Single-Molecule Imaging and Super-Resolution Microscopy of Lipid Domains in Cell Membranes Using Lipid-Binding Proteins and Fluorophore-Conjugated Lipid Analogs

Lipids are spatiotemporally organized in cell membranes, where they play indispensable roles in regulating diverse biological processes. Their distribution and dynamics are intricately coupled to signal transduction, membrane trafficking, and host-pathogen interactions. The past decade has seen substantial progress in the development of lipid probes and imaging techniques, which have greatly advanced our understanding of lipid-mediated regulation in living cells. Chemically optimized lipid analogs conjugated with hydrophilic fluorophores have enabled the faithful visualization of raftophilic lipids, such as sphingomyelin, gangliosides, and cholesterol, while minimizing artifacts. In parallel, genetically encoded lipid sensors derived from lipid-binding protein domains have been established. These sensors selectively report the localization and dynamics of diverse lipid species, including phosphoinositides, cholesterol, sphingomyelin, and phosphatidylserine, in their native contexts. Combined with state-of-the-art advanced microscopy approaches, including ultrafast single-molecule imaging and super-resolution microscopy, these probes facilitate high-resolution and quantitative analyses of lipid organization. This review summarizes recent advances in both synthetic lipid probes and genetically encoded lipid sensors, emphasizing their applications in mechanistic studies of membrane biology. We further discuss current challenges and future directions toward the comprehensive and minimally perturbative visualization of lipids.