Ultrasound contrast agents (UCA) consist of gas-filled coated microbubbles with diameters of 1-10 mu m. Targeted UCA can bind to biomarkers associated with disease through coating-incorporated ligands, making ultrasound molecular imaging possible. The aim of our research was to compare the ligand distribution, binding area, and bound microbubble shape of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) based and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) based lipid-coated microbubbles using super-resolution microscopy. Ligand distribution was studied by conjugating the fluorescent streptavidin Oregon Green 488 to the biotinylated microbubbles. An inhomogeneous streptavidin distribution was found when DSPC was the main coating lipid. When DSPC was replaced by DPPC, a more homogeneous streptavidin distribution was observed. Binding area of targeted microbubbles was studied using biotinylated microbubbles bound to a streptavidin-coated surface. DSPC microbubbles had a significantly smaller binding area than DPPC microbubbles. Whereas the bound DSPC microbubbles remained spherical, the DPPC microbubbles were dome-shaped. This study reveals that lipid-coated microbubbles differ in ligand distribution, binding area, and bound microbubble shape solely on the basis of their main lipid component.