Interaction of Merocyanine 540 with biological membranes studied by polarized fluorescence and lifetime imaging microscopy

摘要

Methods of polarized fluorescence and fluorescence lifetime imaging microscopy were used to characterize the orientation and rotational diffusion capability of merocyanine 540 molecule bind to the model and biological membranes of single isolated cells and multilamellar liposomes. To estimate the binding properties of merocyanine in gel and fluid phases of the lipid bilayer, temperatures below and over phase-transition point of the lipid were used. Using a fluorescence microscope equipped with excitation and emission polarizers an effect of geometrically selective excitation ("photoselection") was observed, indicating that the orientation of merocyanine chromophore is dominantly perpendicular to the membrane surface of synthetic liposomes. Using the microscope setup combined with pulsed laser excitation and gated MCP image amplifier a time-resolved fluorescence images of the phospholipid vesicles stained by merocyanine 540 were obtained, showing no apparent spatial domains of different fluorescence lifetimes in contrast to highly structured images of fluorescence intensity.