Ultrafast tracking of single molecules in cell membrane by coherent brightfield microscopy

Yi-Hung Liao^1*, Chih-Hsiang Lin¹, Chia-Ni Tsai¹, Ching-Ya Cheng¹, Chia-Lung Hsieh¹

¹Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan

* Presenter:Yi-Hung Liao, email:macgyver@gate.sinica.edu.tw

The cell plasma membrane is a membranous boundary serving as a regulating platform to many important cellular events. Detailed membrane dynamics is useful for unveiling the underlying mechanisms of the cell membrane. Single-molecule tracking (SMT) is a powerful tool to acquire high-resolution trajectory of single molecule moving in the membrane [1]. Microscopic (< 100 nm) dynamics and organization, unlike macroscopic ones, are vaguely understood [2]. Acquiring nanoscale dynamic information in the cell membrane is technically challenging because it requires high spatial and temporal resolutions at the same time. Optical scattering-based interferometric microscopy (e.g., coherent brightfield microscopy, COBRI) shows promise of tracking single membrane molecules in model or cellular membrane at ultrahigh spatiotemporal resolutions (up to 500,000 Hz) by using gold nanoparticles (AuNPs) as labels [3-5].

Here, we demonstrate ultrafast SMT in the plasma membrane of live cells by combining COBRI microscopy and monovalent rhizavidin-conjugated AuNPs (rAv-AuNP) probes that have recently been developed in our lab [6]. We emphasize that our monovalent rAv-AuNP probe does not affect the original motion of the membrane molecule via labeling [6], which is critical for resolving the true molecular dynamics in the cell membrane. Using our methods, we characterize the diffusion behaviors of single lipid molecules at 50,000 Hz (20 μs) with a lateral precision of ~6 nm (signal-to-noise ratio of ~ 15). Simultaneous microsecond and nanometer spatiotemporal resolution gives us unprecedented opportunities to investigate membrane dynamics and organization at the mesoscopic length scale (10 – 1000 nm) [6]. We observe that the lipid diffusion deviates from simple Brownian motion, and the diffusion coefficient is around 2 μm²/s at the timescale of 20 μs. Effects of cholesterol and cytoskeleton to the measured molecular diffusion will be presented and discussed.

Keywords: Single-particle tracking, plasma membrane, Gold nanoparticle, Monovalent