Fig. 5: A multi-layer thin-film interference model fit for reflectance spectra of Junonia orithya male blue scale.

A TEM image of a blue scale of J. orithya male. The sample has not been stained with uranyl acetate or lead citrate; only osmium tetroxide is used for the postfixation. The white rectangle indicates the region cropped for the magnified view. The lower lamina shows two brighter layers sandwiched between the darker layers. The scale bar is 200 nm. The same four-layer contrast was observed in 12 images collected from 3 J. orithya male scales from 1 individual; layer thicknesses were quantified from the image shown here. B Experimentally measured reflectance spectrum of a J. orithya male scale (the upper lamina physically removed) placed on top of a glass slide, shown in light blue; solid curve represents the mean spectrum (shaded band = ± 1 SD) of six measurements from n = 2 independent scales. This spectrum is superimposed with four simulated spectra: a single 190 nm-thick layer of chitin placed on a glass slide using chitin’s refractive index from unpigmented glassy scales; a single 190 nm-thick layer of melanin on glass using melanin’s refractive index determined from bird barbules; a four-layer 190 nm-thick film on glass where the refractive indices in each layer are allowed to vary, and taking layer thicknesses from (A); a single 190 nm-thick layer thin film whose refractive indices are allowed to vary. The models account for a thin air gap with variable thickness; however, fitting results suggest its contribution is negligible. Source data for this panel are provided in Supplementary Data 1. C The wavelength-dependent refractive indices (Cauchy coefficients) of melanin, chitin, and the corresponding best-fit indices of the four layers of the lower lamina in the four-layer model in (B). D Schematic of four-layer model: four-layered lower lamina (L₁, L₂, L_3, and L₄) on top of a glass slide with a thin layer of air (L_air) between the lower lamina and the glass. E, F Epi-illumination light microscopy images of the abwing side (E) and the adwing side (F) of the same J. orithya male blue scale imaged with PXCT. The scale bar in panel E represents 20 μm and applies to panels (E–G). G A reflectance map was simulated using the four-layer model and an estimated tilt map of the lower lamina from the PXCT reconstruction. The local tilt affects the color and intensity response that correlates well with the adwing light microscopy image (E).