Understanding the mechanisms of peptide-induced membrane disorder is critical to the design of novel antimicrobial and cell-penetrating peptides. One means of quantifying local structure and order/disorder is through the orientational order parameter, typically obtained using various spectroscopic approaches. We report here on the use of an image-based means of tracking the order parameter in supported lipid bilayers during peptide-induced disordering. By coupling polarized total internal reflection fluorescence microscopy with in situ atomic force microscopy, it is now possible to track changes in order parameter associated with peptide binding and insertion, as well as lipid headgroup and acyl chain reordering, while simultaneously resolving molecular-scale topographical changes. Interactions between the model antimicrobial peptide, indolicidin, and its fluorescent analog, TAMRA-indolicidin, with model eukaryotic (DOPC: DSPC: cholesterol) and prokaryotic (DOPE/DOPG) membranes were tracked using the fluorescent lipid reporters, DiI-C-20 and BODIPY-PC. Changes in the order parameter upon membrane binding and insertion provided insights into the orientation of the peptide and the role of membrane chemistry and composition on insertion dynamics and membrane restructuring. (C) 2009 Elsevier Inc. All rights reserved.