Laser scanning confocal microscopy, advantageous as a non-destructive spatial imaging technique, has been used to probe the internal photonic crystal lattice structure of micron scale core–fluorescein-labeled shell–corona silica microspheres that had been crystallized within anisotropically etched relief patterns in the 100 surface of silicon wafers. Using this method it was possible to determine the three-dimensional positioning of individual microspheres that had been allowed to self-assemble within the confines of square and rectangular shaped pyramidal microwells with sub-micron lateral and vertical spatial resolution. This methodology confirmed that microspheres underwent vectorial crystal growth in the form of a face centered cubic lattice inside the pyramidal microwells in which the 100 face of the photonic crystal was oriented parallel to the 100 surface of the silicon wafer. Furthermore it was feasible to visualize both external and internal defects buried within photonic crystal lattices in the planarized opal-patterned chips.