Characterization of a gold electrode modified with a mixed self-assembled monolayer composed of ssDNA and mercaptohexanol is described using in-situ fluorescence microscopy with particular focus on the study on the homogeneity of the surface in terms of its response to potential perturbation. A change in DNA orientation is driven by the charge on the gold surface, and the extent and the rate of orientation change can be determined using fluorophore modified DNA and fluorescence. Characterization of the surface is typically performed using techniques that determine the average characteristics of the surface. We show that significant differences in the response of various regions of the electrode surface can be observed. These differences can characterized by potential step-imaging measurements or by frequency response analysis of the fluorescence - potential transfer function. Important for high frequency measurements in these low ionic strength electrolytes is the use of a fourth electrode and shunt capacitor which was found to influence the electrochemical impedance results as well as the optical results. The magnitude of the transfer function was found to vary significantly for different regions on the electrode. We demonstrate this for two 95 mu m regions that are on either side of a grain boundary. The phase of the transfer function was found to be more sensitive to variations in the rate of reorientation between these two regions compared to considering only the magnitude. The variation in the response over the electrode surface is an important consideration for the DNA-based biosensing devices based on modified electrode interfaces. (C) 2014 Elsevier Ltd. All rights reserved.