2021 Vol. 7, No. 6
Single-molecule methods have been applied to study the mechanisms of many biophysical systems that occur on the nanometer scale. To probe the dynamics of such systems including vesicle docking, tethering, fusion, traf-ficking, protein-membrane interactions, etc., and to obtain reproducible experimental data; proper methodology and framework are crucial. Here, the authors address this need by developing a protocol for immobilization of vesicles composed of synthetic lipids and measurement using total inter-nal reflection fluorescence (TIRF) microscopy. Furthermore, they demon-strate applications including vesicle clustering mediated by proteins such as alpha-Synuclein (αSyn) and the influence of external ions by using TIRF microscopy. Moreover, they use this method to quantify the dependence of lipid composition and charge on vesicle clustering mediated by αSyn which is based on the methods previously reported.