The nanoscale surface features of lipid-coated microbubbles can dramatically affect how the lipids interact with one another as the microbubble diameter expands and contracts under the influence of ultrasound. During microbubble manufacturing, the different lipid shell species naturally partition forming concentrated lipid islands. In this study the dynamics of how these nanoscale islands accommodate the expansion of the microbubbles are monitored by measuring the fluorescence intensity changes that occur as self-quenching lipophilic dye molecules embedded in the lipid layer change their distance from one another. It was found that when the dye molecules were concentrated in islands, less than 5% of the microbubbles displayed measurable fluorescence intensity modulation indicating the islands were not able to expand sufficiently for the dye molecules to separate from one another. When the microbubbles were heated and cooled rapidly through the lipid transition temperature the islands were melted creating an even distribution of dye about the surface. This resulted in over 50% of the microbubbles displaying the fluorescence-modulated signal indicating that the dye molecules could now separate sufficiently to change their self-quenching efficiency. The separation of the surface lipids in these different formations has significant implications for microbubble development as ultrasound and optical contrast agents.