The surface properties of hydroxyapatite, including electric charge, can influence the biological response, tissue compatibility, and adhesion of biological cells and biomolecules. Results reported here help in understanding this influence by creating charged domains on hydroxyapatite thin films deposited on silicon using electron beam irradiation and investigating their shape, properties, and carbon contamination for different doses of incident injected charge by two methods. Photoluminescence laser scanning microscopy was used to image electrostatic charge trapped at pre-existing and irradiation-induced defects within these domains, while phase imaging in atomic force microscopy was used to image the carbon contamination. Scanning Auger electron spectroscopy and Kelvin probe force microscopy were used as a reference for the atomic force microscopy phase contrast and photoluminescence laser scanning microscopy measurements. Our experiment shows that by combining the two imaging techniques the effects of trapped charge and carbon contamination can be separated. Such separation yields new possibilities for advancing the current understanding of how surface charge influences mediation of cellular and protein interactions in biomaterials.