Exploring Biophysics at the Membrane with Single-Molecule TIRF Microscopy.

Cellular functions result from the action of heteromeric protein machines that typically exhibit greater utility and dynamism than the sum of their parts. Therefore, the structural composition of these multimeric machines must be determined to delineate their functional attributes, including how information is transduced between the extracellular and intracellular environments, and between cells. While evaluating the activity of a protein complex at the population level provides valuable insights, measuring the activity of individual molecules offers a powerful approach for uncovering functional dynamics and reaction kinetics that are often obscured in ensemble measurements. Here we describe the background and application of Total Internal Reflection Fluorescence, or TIRF microscopy. We introduce the photo-physics that underpin TIRF and describe its utility in the study of membrane protein biophysics. We focus on applications of TIRF designed to determine the stoichiometry and real-time movement of protein complexes, especially ion channels and signaling receptors, in biological membranes.