Fluorescence-based single molecule techniques, mainly including fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET), are able to analyze the conformational dynamics and diversity of biological macromolecules. They have been applied to analysis of the dynamics of membrane proteins, such as membrane receptors and membrane transport proteins, due to their superior ability in resolving spatio-temporal heterogeneity and the demand of trace amounts of analytes. In this review, we first introduced the basic principle involved in FCS and smFRET. Then we summarized the labelling and immobilization strategies of membrane protein molecules, the confocal-based and TIRF-based instrumental configuration, and the data processing methods. The applications to membrane protein dynamics analysis are described in detail with the focus on how to select suitable fluorophores, labelling sites, experimental setup and analysis methods. In the last part, the remaining challenges to be addressed and further development in this field are also briefly discussed.

基于荧光的单分子技术，主要包括荧光相关光谱（FCS）和单分子荧光共振能量转移（smFRET），能够分析生物大分子的构象动力学和多样性。由于它们在解决时空异质性和对痕量分析物的需求方面具有卓越的能力，因此它们已被应用于分析膜蛋白（如膜受体和膜转运蛋白）的动力学。在这篇综述中，我们首先介绍了 FCS 和 smFRET 涉及的基本原理。然后我们总结了膜蛋白分子的标记和固定策略，基于共焦和基于TIRF的仪器配置以及数据处理方法。详细描述了膜蛋白动力学分析的应用，重点介绍了如何选择合适的荧光团、标记位点、实验设置和分析方法。在最后一部分，还简要讨论了该领域尚待解决的挑战和进一步发展。