Elucidating nanostructural organization and photonic properties of butterfly wing scales using hyperspectral microscopy

Biophotonic nanostructures in butterfly wing scales remain fascinating examples of biological functional materials, with intriguing open questions with regard to formation and evolutionary function. One particularly interesting butterfly species, <i>Erora opisena</i> (Lycaenidae: Theclinae), develops wing scales that contain three-dimensional photonic crystals that closely resemble a single gyroid geometry. Unlike most other gyroid-forming butterflies, <i>E. opisena</i> develops discrete gyroid crystallites with a pronounced size gradient hinting at a developmental sequence frozen in time. Here, we present a novel application of a hyperspectral (wavelength-resolved) microscopy technique to investigate the ultrastructural organization of these gyroid crystallites in dry, adult wing scales. We show that reflectance corresponds to crystallite size, where larger crystallites reflect green wavelengths more intensely; this relationship could be used to infer size from the optical signal. We further successfully resolve the red-shifted reflectance signal from wing scales immersed in refractive index liquids with varying refractive index, including values similar to water or cytosol. Such photonic crystals with lower refractive index contrast may be similar to the hypothesized nanostructural forms in the developing butterfly scales. The ability to resolve these fainter signals hints at the potential of this facile light microscopy method for <i>in vivo</i> analysis of nanostructure formation in developing butterflies.