Brush border microvilli (MV) are highly specialized finger-like protrusions emanating from the apical surfaces of certain types of epithelial cells. In the kidney proximal tubule they provide an increase in the surface area for the absorption of nutrients. Renal tubular reabsorption of inorganic phosphate (Pi) occurs via the sodium gradient-dependent Pi (NaPi) cotransport proteins localized in the MV membrane. The localization and the regulation of the NaPi transporters on the MV are related to a set of specific protein-protein interactions. These include interaction with the PDZ proteins, a family of proteins generally acting as scaffolders of subcellular structures, and mediating the interaction with the actin-based MV cytoskeleton.Here we use the Modulation Tracking (MT) optical method to image small protrusions in live cells and apply it to the study of MV in live cultured proximal tubule cells. MV are several microns long with a lateral size of the order of 100nm, and are mobile. We image them by using the MT feedback-based scanning algorithm that, generating a light envelope around the MV, provides high-resolution 3D images of the moving MV in real time. We observe the distribution and dynamics of different proteins within the MV and discuss the implications for their functionality.