Plasma membranes play important roles in many metabolic processes. They control the information flow and substance trafficking inward or outward the cells and serve as the stage for many biochemical reactions (Almen
et al. 2009; Bretscher and Raff
1975). The functions of plasma membranes rely on membrane-based biomolecules, especially membrane proteins. Involved in processes such as ligand–receptor interactions (Latorraca
et al. 2017), transmembrane transport (Bennett
et al. 2019; de Lera Ruiz and Kraus
2015) and lipid organization (Lingwood and Simons
2010; Sonnino and Prinetti
2013), these proteins undergo multiple conformational changes upon activation and are precisely regulated (Contreras
et al. 2010; Latorraca
et al. 2017; Leth-Larsen
et al. 2010; Nishida
et al. 2014). Therefore, probing the dynamics of membrane-bound proteins as well as the interaction between the proteins and the membranes are crucial for understanding their functions and the underlying mechanisms. Over years, structural analysis techniques such as X-ray crystallography (Andersson
et al. 2019; Lieberman
et al. 2013), nuclear magnetic resonance (Bibow and Hiller
2019; Nishida
et al. 2014; Radic and Pattanaik
2018), neuron reflectivity (Hellstrand
et al. 2013), and the fast-developing electron microscopy techniques (Cheng
2018; Garcia-Nafria and Tate
2020; Yao
et al. 2020) have provided valuable information about the structures of membrane-interacting macromolecules. However, these methods either acquire only static conformations, or work with ensembles of molecules. Because the macromolecular machines often work in the timescale below seconds and may switch among multiple conformational states (Haberl
et al. 2009; Prakash and Gorfe
2019; Volkman
et al. 2001), the time- or ensemble-averaged results could not fully uncover the mechanism of membrane proteins. Single-molecule methods are able to explore the molecular details of various physiological processes in real time, including immune responses, membrane fusion and fission, phase separation and lipid raft formation.