Abstract: The chemical composition and method of the invention enable characterization of microscopic defects in membranes such as pinholes, cracks or fissures. The present invention, however, can be used to characterize defects on different types of porous and non-porous membranes used for diverse applications in various industries. It uses brightly fluorescing silica or silsesquioxane spheres prepared with pre-determined definitive and uniform sizes (15 nm-50 microns). The spheres' uniform, controlled size allows them to be used to characterize defects or holes in membranes based on a size exclusion mechanism. The spheres used are engineered to glow brightly when exposed to ultraviolet light in order to allow visual or highly sensitive fluorescence spectroscopy or microscopy to characterize the passage of the particles through defects or holes in a membrane and even identify where the defect is located.