We report the use of total internal reflection fluorescence microscopy (TIRFM) to study cell adhesion on patterned self-assembled monolayers (SAMs) on gold surfaces. Microcontact printing was used to pattern hydrophobic features to which the extracellular protein fibronectin was adsorbed, while dip-pen nanolithography was used to produce electroactive nanoarrays of hydroquinone-terminated alkanethiol on gold-coated quartz substrates. The hydroquinone was electrochemically oxidized to the corresponding quinone, and an oxyamine-tethered linear Arg-Gly-Asp (RGD) peptide was chemoselectively immobilized. A prism-based method of TIRFM was used to examine adhered cells on both the microscale and nanoscale features. We also demonstrate that, following imaging with TIRFM, the substrates can be visualized using conventional fluorescence microscopy.