For molecular imaging using ultrasound contrast agents, targeted microbubbles are designed with specific ligands linked to the coated shell. Research is ongoing to determine the binding force of targeted microbubbles and to distinguish bound from unbound targeted microbubbles using ultrasound. For this, the actual surface of the targeted microbubbles that binds to a pathology is important. This study focuses on determining the surface contact of bound targeted microbubbles by fluorescence microscopy. Biotinylated lipid-coated microbubbles (3-7 μm in diameter) with either DSPC or DPPC as the main lipid were targeted to a streptavidin-coated surface. The binding area of targeted microbubbles was found to be 6 ± 4% of the total microbubble surface for microbubbles with DSPC as the main lipid (n=22) and 11 ± 4% for microbubbles with DPPC as the main lipid (n=24). The difference can be explained by the heterogeneous distribution of the ligand for DSPC microbubbles whereas the ligand is homogeneously distributed for DPPC microbubbles. These findings can be used to improve the binding of targeted microbubbles and for the ongoing research to distinguish bound from unbound microbubbles.