Understanding the spatial organization of nano-and micro-sized particle is very important in the fabrication of complex structures having unprecedented properties. Study on self-assembly of submicroscopic colloidal particles at high ionic strength solution at single particle resolution can provide new insight into the nanoscale interactions. In this study, we studied the self assembly behavior of negatively charged 0.2 and 1 μm colloidal particles at high ionic strength on glass-solution interface that is, in situ environment. The self-assembled 0.2 μm particles could not be resolved with conventional confocal and epi-fluorescent microscopy, so a home-built continuous wave stimulated emission depletion (STED) microscope was used for the study. We found that particles self-assemble into ordered and disordered structures at higher and lower ionic strength solution, respectively. The optical imaging methods allowed us to measure inter-particle gap at second energy minimum directly. Interestingly, we found that the inter-particle gap in the wet self-assembly higher than the classical Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory predicted. The in situ investigation of particle self-assembly at high ionic strength will provide more insight for the understanding nanoscale interactions.