Polarized iridescence of the tropical carpenter bee, Xylocopa latipes.

The tropical carpenter bee, Xylocopa latipes, has metallic-reflecting, iridescent wings. The wing reflectance spectra for TE- and TM-polarized light depend on the angle of light incidence in a way characteristic for dielectric multilayers. Anatomy indicates the presence of melanin multilayers in the wing's chitinous matrix. A simple optical model of melanin multilayers explains the angle dependence of the wing reflectance spectra. The wing reflections that occur upon oblique illumination exhibit colourful and strongly polarized light patterns, which may mediate intraspecific signaling and mutual recognition by conspecifics.

Coloration principles of the Great purple emperor butterfly (Sasakia charonda).

The dorsal wings of male Sasakia charonda butterflies display a striking blue iridescent coloration, which is accentuated by white, orange-yellow and red spots, as well as by brown margins. The ventral wings also have a variegated, but more subdued, pattern. We investigated the optical basis of the various colors of intact wings as well as isolated wing scales by applying light and electron microscopy, imaging scatterometry and (micro)spectrophotometry. The prominent blue iridescence is due to scales with tightly packed, multilayered ridges that contain melanin pigment. The scales in the brown wing margins also contain melanin. Pigments extracted from the orange-yellow and red spots indicate the presence of 3-OH-kynurenine and ommochrome pigment. The scales in the white spots also have multilayered ridges but lack pigment. The lower lamina of the scales plays a so-far undervalued but often crucial role. Its thin-film properties color the majority of the ventral wing scales, which are unpigmented and have large windows. The lower lamina acting as a thin-film reflector generally contributes to the reflectance of the various scale types.

Polymorphism of Colias croceus from the Azores caused by differential pterin expression in the wing scales.

The pierid butterfly Colias croceus (Geoffroy in Fourcroy, 1785), established in the Azores archipelago, is polymorphic with six forms, C. croceus f. croceus ♂ and ♀, C. c. f. cremonae ♂ and ♀, C. c. f. helice ♀, and C. c. f. cremonaehelice ♀. We investigated the optical mechanisms underlying the wing colouration of the butterflies by performing spectrophotometry and imaging scatterometry of the variously coloured wing areas and scales. The scale colouration is primarily due to wavelength-selective absorption of incident light by pterins expressed in granular beads in the wing scales, but thin film reflections of the scales' lower lamina and scale stacking also contribute. Three forms (croceus ♂ and ♀ and helice ♀) are consistent with the patterns of the well-known 'alba' polymorphism. We postulate the coexistence of a second polymorphism, 'cremonae', to understand the three other forms (cremonae ♂ and ♀, and cremonaehelice ♀), which are characterized by the absence of red pigment, presumably due to the differential blocking of erythropterin expression.

Coloration of Flowers by Flavonoids and Consequences of pH Dependent Absorption.

Flavonoid pigments are key determinants of flower colors. As absorption spectra of flavonoids are known to be severely pH-dependent, cellular pH will play a crucial role in flower coloration. The flavonoids are concentrated in the vacuoles of the flowers' epidermal cells, and thus the pigments' absorption spectra are modulated by the vacuolar pH. Here we study the pH dependence of flavonoid absorption spectra in extracts from flowers of two poppy species Papaver dubium (red) and Meconopsis cambrica (orange), and a white and red Mandevilla sanderi variety. In the red poppy and Mandevilla flowers, absorption spectra of the cyanidin- and pelargonidin-based anthocyanins peak in the blue-green-wavelength range at low pH, but exhibit a distinct bathochromic shift at higher pH. This shift to longer wavelengths is not found for the blue-absorbing nudicaulin derivatives of M. cambrica, which have a similar absorption spectrum at low and high pH. The pH-dependent absorption changes of the white M. sanderi's flavonoid remained restricted to the UV. An analysis of the spectra with logistic functions suggests that the pH-dependent characteristics of the basic states of flavonols and anthocyanins are related. The implications of tuning of pH and pigment absorption spectra for studies on flower color evolution are discussed.

Classical lepidopteran wing scale colouration in the giant butterfly-moth Paysandisia archon.

The palm borer moth Paysandisia archon (Castniidae; giant butterfly-moths) has brown dorsal forewings and strikingly orange-coloured dorsal hindwings with white spots surrounded by black margins. Here, we have studied the structure and pigments of the wing scales in the various coloured wing areas, applying light and electron microscopy and (micro)spectrophotometry, and we analysed the spatial reflection properties with imaging scatterometry. The scales in the white spots are unpigmented, those in the black and brown wing areas contain various amounts of melanin, and the orange wing scales contain a blue-absorbing ommochrome pigment. In all scale types, the upper lamina acts as a diffuser and the lower lamina as a thin film interference reflector, with thickness of about 200 nm. Scale stacking plays an important role in creating the strong visual signals: the colour of the white eyespots is created by stacks of unpigmented blue scales, while the orange wing colour is strongly intensified by stacking the orange scales.

Magnificent magpie colours by feathers with layers of hollow melanosomes.

The blue secondary and purple-to-green tail feathers of magpies are structurally coloured owing to stacks of hollow, air-containing melanosomes embedded in the keratin matrix of the barbules. We investigated the spectral and spatial reflection characteristics of the feathers by applying (micro)spectrophotometry and imaging scatterometry. To interpret the spectral data, we performed optical modelling, applying the finite-difference time domain (FDTD) method as well as an effective media approach, treating the melanosome stacks as multi-layers with effective refractive indices dependent on the component media. The differently coloured magpie feathers are realised by adjusting the melanosome size, with the diameter of the melanosomes as well as their hollowness being the most sensitive parameters that influence the appearance of the feathers.

Iridescent flowers? Contribution of surface structures to optical signaling.

The color of natural objects depends on how they are structured and pigmented. In flowers, both the surface structure of the petals and the pigments they contain determine coloration. The aim of the present study was to assess the contribution of structural coloration, including iridescence, to overall floral coloration. We studied the reflection characteristics of flower petals of various plant species with an imaging scatterometer, which allows direct visualization of the angle dependence of the reflected light in the hemisphere above the petal. To separate the light reflected by the flower surface from the light backscattered by the components inside (e.g. the vacuoles), we also investigated surface casts. A survey among angiosperms revealed three different types of floral surface structure, each with distinct reflections. Petals with a smooth and very flat surface had mirror-like reflections and petal surfaces with cones yielded diffuse reflections. Petals with striations yielded diffraction patterns when single cells were illuminated. The iridescent signal, however, vanished when illumination similar to that found in natural conditions was applied. Pigmentary rather than structural coloration determines the optical appearance of flowers. Therefore, the hypothesized signaling by flowers with striated surfaces to attract potential pollinators presently seems untenable.

The colouration toolkit of the Pipevine Swallowtail butterfly, Battus philenor: thin films, papiliochromes, and melanin.

The ventral hindwings of Pipevine Swallowtail butterflies, Battus philenor, display a colourful pattern, created by variously coloured wing scales. Reflectance and transmittance measurements of single scales indicate that the cream and orange scales contain papiliochrome pigments, while brown, black and blue scales contain melanin. Microspectrophotometry and scatterometry of both sides of the wing scales show that the lower lamina acts as a thin film, with reflection properties dependent on the scale's pigmentation. Notably in the orange scales, the reflectance spectrum of the lower lamina is tuned to the pigment's absorbance spectrum. The dorsal hindwings of the male (but not the female) B. philenor are blue-green iridescent. At oblique illumination, the light reflected by the male's dorsal hindwings can be highly polarised, which may have a function in intersexual signalling.

Coloration principles of nymphaline butterflies - thin films, melanin, ommochromes and wing scale stacking.

The coloration of the common butterflies Aglais urticae (small tortoiseshell), Aglais io (peacock) and Vanessa atalanta (red admiral), belonging to the butterfly subfamily Nymphalinae, is due to the species-specific patterning of differently coloured scales on their wings. We investigated the scales' structural and pigmentary properties by applying scanning electron microscopy, (micro)spectrophotometry and imaging scatterometry. The anatomy of the wing scales appears to be basically identical, with an approximately flat lower lamina connected by trabeculae to a highly structured upper lamina, which consists of an array of longitudinal, parallel ridges and transversal crossribs. Isolated scales observed at the abwing (upper) side are blue, yellow, orange, red, brown or black, depending on their pigmentation. The yellow, orange and red scales contain various amounts of 3-OH-kynurenine and ommochrome pigment, black scales contain a high density of melanin, and blue scales have a minor amount of melanin pigment. Observing the scales from their adwing (lower) side always revealed a structural colour, which is blue in the case of blue, red and black scales, but orange for orange scales. The structural colours are created by the lower lamina, which acts as an optical thin film. Its reflectance spectrum, crucially determined by the lamina thickness, appears to be well tuned to the scales' pigmentary spectrum. The colours observed locally on the wing are also due to the degree of scale stacking. Thin films, tuned pigments and combinations of stacked scales together determine the wing coloration of nymphaline butterflies.

Sexual dichromatism of the damselfly Calopteryx japonica caused by a melanin-chitin multilayer in the male wing veins.

Mature male Calopteryx japonica damselflies have dark-blue wings, due to darkly coloured wing membranes and blue reflecting veins. The membranes contain a high melanin concentration and the veins have a multilayer of melanin and chitin. Female and immature C. japonica damselflies have brown wings. We have determined the refractive index of melanin by comparing the differently pigmented wing membranes and applying Jamin-Lebedeff interference microscopy. Together with the previously measured refractive index of chitin the blue, structural colour of the male wing veins could be quantitatively explained by an optical multilayer model. The obtained melanin refractive index data will be useful in optical studies on melanized tissues, especially where melanin is concentrated in layers, thus causing iridescence.

Glass scales on the wing of the swordtail butterfly Graphium sarpedon act as thin film polarizing reflectors.

The wings of the swordtail butterfly Graphium sarpedon (the Common Bluebottle) have blue/green-colored patches that are covered on the underside by two types of scales: white and glass scales. Transmission and scanning electron microscopy revealed that the white scales are classically structured: the upper lamina, with prominent ridges and large open windows, is well separated by trabeculae from a flat, continuous lower lamina. In the glass scales, the upper lamina, with inconspicuous ridges and windows, is almost flat and closely apposed to the equally flat lower lamina. The glass scales thus approximate ideal thin films, in agreement with the observation that they reflect light directionally and are iridescent. Reflectance and transmittance spectra measured from the glass scales with a microspectrophotometer agree with spectra calculated for an ideal non-absorbing thin film. Imaging scatterometry of single, isolated glass scales demonstrated that the reflected light can be strongly polarized, indicating that they function as polarizing reflectors.

Kingfisher feathers--colouration by pigments, spongy nanostructures and thin films.

The colours of the common kingfisher, Alcedo atthis, reside in the barbs of the three main types of feather: the orange breast feathers, the cyan back feathers and the blue tail feathers. Scanning electron microscopy showed that the orange barbs contain small pigment granules. The cyan and blue barbs contain spongy nanostructures with slightly different dimensions, causing different reflectance spectra. Imaging scatterometry showed that the pigmented barbs create a diffuse orange scattering and the spongy barb structures create iridescence. The extent of the angle-dependent light scattering increases with decreasing wavelength. All barbs have a cortical envelope with a thickness of a few micrometres. The reflectance spectra of the cortex of the barbs show oscillations when measured from small areas, but when measured from larger areas the spectra become wavelength independent. This can be directly understood with thin film modelling, assuming a somewhat variable cortex thickness. The cortex reflectance appears to be small but not negligible with respect to the pigmentary and structural barb reflectance.

Refractive index and dispersion of butterfly chitin and bird keratin measured by polarizing interference microscopy.

Using Jamin-Lebedeff interference microscopy, we measured the wavelength dependence of the refractive index of butterfly wing scales and bird feathers. The refractive index values of the glass scales of the butterfly Graphium sarpedon are, at wavelengths 400, 500 and 600 nm, 1.572, 1.552 and 1.541, and those of the feather barbules of the white goose Anas anas domestica are 1.569, 1.556 and 1.548, respectively. The dispersion spectra of the chitin in the butterfly scales and the keratin in the bird barbules are well described by the Cauchy equation n(λ) = A + B/λ(2), with A = 1.517 and B = 8.80·10(3) nm(2) for the butterfly chitin and A = 1.532 and B = 5.89·10(3) nm(2) for the bird keratin.

Polarized iridescence of the multilayered elytra of the Japanese jewel beetle, Chrysochroa fulgidissima.

The elytra of the Japanese jewel beetle Chrysochroa fulgidissima are metallic green with purple stripes. Scanning electron microscopy and atomic force microscopy demonstrated that the elytral surface is approximately flat. The accordingly specular green and purple areas have, with normal illumination, 100-150 nm broad reflectance bands, peaking at about 530 and 700 nm. The bands shift progressively towards shorter wavelengths with increasing oblique illumination, and the reflection then becomes highly polarized. Transmission electron microscopy revealed that the epicuticle of the green and purple areas consists of stacks of 16 and 12 layers, respectively. Assuming gradient refractive index values of the layers between 1.6 and 1.7 and applying the classical multilayer theory allowed modelling of the measured polarization- and angle-dependent reflectance spectra. The extreme polarized iridescence exhibited by the elytra of the jewel beetle may have a function in intraspecific recognition.

Dramatic colour changes in a bird of paradise caused by uniquely structured breast feather barbules.

The breast-plate plumage of male Lawes' parotia (Parotia lawesii) produces dramatic colour changes when this bird of paradise displays on its forest-floor lek. We show that this effect is achieved not solely by the iridescence--that is an angular-dependent spectral shift of the reflected light--which is inherent in structural coloration, but is based on a unique anatomical modification of the breast-feather barbule. The barbules have a segmental structure, and in common with many other iridescent feathers, they contain stacked melanin rodlets surrounded by a keratin film. The unique property of the parotia barbules is their boomerang-like cross section. This allows each barbule to work as three coloured mirrors: a yellow-orange reflector in the plane of the feather, and two symmetrically positioned bluish reflectors at respective angles of about 30°. Movement during the parotia's courtship displays thereby achieves much larger and more abrupt colour changes than is possible with ordinary iridescent plumage. To our knowledge, this is the first example of multiple thin film or multi-layer reflectors incorporated in a single structure (engineered or biological). It nicely illustrates how subtle modification of the basic feather structure can achieve novel visual effects. The fact that the parotia's breast feathers seem to be specifically adapted to give much stronger colour changes than normal structural coloration implies that colour change is important in their courtship display.

Butterfly wing colors: glass scales of Graphium sarpedon cause polarized iridescence and enhance blue/green pigment coloration of the wing membrane.

The wings of the swordtail butterfly Graphium sarpedon nipponum contain the bile pigment sarpedobilin, which causes blue/green colored wing patches. Locally the bile pigment is combined with the strongly blue-absorbing carotenoid lutein, resulting in green wing patches and thus improving camouflage. In the dorsal forewings, the colored patches lack the usual wing scales, but instead have bristles. We have found that on the ventral side most of these patches have very transparent scales that enhance, by reflection, the wing coloration when illuminated from the dorsal side. These glass scales furthermore create a strongly polarized iridescence when illuminated by obliquely incident light from the ventral side, presumably for intraspecific signaling. A few ventral forewing patches have diffusely scattering, white scales that also enhance the blue/green wing coloration when observed from the dorsal side.

Imaging scatterometry and microspectrophotometry of lycaenid butterfly wing scales with perforated multilayers.

We studied the structural as well as spatial and spectral reflectance characteristics of the wing scales of lycaenid butterfly species, where the scale bodies consist of perforated multilayers. The extent of the spatial scattering profiles was measured with a newly built scatterometer. The width of the reflectance spectra, measured with a microspectrophotometer, decreased with the degree of perforation, in agreement with the calculations based on multilayer theory.