Concurrent diffusion of nicotinic acetylcholine receptors and fluorescent cholesterol disclosed by two-colour sub-millisecond MINFLUX-based single-molecule tracking.

The diffusion and interaction dynamics of membrane proteins and lipids are key for cell function, but their disclosure is hampered by limited temporal and spatial resolution of conventional observation technologies. Here we exploit the capabilities of minimal fluorescence emission photon fluxes (MINFLUX) microscopy in single-molecule co-tracking experiments of an important membrane protein and cholesterol with enhanced spatiotemporal resolution. Specifically, we interrogate the 2D translational mobility of a ubiquitous cell-surface protein, the nicotinic acetylcholine receptor, in tandem with a fluorescent cholesterol analogue for minute-long periods, reaching nanometric precision and sub-millisecond time resolution. To this end, we implement a multiplexing procedure that enables the simultaneous excitation of the two fluorescent-labelled molecules using a single wavelength, followed by discrimination of their emissions via differential ratiometric recording. We disclose a cholesterol-dependent heterogeneous spectrum of diffusive behaviours with regions of joint translational motion.