Direct Observation and Control of Supported Lipid Bilayer Formation with Interferometric Scattering Microscopy

Abstract

Supported lipid bilayers (SLB) are frequently used to study processes associated with or mediated by lipid membranes. The mechanism by which SLBs form is a matter of debate, largely due to the experimental difficulty associated with observing the adsorption and rupture of individual vesicles. Here, we used interferometric scattering microscopy (iSCAT) to directly visualize membrane formation from nanoscopic vesicles in real time. We observed a number of previously proposed phenomena such as vesicle adsorption, rupture, movement, and a wave-like bilayer spreading. By varying the vesicle size and the lipid-surface interaction strength, we rationalized and tuned the relative contributions of these phenomena to bilayer formation. Our results support a model where the interplay between bilayer edge tension and the overall interaction energy with the surface determine the mechanism of SLB formation. The unique combination of sensitivity, speed, and label-free imaging capability of iSCAT provides exciting prospects not only for investigations of SLB formation, but also for studies of assembly and disassembly processes on the nanoscale with previously unattainable accuracy and sensitivity.

Visualization of the spontaneous emergence of a complex, dynamic, and autocatalytic system

“…The availability of binding sites itself depends on several competing dynamic processes such as unbinding events, formation of a supported lipid bilayer, deformation of individual aggregate structures, and the size of the aggregate products. Some of these processes have been reported to be dependent on the local density of particles on the substrate (19). Hence variation in the maximum landing rate is not unexpected.…”

Hydrolysis of a Lipid Membrane by Single Enzyme Molecules: Accurate Determination of Kinetic Parameters

“…Concerning the precision of the parameter estimates, further improved statistics may aid a more detailed analysis of the observed membrane-curvature dependence of the enzyme-specific Michaelis-Menten parameters. Such studies would also benefit from direct label-free visualization of individual liposomes, [20] which could offer a means to deduce whether there is an influence on the measured enzymatic kinetics of the dye-labeled lipids and also open up for extensions including studies addressing the influence of lipid composition on digestion kinetics, as previously addressed using ensemble-averaging approaches. [9] Regarding these aspects, it is interesting to note that PLA2 and related membraneassociated enzymes are biomarkers for a range of diseases, [1b, 21] and have emerged as potential drug targets, [22] and also participate in the innate immune response by acting as potent antimicrobial agents.…”

Detection and Digital Resolution Counting of Nanoparticles with Optical Resonators and Applications in Biosensing

“…Kukura et al [162] combined single molecule fluorescence microscopy and iSCAT and were able to visualize the location and orientation of single quantum dot-labeled Simian virus 40 (40 nm diameter) particles in real time. iSCAT has also been used to visualize the mechanisms involved in the formation of supported lipid bilayers (SLB) which has been difficult to observe [164]. iSCAT enabled direct observation of single vesicle adsorption and the subsequent transformation into a planar bilayer.…”