Nanofabricated and nanopatterned surfaces have revealed the sensitivity of cell adhesion to nanoscale variations in the spacing of adhesive ligands such as the tripeptide arginine-glycine-aspartic acid (RGD). To date， surface characterisation and cell adhesion are often examined in two separate experiments so that the localisation of ligands and adhesion proteins cannot be combined in the same image. Here we developed self-assembled monolayer chemistry for indium tin oxide (ITO) surfaces for single molecule localisation microscopy (SMLM). Cell adhesion and spreading were sensitive to average RGD spacing. At low average RGD spacing， a threshold exists of 0.8 RGD peptides per µm that tether cells to the substratum but this does not enable formation of focal adhesions. These findings suggest that cells can sense and engage single adhesive ligands but ligand clustering is required for cell spreading. Thus， our data reveal subtle differences in adhesion biology that may be obscured in ensemble measurements.