Peptide Nucleic Acid (PNA) is an important alternative nucleic acid, which exhibits more effective DNA/RNA detection capabilities compared to DNA. Its potential utility in nucleic acid based detection technologies warrants detail understanding of it's self-assembly behavior on solid substrates, e.g., gold. In the present study, we have applied high-resolution Atomic Force Microscopy (AFM) for obtaining direct visual information on PNA adsorption and formation of a self-assembled monolayer of PNA on gold(111) surface. We show from the molecularly resolved AFM data that PNA molecules form a well-defined 1-dimensional molecule-by-molecule ordering, over a considerable length scale (few hundred nm), as well as 2-dimensional ordering over a wide area of about 10 mu m x 10 mu m, due to parallel positioning of the 1-dimensional ordered arrangements. The way the parameters like PNA concentration, incubation time, incubation temperature and PNA deposition methods can affect the formation of such ordered self-assembled PNA structures has been investigated. Some of the primary observations are that the minimum PNA concentration and incubation time for large scale (10 mu m x 10 mu m) 2-dimensional order formation are 0.51 mu M and 4 h, respectively. Furthermore, a dense and well-ordered layer over a large area could be better formed in case of immersion method compared to the droplet contact and droplet deposition methods. From the Reflection Absorption Infra Red Spectroscopy (RAIRS) data, indications for PNA concentration-driven reorientation of the PNA backbone towards more upright configuration on gold(111) surface, were obtained. (C) 2011 Elsevier Inc. All rights reserved.