Biomembranes are complex systems that regulate numerous biological processes. Lipid phases that constitute these membranes influence their properties and transport characteristics. Here, we demonstrate the potential of short-range dynamics imaging (excited-state lifetime, rotational diffusion, and order parameter) as a sensitive probe of lipid phases in giant unilamellar vesicles (GUVs). Liquid-disordered and gel phases were labeled with Bodipy-PC at room temperature. Two-photon fluorescence lifetime imaging microscopy of single-phase GUVs reveals more heterogeneity in fluorescence lifetimes of Bodipy in the gel phase (DPPC: 3.8+/-0.6 ns) as compared with the fluid phase (DOPC: 5.2+/-0.2 ns). The phase-specificity of excited-state lifetime of Bodipy-PC is attributed to the stacking of ordered lipid molecules that possibly enhances homo-FRET. Fluorescence polarization anisotropy imaging also reveals distinctive molecular order that is phase specific. The results are compared with DiI-C12-labeled fluid GUVs to investigate the sensitivity of our fluorescence dynamics assay to different lipid-marker interactions. Our results provide a molecular perspective of lipid phase dynamics and the nature of their microenvironments that will ultimately help our understanding of the structure-function relationship of biomembranes in vivo. Furthermore, these ultrafast excited-state dynamics will be used for molecular dynamics simulation of lipid-lipid, lipid-marker and lipid-protein interactions.

Cell adhesion to extracellular matrix (ECM) is fundamental to many distinct aspects of cell biology, and has been an active topic for label-free biosensors. However, little attention has been paid to study the impact of receptor signaling on the cell adhesion process. We here report the development of resonant waveguide grating biosensor-enabled label-free and fluorescent approaches, and their use for investigating the adhesion of an engineered HEK-293 cell line stably expressing green fluorescent protein (GFP) tagged β2-adrenergic receptor (β2-AR) onto distinct surfaces under both ambient and physiological conditions. Results showed that cell adhesion is sensitive to both temperature and ECM coating, and distinct mechanisms govern the cell adhesion process under different conditions. The β2-AR agonists, but not its antagonists or partial agonists, were found to be capable of triggering signaling during the adhesion process, leading to an increase in the adhesion of the engineered cells onto fibronectin-coated biosensor surfaces. These results suggest that the dual approach presented is useful to investigate the mechanism of cell adhesion, and to identify drug molecules and receptor signaling that interfere with cell adhesion.