High-resolution fluorescence microscopy is increasingly contributing to our understanding of molecular processes. By utilizing single-molecule intensity information, imaging experiments can be rendered quantitative, yielding insights into the stoichiometry and kinetics of the components of a molecular assembly. Here, we describe the experimental and analytical steps needed to study the assembly of clathrin-coated vesicles with single-molecule resolution, using total internal reflection fluorescence microscopy. Many components of the protocol are broadly applicable to the characterization of other molecular processes.
Keywords: Clathrin-coated vesicles; Imaging; Single molecule; TIRF.