Single-Molecule Fluorescence Microscopy to Determine Phospholipid Lateral Diffusion
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The single-molecule detection (SMD) of individual fluorophores represents a powerful experimental technique that allows for the observation of individual membrane molecules in their different dynamic states without having to average over a large number of molecules. Spatial resolution in ensemble-averaging techniques such as fluorescence recovery after photobleaching, is limited by the diffraction limit of light (∼250 nm). In contrast, SMD (as well as single-molecule tracking of gold-labeled biomolecules through Nanovid microscopy) provides a tracking accuracy of approx 10–30 nm (dependent on experimental conditions). This level of accuracy makes single-molecule tracking techniques much better suited to detect nanometer-size heterogeneous structures in membranes. SMD can be easily applied to model and cellular membranes using a variety of fluorescent labels including organic dyes, quantum dots, and dye/quantum dots-doped nanoparticles.
The main focus of this chapter is to outline the SMD methodology to study lateral diffusion of lipids in model membranes. Subheading 1. provides an overview about the development of single-molecule tracking techniques, and explains the basic concept of single-molecule tracking. Subheading 2. lists all relevant chemicals necessary to successfully conduct lipid lateral diffusion studies on model membranes. Subheading 3. describes a typical experimental setup for SMD using wide-field illumination; thus, this setup can be utilized to track single-lipid tracers in solid-supported phospholipid bilayers and phospholipid monolayers at the air-water interface. Furthermore, some general considerations are included about different fluorescent labels for lipid-tracking studies. In addition a description of sample preparation procedures for the design of solid-supported phospholipid bilayers and Langmuir monolayers of phospholipids are described. Finally, Subheading 4. lists additional relevant information helpful for conducting SMD experiments on lipid membranes.
