Characterizing single molecule probes of lipid membrane structure and dynamics

Biological membranes are rich in structural and dynamic heterogeneities that enable the various cellular processes to take place. However, understanding how these local heterogeneities affect membrane function has been difficult to address experimentally because of the small size of the features and the local nature of the dynamic motions. In this work, research is presented that is directed toward developing techniques capable of evaluating these heterogeneities in model membrane systems. First the single molecule fluorescence intensity fluctuations from the membrane probe diIC18 are characterized in monolayer films of the lipid DPPC. These studies reveal that the timescales of the intensity fluctuations track the local order in the lipid film. The intensity fluctuations are assigned to a mechanism which alters the emission yield of the molecule through spontaneous conformational motions. This mechanism is extremely sensitive to motions in the tail group region of the lipids. The intensity fluctuations are then used to evaluate the effect of cholesterol on the dynamic properties of the monolayer lipid films. These studies reveal that cholesterol has a concentration dependent effect on the dynamic properties of the lipid films that results from breaking the films into small lipid rich domains. Finally, a novel probe design for near-field scanning optical microscopy (NSOM) is presented and characterized. The cantilevered probes developed here are capable of scanning over large areas of the sample surface and can track larger topography changes compared to conventional NSOM probes. Additionally, they are capable of imaging fragile biological samples in aqueous solution while retaining the high spatial resolution characteristic of NSOM. Although the cantilevered probes suffer in polarization preservation and are much less efficient at delivering light to the sample surface, they are still capable of single molecule fluorescence detection.