Synchrotron‐source micro‐x‐ray computed tomography for examining butterfly eyes

• Published in: Ecology and evolution Vol. 14; no. 4; pp. e11137 - n/a
• Main Authors: Paukner, Dawn, Wildenberg, Gregg A., Badalamente, Griffin S., Littlewood, Peter B., Kronforst, Marcus R., Palmer, Stephanie E., Kasthuri, Narayanan
• Format: Journal Article
• Language: English
• Published: England John Wiley & Sons, Inc 01.04.2024; John Wiley and Sons Inc; Wiley
• Subjects: Acuity; Adaptation; Algorithms; Anatomy; Butterflies & moths; butterfly; Cell membranes; Compound eye; Computed tomography; Cones; Datasets; Ecological adaptation; Electron microscopy; Entomology; Evolution; Eye; Image contrast; Insects; interommatidial angle; Labels; Machine learning; Microscopy; micro‐CT; Morphology; Ommatidia; Open source software; Osmium; Osmium tetroxide; Potassium; Resolution; Species; synchrotron; Tomography; Visual system; X-rays; United States > US; Chicago Illinois; compound eye; ommatidia; interommatidial angle; micro‐CT; synchrotron; butterfly
• ISSN: 2045-7758; 2045-7758
• DOI: 10.1002/ece3.11137

Comparative anatomy is an important tool for investigating evolutionary relationships among species, but the lack of scalable imaging tools and stains for rapidly mapping the microscale anatomies of related species poses a major impediment to using comparative anatomy approaches for identifying evolutionary adaptations. We describe a method using synchrotron source micro‐x‐ray computed tomography (syn‐μXCT) combined with machine learning algorithms for high‐throughput imaging of Lepidoptera (i.e., butterfly and moth) eyes. Our pipeline allows for imaging at rates of ~15 min/mm3 at 600 nm3 resolution. Image contrast is generated using standard electron microscopy labeling approaches (e.g., osmium tetroxide) that unbiasedly labels all cellular membranes in a species‐independent manner thus removing any barrier to imaging any species of interest. To demonstrate the power of the method, we analyzed the 3D morphologies of butterfly crystalline cones, a part of the visual system associated with acuity and sensitivity and found significant variation within six butterfly individuals. Despite this variation, a classic measure of optimization, the ratio of interommatidial angle to resolving power of ommatidia, largely agrees with early work on eye geometry across species. We show that this method can successfully be used to determine compound eye organization and crystalline cone morphology. Our novel pipeline provides for fast, scalable visualization and analysis of eye anatomies that can be applied to any arthropod species, enabling new questions about evolutionary adaptations of compound eyes and beyond. We describe a novel method for measuring the organization of compound eyes using synchrotron source micro‐CT. Using a semi‐automated analysis pipeline, we measure ommatidial diameter, interommatidial angle, and crystalline cone morphology and show how these features could be compared to theoretical optima to better understand compound eye optics.