In this study we describe the first direct, real-space characterization of a novel type of poly(vinyl alcohol) (PVA) based microballoons in aqueous environment using scanning transmission X-ray microscopy (STXM). From the oxygen K-edge near-edge X-ray absorption fine structure (NEXAFS) spectra taken from the microballoon interiors we could unambiguously distinguish between water- and air-filled particles. We also demonstrate that STXM imaging below and above the O K-edge (520 eV and 550 eV) can provide unique information on the composition of microballoons in water. It was found that the particular microballoon system investigated here has a remarkable high stability and is able to contain gases for more than 6 months, making it well suited for biomedical applications.