2021 Vol. 7, No. 6

Cover Story

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.

Quantitation of nucleoprotein complexes by UV absorbance and Bradford assay
Lipid species dependent vesicles clustering caused by alpha-synuclein as revealed by single-vesicle imaging with total internal reflection fluorescence microscopy
Optical tweezer and TIRF microscopy for single molecule manipulation of RNA/DNA nanostructures including their rubbery property and single molecule counting
Molecular mechanisms of Streptococcus pyogenes Cas9: a single-molecule perspective
Real-time imaging of structure and dynamics of transmembrane biomolecules by FRET-induced single-molecule fluorescence attenuation
A review for cell-based screening methods in drug discovery