We introduce the use of second-harmonic generation microscopy to investigate individual persistent luminescent microparticles that are either embedded in glass or as prepared. Three-dimensional mapping of the second-harmonic generation from monoclinic dysprosium- and europium-doped strontium aluminates, a popular persistent luminescent material, allows us to unambiguously visualize and reveal for the first time the presence of micrometer-sized structured domains from such microparticles. The technique was found to have high potential for studying noninvasively a wide range of individual persistent luminescent entities that are embedded in a variety of glass matrices.