Interfacial Stresses on Droplet Interface Bilayers Using Two Photon Fluorescence Lifetime Imaging Microscopy

Response of lipid bilayers to external mechanical stimuli is an active area of research with implications for fundamental and synthetic cell biology. However, there is a lack of tools for systematically imposing mechanical strains and non-invasively mapping out interfacial (membrane) stress distributions on lipid bilayers. In this article, we report a miniature platform to manipulate model cell membranes in the form of droplet interface bilayers (DIBs), and non-invasively measure spatio-temporally resolved interfacial stresses using two photon fluorescence lifetime imaging of an interfacially active molecular flipper (Flipper-TR). We established the effectiveness of the developed framework by investigating interfacial stresses accompanying three key processes associated with DIBs: thin film drainage between lipid monolayer coated droplets, bilayer formation, and bilayer separation. Interestingly, the measurements also revealed fundamental aspects of DIBs including the existence of a radially decaying interfacial stress distribution post bilayer formation, and the simultaneous build up and decay of stress respectively at the bilayer corner and center during bilayer separation. Finally, utilizing interfacial rheology measurements and MD simulations, we also reveal that the tested molecular flipper is sensitive to membrane fluidity that changes with interfacial stress - expanding the scientific understanding of how molecular motors sense stress.