Fluorescence Lifetime-Based Line-Scanning FRET-FCCS for Simultaneous Analysis of Protein Binding and Diffusion on Membranes

Probing protein dynamics and interactions in cellular or subcellular membranes is vital to understanding life processes and regulatory mechanisms. However, this remains challenging due to the temporal drift of the dynamic membrane and the inability to measure both accurate protein dynamics and protein–protein interactions. To address this challenge, we developed line-scanning fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy (termed ls-FRET-FCCS), enabling the study of biomolecular dynamics and interactions on mitochondrial membranes simultaneously. By repeatedly scanning across a membrane, this approach compensates for membrane motion while providing multidimensional readouts, including molecular diameter, concentration, diffusion coefficient, and intermolecular distance. We also demonstrate the proposed technique with a set of measurements, including, notably, the study of molecular interactions on contacted lysosomal and mitochondrial membranes. These results demonstrate that ls-FRET-FCCS is a versatile tool for simultaneously probing molecular interactions and dynamic processes on the membranes of living cells and subcellular organelles, offering both high precision and experimental flexibility.