TIRF Imaging of Lipid Bilayer Arrays

Abstract

A TIRF imaging platform for the simultaneous interrogation of multiple lipid bilayers in an arrayed format has been developed. Here, a proof of principle demonstration is made with the quantification of Calcium flux through the trans membrane pore, alpha-hemolysin. The effect of pore blocking by cyclodextrins is also demonstrated. Utilising a droplet on hydrogel method of lipid bilayer formation, an array of multiple lipid bilayers are illuminated simultaneously by the evanescent field of laser light undergoing total internal reflection at the surface of the glass hydrogel support layer. The droplet based method of bilayer production is ideally suited to an array platform, with the segregated contents of each droplet affording the opportunity to incorporate various membrane proteins, or simultaneously screen a variety of molecular candidates for their interaction with incorporated membrane components. The system offers the opportunity of asymmetric bilayer generation and the option of rapidly screening environmental dependencies for protein function. The scalable and automatable nature of the approach, together with the ability to tailor droplet contents, creates many exciting opportunities for characterisation of membrane protein behaviour and ligand interactions, the focus of future studies with the platform.