Tracking Single Proteins in Lipid Bilayers Using Fluorescence Microscopy

Direct observation is a fundamental method for scientific discovery and understanding. Time-lapse single-molecule fluorescence imaging allows the observation of individual proteins as they move and interact with their surroundings, making states that would normally be hidden in ensemble measurements visible. Single-protein tracking is especially suited for studying membrane proteins, where its two-dimensional nature enables fast, wide-field imaging with single-fluorophore sensitivity. Using artificial lipid bilayers that mimic membranes where transmembrane or peripheral membrane proteins are found (such as plasma membranes or membranes of intracellular organelles from various cell types) permits measurement of specific interactions in near-native environments. It also allows for the gradual addition of complexity, one component at a time, while suppressing background interference from fluorescence and Raman scattering. By adding temperature control, it becomes possible to determine the thermodynamic properties of single-particle interactions as well. In this work, we describe a protocol for sample preparation, data collection, single-molecule fluorescence tracking, and analysis of membrane protein trajectories in lipid bilayers using Aquaporin-4. This work serves as a foundational example and can be adapted to different proteins and bilayer compositions.