Fluorescent silica nanoparticles are widely used for various applications from mechanical reinforcement to biology. In many cases, their surface has to be tailored. Herein fluorescent silica nanoparticles are synthesized by a reverse micro-emulsion process and functionalized by silane coupling agents owning amino and thiol groups. The functionalization is then characterized by macroscopic well-known methods (zeta potential, hydrophilic to hydrophobic ratio, etc.) and an original method based onto TEM observations of the contrast between the silica core and the metallic ions chelated by the functional groups grafted onto the surface is also introduced. This method reveals that the functionalization is effective and that it occurs by ''nano domains.'' It is therefore possible to characterize the functionalization by in situ observations. Finally, the characterized nanoparticles are incorporated into a PMMA thin film. The fluorescence of the nanoparticles allows the monitoring of the level of dispersion of the nanoparticles within the polymer and confirms all the other characterizations.