In this report, helium ion microscopy (HIM) is used to study the micro and nanostructures responsible for structural color in the wings of two species of Lepidotera from the Papilionidae family: Papilio ulysses (Blue Mountain Butterfly) and Parides sesostris (Emerald-patched Cattleheart). Electronic charging of uncoated scales from the wings of these butterflies, due to the incident ion beam, is successfully neutralized, leading to images displaying a large depth-of-field and a high level of surface detail, which would normally be obscured by traditional coating methods used for scanning electron microscopy (SEM). The images are compared with those from variable pressure SEM, demonstrating the superiority of HIM at high magnifications. In addition, the large depth-of-field capabilities of HIM are exploited through the creation of stereo pairs that allows the exploration of the third dimension. Furthermore, the extraction of quantitative height information which matches well with cross-sectional transmission electron microscopy measurements from the literature is demonstrated. SCANNING 33: 107–120, 2012. © 2011 Wiley Periodicals, Inc.