- Review
Single-Molecule Imaging and Super-Resolution Microscopy of Lipid Domains in Cell Membranes Using Lipid-Binding Proteins and Fluorophore-Conjugated Lipid Analogs
- Toshiki Mori and
- Kenichi G. N. Suzuki
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.
16 October 2025
![Raftophilic lipid probes conjugated with fluorescent organic dyes. (a). Schematic diagram of the partitioning of lipid probes into Lo (raft)-like (magenta) and Ld (non-raft)-like domains (gray). GM3 or SM probes conjugated with hydrophilic dyes at the sialic acid via nonaethyleneglycol (neg) preferentially partitioned into Lo-like phase, whereas probes conjugated with hydrophobic dyes such as ATTO647N partitioned into Ld-like phase [27,34]. (b). Chemical structures of 488neg-SM and 594neg-SM [26] (top), and 488-S9-GM3 and 594-S9-GM3 [27] (bottom). (c). Representative fluorescence images from simultaneous dual-color video sequences of the probes in membrane blebs of RBL-2H3 cells at 8 °C [26,27]. Probes in blue and red denote those localized in raft-like and non-raft-like phases, respectively. “S9” indicates that fluorescent dyes were conjugated to the C9 position of the terminal sialic acid of GM3 or GM1 [27]. “Neg” denotes that nonaethyleneglycol was used as a spacer between the choline moiety of SM or PC and the dyes [26]. Adapted from Figure 1, 5 in [26] and Figure 2 in [27] with permission.](/_ipx/b_%23fff&f_webp&q_100&fit_outside&s_470x317/https://mdpi-res.com/membranes/membranes-15-00317/article_deploy/html/images/membranes-15-00317-g001-550.jpg)
