Investigation of Membrane Domains and Single-molecule Dynamics in Lipid Bilayers Using Interferometric Scattering (iSCAT) Optical Microscopy
Chun-Yu Chou1*, Hsiao-Mei Wu1, Ying-Hsiu Lin1, Tsu-Chi Yan1, Chia-Lung Hsieh1
1Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan
* presenting author:Chun-Yu Chou,
Membrane domains of different phases in lipid bilayer membranes are of great research interests because they participate in many cell functions. For example, the liquid-ordered (Lo) phase has been thought to underlie the concept of ”lipid rafts” in cell membranes which is essential for endocytosis, signaling and membrane trafficking. While the biological significance of raft domains has been generally recognized, the nature and fundamental mechanisms of rafts are still poorly understood because of the lack of technology to resolve the dynamic molecular interaction of rafts at the nanoscale. Here, we use a novel optical imaging technique, called interferometric scattering (iSCAT) optical microscopy, to study the phase transition and single-molecule dynamics in raft-containing reconstituted bilayer membranes. Small gold nanoparticle, 20 nm in diameter, is attached to the head group of single lipid molecule and its motion is precisely recorded at high speed. The linear scattering signal from the nanoparticles was detected by optical interference where optimal spatial localization precision down to 3 nm is achieved at 50,000 frames per second. Furthermore, the photostable linear scattering signal offers unlimited observation time. Using this technique, we recorded accurate and long diffusion trajectories of single lipid molecules in raft-containing supported lipid bilayers. The processes of single molecules partitioning into and escaping from the raft-mimetic Lo domains are directly visualized in a continuous manner with unprecedented clarity. Surprisingly, we observe anomalous subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, that transiently trap the saturated lipids. Our data provide the first experimental evidence of non-uniform molecular organization of the Lo phase and show its critical role in determining molecular dynamics in the Lo phase. In addition to single-molecule dynamics, we show that the high sensitivity offered by iSCAT optical microscopy allows us to directly visualize the membrane domains of liquid-ordered, liquid-disordered, and solid-ordered phases in a phase coexistence membrane via intrinsic scattering without any labeling. Such label-free imaging provides the unique opportunity to study rapid phase transition and domain formation in biological membranes.

Keywords: iSCAT, single particle tracking, supported lipid bilayers, raft membrane