DOI: 10.5061/dryad.w0vt4b912
title: Erora opisena optical and tomography data and associated MATLAB scripts

description: 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.
description: 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.
description: # *Erora opisena* optical and tomography data and associated MATLAB
 scripts
 [https://doi.org/10.5061/dryad.w0vt4b912](https://doi.org/10.5061/dryad.w0vt4b912) This data set contains reflectance data of single-wing scales measured using hyperspectral microscopy and microspectrophotometry as well as two tomography data sets of a single-wing scale. ## Description of the data and file structure Reflectance data measured using hyperspectral microscopy presented in Figure 2 is calculated using the dark data, the sample data, and the reference data which are all included in MATLAB files each containing one variable that is a three-dimensional matrix where each page is an image taken under a specific wavelength of light. Dark data is contained within the following files: dark_10x_HSM.mat dark_20x_1_HSM.mat dark_50x_1_HSM.mat Sample data is contained within the following files: scales_10x_HSM.mat scales_20x_1_HSM.mat scales_50x_1_HSM.mat Reference data is contained within the following files: mirror_10x_HSM.mat mirror_20x_1_HSM.mat mirror_50x_1_HSM.mat  Microspectrophotometry data presented in Figure 2 is contained within the files:  opisena1_sample1_10x_MSP.mat opisena1_sample1_20x_MSP.mat opisena1_sample1_50x_MSP.mat Reflectance data presented in Figure 3 is calculated using the dark data (dark_50x_SEM.mat), the sample data (scales_50x_SEM.mat), and the reference data (mirror_50x_SEM.mat). Each file contains a single variable that is a three-dimensional matrix where each page is an image taken under a specific wavelength of light. Optical model data is contained within the file named area_thickness_models.mat. This file contains a MATLAB structure containing all the data presented in the grey shading of Figure 3G. Reflectance data presented in Figure 4 was measured under differing immersion conditions. All conditions used the same reference and dark measurements contained within the files: mirror_standard.mat (reference), and blackfoil_oil.mat (dark). For scales measured in air, the sample data is within the file air.mat. Sample data for the following immersion conditions are contained within the file names in brackets: 1.3 (13_immersion.mat), 1.4 (14_immersion.mat), 1.45 (145_immersion.mat), and 1.5 (15_immersion.mat).  X-ray tomography data from a single scale has been stitched together using the data contained within the files:  ZEISS_810Ultra_Erlangen_Butterfly4_LFOV_top_Reconstruction.tif ZEISS_810Ultra_Erlangen_Butterfly4_LFOV_bottom_Reconstruction.tif  Visualizations of this tomography data were previously published in science.org/doi/10.1126/sciadv.1603119. ## Code/Software The MATLAB code included here is a custom-made application that controls the liquid crystal filter and monochromatic camera via a serial interface. The software files include kurios.m, hyperspectral_image_acquisition.mlapp, and calibration_app.mlapp. The two .mlapp files are MATLAB app files that can be opened in MATLAB and generate a user interface that allows the user to control different parameters of the Kurios liquid crystal filter. The kurios.m file defines the serial and communication ports for the Kurios liquid crystal filter.