Far field scattering pattern of differently structured butterfly scales
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- Giraldo, M.A., Yoshioka, S. & Stavenga, D.G. J Comp Physiol A (2008) 194: 201. doi:10.1007/s00359-007-0297-8
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The angular and spectral reflectance of single scales of five different butterfly species was measured and related to the scale anatomy. The scales of the pierids Pieris rapae and Delias nigrina scatter white light randomly, in close agreement with Lambert’s cosine law, which can be well understood from the randomly organized beads on the scale crossribs. The reflectance of the iridescent blue scales of Morpho aega is determined by multilayer structures in the scale ridges, causing diffraction in approximately a plane. The purple scales in the dorsal wing tips of the male Colotis regina act similarly as the Morpho scale in the blue, due to multilayers in the ridges, but the scattering in the red occurs as in the Pieris scale, because the scales contain beads with pigment that does not absorb in the red wavelength range. The green–yellow scales of Urania fulgens backscatter light in a narrow spatial angle, because of a multilayer structure in the scale body.
KeywordsSingle butterfly scale Structural colour Lambertian reflector Scattering Angular reflectance
Many butterflies have vivid, colourful wing patterns, created by rows of partly overlapping scales with intricate spatial structures that scatter incident light. The scattered light can interfere coherently or incoherently. When the scale structures have spatial periodicity, the interference of coherent light waves often results in striking iridescences. The displayed colours are then called structural or physical. Without periodicity, light scattering is incoherent or random, resulting in white scales, unless they contain pigment that selectively absorbs in a certain wavelength range. In the latter case a pigmentary or chemical colour results (Fox and Vevers 1960; Vukusic and Sambles 2003; Kinoshita and Yoshioka 2005). Many butterfly species feature structural as well as pigmentary colours (Rutowski et al. 2005; Yoshioka and Kinoshita 2006a).
Each butterfly wing scale is the cuticular product of a single cell, with a rather flat, unstructured lower scale lamina and a highly structured upper lamina, typically consisting of longitudinal ridges connected by crossribs (Ghiradella 1998). The fine structure of butterfly scales is highly variable (Vukusic et al. 2000). For instance, the crossribs of the white scales of pierids are adorned with granules (Yagi 1954; Giraldo and Stavenga 2007; Morehouse et al. 2007), the ridges of Morpho scales and many male Pieridae are elaborated into multilayer structures (Ghiradella et al. 1972; Vukusic et al. 1999; Kinoshita et al. 2002), and many papilionids and lycaenids have scales with photonic crystal properties (Vukusic et al. 2002; Vukusic and Sambles 2003; Kertesz et al. 2006).
The scales reflect only part of the incident light flux, and the remaining part is transmitted unless it is absorbed by pigment. Because the scales are arranged in layers on both sides of the wing, incident light suffers reflection and transmission in each layer of the scale stacks, and therefore the wing reflectance is not solely due to backscattering by the top layer, the cover scales, but it is the cumulative effect of the scale stacks on both wing sides. Yoshioka and Kinoshita (2006b) investigated this phenomenon in Morpho wings, and to explain the reflectance spectra of intact wings from the spectra of individual scales they used a simplified scale stack model, assuming that normally incident light rays are also reflected and transmitted normally. Stavenga et al. (2006) developed a more general model for the reflectance of butterfly wings to interpret reflectance measurements performed on intact as well as partly or completely denuded wings of the small white butterfly, Pieris rapae. The basic assumption of the latter model, that the scales scatter randomly, was however not specifically demonstrated.
Knowledge of the spatial and spectral reflectance characteristics of single scales is essential to further develop our understanding of the coloration principles applied by butterflies. In the present study we investigate the scattering properties of wing scales of a variety of butterflies, and we correlate the scattering diagrams with the electron micrographs of the scale structure. We show that white scales of pierid butterflies are approximately random scatterers. Iridescent scales, with multilayer structures, exhibit directional reflection. Whereas some butterfly species appear to have scales with dominant iridescence, other species combine iridescence and scattering properties.
Materials and methods
We investigated the scales of a variety of butterflies. The small white, P. rapae, was obtained from a continuous culture maintained by Dr J. J. van Loon, Entomology Department, University of Wageningen (the Netherlands). The black jezebel, Delias nigrina, was captured near Bateman’s Bay, Australia. The Morpho aega was purchased. The purple tip, Colotis regina, was received from the butterfly collection of the Royal Museum for Central Africa, Brussels (Belgium; curator Dr U. Dall’Asta). The moth Urania fulgens was a gift from Dr Marta Wolff, Entomology group, University of Antioquia (Medellín, Colombia).
Angular distribution of scattering by single scales
The angular dependence of the light scattering by the scale was measured with a lightguide, which was mounted on a rotating motor and connected to a spectrometer (Yoshioka and Kinoshita 2006b). The scales were adjusted so that the scale plane was perpendicular to the light beam, as judged by the symmetrical reflection pattern with respect to the axial direction. The experiments were run using a Labview interface, which allowed the measurement of reflectance spectra in angular steps of 1° over a 180° angle (Fig. 1b). Angular reflectance curves were calculated for a series of wavelengths with 10 nm interval by sequentially averaging the reflectance spectra over 10 nm wavelength ranges. In addition, the spectral reflectances of single scales were measured with a microspectrophotometer, consisting of a xenon light source, a Leitz Ortholux microscope, and a fiber optic spectrometer. The microscope objective was an Olympus 20×, NA 0.46. A white reflectance standard (Spectralon, Labsphere, North Sutton, NH, USA) served as the reference in all cases.
Subsequent to the microspectrophotometry, the single scales were prepared for scanning electron microscopy (SEM). Additionally, pieces of wing were cut and put on the SEM-holder in different positions to visualize the upper surface as well as cross-section of the scales. Samples were sputtered with palladium for 5 min with 800 V and 200 mTorr (Hummer, Alexandria, VA, USA). A Philips XL-30 scanning electron microscope with a voltage of 3 kV was used to investigate the scale anatomy. For transmission electron microscopy, wing pieces were prepared as usual. In brief, samples were embedded in agar for better handling, prefixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, and postfixed in 1% OsO4/1.5% K4 Fe(CN)6 in 0.1 M cacodylate. Subsequent washing with double-distilled water and dehydration with an alcohol series that ended with 100%, were followed by propylene oxide during 30 min and embedding in Epon. Post-microtomed samples were contrasted with uranyl acetate in methanol for 2 min and lead in water for 1 min, and were then examined with a Philips 201 transmission electron microscope.
The white scales of the small white, P. rapae, are marked by ovoid beads that adorn the crossribs (Fig. 2a). The angular light scattering of a single scale can be readily visualized with the setup of Fig. 1a, where a beam of white light is focused on a scale via a small hole in a screen. Figure 2b is a photograph of the screen, showing the angular distribution of the light reflected by a white scale taken from the dorsal wing of a male small white, P. rapae. The scale scatters light approximately circular-symmetrically, suggesting that the scale acts as a diffuse scatterer.
The angular distribution of the scattering was quantitatively investigated with the setup of Fig. 1b, where the light reflected by the scale is measured as a function of angle in the horizontal plane. Figure 2c presents the reflectance of the scale of Fig. 2b as a function of angle for a number of wavelengths, normalized to the maximal reflectance value; the angle is 0° for the normal to the scale. The scale’s reflectance spectrum for normally incident light is given in Fig. 2d. The reflectance is high in the visible wavelength range, but it is low in the ultraviolet, because of an ultraviolet-absorbing pigment, presumably leucopterin (Wijnen et al. 2007), which is concentrated in pigment granules, the ovoid beads (Fig. 2a). The beads act as strong scatterers at wavelengths where the pigment absorption is negligible (Stavenga et al. 2004; Giraldo and Stavenga 2007; Morehouse et al. 2007). In addition to the beads, the other structures of the scale, that is the ridges and crossribs of the upper lamina of the scale as well as the lower lamina, contribute to the scattering (Fig. 2c).
Figure 2e–h presents a similar set of data for a white scale of the dorsal forewing of a male black jezebel, D. nigrina. This case is obviously very similar to that of the white P. rapae scale. The scale anatomy with crossribs studded with beads is very similar (Fig. 2e), the scattering is again approximately random (Fig. 2f, g), and the reflectance spectrum is also high in the visible and low in the UV (Fig. 2h).
The strikingly blue M. aega has scales where the lamellae of the ridges form multilayers (Fig. 2i). Different from Morpho didius, for example, which has glass cover scales and strongly pigmented ground scales (Vukusic et al. 1999), cover and ground scales of M. aega cannot be distinguished. The scattering diagram of a scale of a M. aega is a horizontal stripe (Fig. 2j), perpendicular to the vertically oriented scale ridges. The angular dependence of the reflectance varies strongly with the wavelength (Fig. 2k), and the reflectance measured with normally incident light features a distinct peak in the blue (Fig. 2l), which is due to the multilayered structure of the scale ridges (Fig. 2i; see also Vukusic et al. 1999; Kinoshita et al. 2002; Yoshioka and Kinoshita 2004). A reflectance peak value of more than two results, because the scale’s scattering is highly directional and the reflectance was measured relative to the diffusely scattering white standard (Fig. 2l).
The purple scales at the dorsal wing tips of the male purple tip, C. regina, have ridges that are fine-structured similarly as in the case of M. aega (Fig. 2m). The purple scale features a scattering diagram with a blue stripe and a red diffuse pattern (Fig. 2n), which is a mixture of the stripe phenomenon of Fig. 2j and the diffuse patterns of Fig. 2b, f. The blue stripe is due to light backscattered by the fine-structured ridges, and the red diffuse pattern results from randomly scattered light, filtered by a pigment contained in granules below the multilayered ridges (Fig. 2m). The blue peaking reflectance spectrum, shown in Fig. 2p, is mainly due to reflection by the ridges, and the red band, above 600 nm, is mainly caused by the light scattering pigment granules (Fig. 2p).
A green–yellow reflecting scale of the moth U. fulgens has between the upper and lower scale laminae (Fig. 2q) an elaborate multilayer system, which yields a spatially restricted, directional scattering diagram (Fig. 2r, s). The high amplitude of three of its reflectance spectrum is again due to the directionality of the scale reflectance (Fig. 2t). The reflectance spectrum features a distinct band, peaking at 590 nm, with halfwidth about 120 nm, indicating that an interference reflector is involved.
The relationship between the optical properties of butterfly scales and their structure has been the topic of several studies (e.g., Vukusic et al. 1999, 2002; Kinoshita et al. 2002). Most of the previous investigations have focused on iridescent scales. Here, we have compared the reflection pattern of five differently structured single butterfly scales that scatter light coherently or incoherently, or both.
We started with a simple white scale common to many species of the pierid subfamily Pierinae. Due to the characteristic beaded structure light scattering is strong, thus causing the intense white colour. We find that the white pierid scales approximate the properties of a Lambertian diffuser, at least in the wavelength range where pigment absorption is negligible (Fig. 3).
The ridges of the scales of Morpho butterflies cause a blue colour. Melanin pigment below the multilayered ridges absorbs stray light over the whole visible wavelength range, including the ultraviolet, thus supporting the strikingly blue wing colour (Yoshioka and Kinoshita 2006a). The pigment of the scales in the dorsal wing tips of C. regina also absorbs stray light, but not in the long-wavelength range. The remaining red light together with the blue iridescence causes the purple colour.
The scattering diagram of U. fulgens is not perfectly directional (Figs. 2r, s), which should have been the case when the scales consisted of an ideal multilayer. Transmission electron microscopy shows that between the multilayers exist pillars (Fig. 4b), which presumably cause the spread in the scattering diagram. Of course, the scales—and therefore the multilayers—are also not perfectly flat. The ridges (see Figs. 2q, 4b) will further contribute to some diffuse scattering.
Prof. J. T. M. de Hosson and G. ten Brink (Materials Science Department, University of Groningen) provided essential facilities for scanning electron microscopy, and Dr. H. van der Want together with D. Kalicharan and H. Blaauw (Electron Microscopy Department, Cellular Biology, University of Groningen) provided the support for the transmission electron microscopy. We thank Dr. H. Ghiradella for reading the manuscript and for valuable suggestions. A grant from the University of Osaka, Japan, enabled the research stay of M. A. G. in the Graduate School of Frontier Biosciences in Osaka. A visit to the Royal Museum for Central Africa, Brussels, was supported by the EU via SYNTHESYS. Further financial support was given by the EOARD (Grant 063027).
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