High resolution tracking of individual cell surface proteins is limited by several factors, including probe size that can impair protein trafficking and restrict their access to crowded cellular environments, ligand multivalency that induces protein cross-linking, and potential loss of function of recombinant proteins fused to large tags. To overcome these issues, we developed a labeling method that uses monomeric streptavidin (mStrav) conjugated to highly photo-robust organic Atto dyes. mStrav is a 3-nm-molecule, stable and easy to produce, that binds biotinylated proteins with high affinity (Lim et al., Biochemistry 2011). Atto-conjugated mStrav is then used as a probe to label cells co-transfected with a membrane molecule of interest carrying a 15 amino acid extra-cellular acceptor peptide (AP), and the biotin ligase BirA that covalently adds biotin to the tag during protein maturation. We used this technique to study membrane diffusion and nanoscale organization of the synaptic adhesion molecules neurexin-1β and neuroligin-1 in hippocampal neurons and β3-integrins in fibroblasts. We show that the monovalency of mStrav prevents protein cross-linking, allowing more accurate diffusion measurements. The small sizes of the AP tag (