Biophotonic nanostructures in butterfly wing scales remain fascinating examples of biological functional materials, with intriguing open questions regarding formation and evolutionary function. One particularly interesting butterfly species, Erora opisena (Lycaenidae: Theclinae), develops wing scales that contain three-dimensional photonic crystals that closely resemble a single gyroid geometry. Unlike most other gyroid-forming butterflies, E. opisena 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 organisation 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 in vivo analysis of nanostructure formation in developing butterflies. Methods These data sets were collected using hyperspectral microscopy, microspectrophotometry and x-ray tomography. For hyperspectral microscopy, illumination was provided by a Zeiss Axioscope 5 microscope collimated white LED source or a halogen light source (OSL2, Thorlabs) that was passed through a collimating lens (OSL2COL, Thorlabs) and a shortpass filter (FESH0750, Thorlabs). The collimated light was passed through a tunable bandpass filter (Kurios, Thorlabs Inc.), and a quartz depolariser to counteract any polarisation effect of the liquid crystal filter (DPU-25, Thorlabs; a schematic diagram is shown in Figure 1A). The tunable bandpass filter was controlled over the serial interface by custom software written in MATLAB 2023a (The MathWorks Inc., USA). The bandwidth was set to medium, delivering a restricted spectrum of light with a full width at half-maximum (FWHM) of approximately 18 nm (Figure 1B). Samples were illuminated with wavelengths between 430–720 nm in 10 nm steps. For each illumination wavelength, an image was captured with a 20 MP monochromatic camera with pixel size 2.4 μm (BFS-U3-200S6M-C, Teledyne FLIR) mounted to the camera port of the microscope. Because the illumination intensities were not equal across all wavelengths (Figure 1B), a calibration run was initially conducted on a reference aluminium mirror (PF10-03-F01, Thorlabs) and the exposure times and gain values of the camera were adjusted such that the average pixel intensity of each image was approximately equal. This ensured that pixels were neither overexposed nor under-exposed for any one wavelength. Subsequently, the sample was measured using the calibrated exposure and gain values and the reflectance at each wavelength was calculated by averaging the pixels in the area of interest, subtracting the dark background from the sample data, and then dividing the sample data by the reference mirror to obtain relative reflectance.

For microspectrophotometry, illumination was provided by a halogen light source (OSL2, Thorlabs Inc.) passed through a collimating lens (COP4-A, Thorlabs). Relative reflectance spectra were collected via a microscope side port below the tube lens, with the light path consisting of a mirror, a focusing quartz lens and a 200 μm quartz fibre (FC-UVIR200, Avantes) attached to a spectrometer (AvaSpec-ULS2048XL-EVO, Avantes). The measurement spot diameter of 4, 10, or 20 μm using the 50×, 20× and 10× objectives, respectively, was set to the centre of the image using a mirror in the object plane. An aluminium mirror (PF10-03-F01, Thorlabs) served as the reference.

Details of the x-ray tomography data collection can be found in Wilts et al. (2017). Briefly, an isolated wing scale was imaged using a Zeiss Xradia 810 Ultra X-ray microscope producing two tomography datasets with voxel sizes of 150 nm, one dataset of the base of the wing scale and a second of the tip of the wing scale.