Dynamic Three-dimensional Microscopy for Measuring Membrane Bound Protein Self-Assembly

Dynamic characterization of protein structure lags that of static characterization due to added complexity and lower signal levels. The dynamic transitions of proteins represent an untapped well of information in cellular development and physiological balance. We have developed a new fluorescence microscopy tool based upon Time-Resolved Fluorescence Anisotropy (TRFA) that allows us to monitor orientation changes of molecules within a lipid monolayer at the air-water interface. In contrast to confocal microscopy, our device is not limited to collinear excitation and fluorescence collection; we are able to excite fluorescence at a variable angle from grazing incident to the more traditional, perpendicular to the layer excitation. This allows us to measure not only the in-plane rotation of membrane molecules, but also changes in their wobbling and tilt orientations. For a DPPC monolayer labeled with 1{\%} fluorescently labeled lipid molecules we were able to identify the phase transition from the liquid-expanded to the liquid-condensed phase. In addition we followed and characterized for the first time a kinetic phase transition at high pressures where the fluorescent lipids phase separate from the DPPC and form a network of small nanoclusters. We envision this microscope being implemented to observe dynamic adsorption and self assembly of membrane bound proteins as well as observing the changes in lipid domain heterogeneity and dynamics when perturbed by the presence of nano-particulant pollutants.