Analysis of Membrane‐Protein Complexes by Single‐Molecule Methods

Fluorescence correlation spectroscopy (FCS) and single-particle tracking (SPT) methodologies are powerful techniques to study the diffusion and interaction of membrane proteins in artificial or cellular membranes. In this chapter, the two methods are introduced, compared, and examples of their application to the study of membrane-protein complexes are given. The concept of total internal reflection fluorescence (TIRF) is described in detail. The chapter explains the principles of TIRF microscopy for single-molecule imaging and the theory behind single-particle detection and tracking, together with the methods of analysis currently used. It then talks about some key applications of single-particle imaging to the study of protein stoichiometry and kinetics in membranes. The single-molecule brightness analysis, originally introduced by Schmidt et al., is based on the comparison of the fluorescent intensity of a cluster of fluorophores with the brightness of a single fluorophore.