Academy of Sciences, Beijing 100049, China.
Annu Rev Anal Chem (Palo Alto Calif). 2020 Jun 12;13(1):337-361. doi: 10.1146/annurev-anchem-091619-094308. Epub 2020 Mar 30.
Live-cell single-molecule fluorescence imaging has become a powerful analytical tool to investigate cellular processes that are not accessible to conventional biochemical approaches. This has greatly enriched our understanding of the behaviors of single biomolecules in their native environments and their roles in cellular events. Here, we review recent advances in fluorescence-based single-molecule bioimaging of proteins in living cells. We begin with practical considerations of the design of single-molecule fluorescence imaging experiments such as the choice of imaging modalities, fluorescent probes, and labeling methods. We then describe analytical observables from single-molecule data and the associated molecular parameters along with examples of live-cell single-molecule studies. Lastly, we discuss computational algorithms developed for single-molecule data analysis.
活细胞单分子荧光成像已成为一种强大的分析工具，可用于研究传统生化方法无法研究的细胞过程。这极大地丰富了我们对单个生物分子在其天然环境中的行为及其在细胞事件中的作用的理解。在这里，我们综述了近年来在活细胞中基于荧光的蛋白质单分子生物成像的进展。我们首先考虑了单分子荧光成像实验的设计的实际问题，例如成像模式、荧光探针和标记方法的选择。然后，我们描述了单分子数据的分析可观察量以及相关的分子参数，并举例说明了活细胞中单分子研究。最后，我们讨论了为单分子数据分析开发的计算算法。
