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Butterfly scale form birefringence related to photonics
Micron 42 (2011) 801–807
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Butterfly scale form birefringence related to photonics Benedicto de Campos Vidal ∗ Departamento de Anatomia, Biologia Celular e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas, CEP, 13083-863 Campinas, SP, Brazil
a r t i c l e
i n f o
Article history: Received 17 February 2011 Received in revised form 28 April 2011 Accepted 29 April 2011 Keywords: Butterfly Wing scale Optical anisotropy Photonics FT-IR
a b s t r a c t Wings of the butterflies Morpho aega and Eryphanis reevesi were investigated in the present study by fluorescence, polarization and infra-red (IR) spectroscopic microscopy with the aim of identifying the oriented organization of their components and morphological details of their substructures. These wings were found to exhibit a strong iridescent glow depending on the angle of the incident light; their isolated scales exhibited blue fluorescence. Parallel columns or ridges extend from the pad and sockets to the dented apical scale’s region, and they are perpendicular to the ribs that connect the columnar ridges. The scales reveal linear dichroism (LD) visually, when attached on the wing matrix or isolated on slides. The LD was inferred to be textural and positive and was also demonstrated with IR microscopy. The scale columns and ribs are birefringent structures. Images obtained before and after birefringence compensation allowed a detailed study of the scale morphology. Form and intrinsic birefringence findings here estimated and discussed in the context of nonlinear optical properties, bring to the level of morphology the state of molecular order and periodicity of the wing structure. FT-IR absorption peaks were found at wavenumbers which correspond to symmetric and asymmetric (–N–H) stretching, symmetric (–C–H) stretching, amide I (–C O) stretching, amide II(–N–H), and -linking. Based on LD results obtained with polarized IR the molecular vibrations of the wing scales of M. aega and E. reevesi are assumed to be oriented with respect to the long axis of these structures. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Butterfly wings are composed of scales of photonic properties of which are well characterized. These structures are often used as templates to achieve manmade photonic structures. An examination of four families and twelve species of lepidopterans concluded that all the butterfly scales observed exhibit coherent scattered visible wavelengths (Prum et al., 2006). The scales of swallowtail Papilionidae butterflies exhibit a nanostructure characterized by a 2D photonic crystal cylinder of hollow air (Vukusic and van Hooper, 2005). The air cylinders are arranged in the photonic crystal quasiperiodically and are infused with a highly fluorescent pigment (Vukusic and van Hooper, 2005). Scanning electron microscopy aspects of the photonic scales of Sasakia charonda revealed the detailed morphology of these structures and were suggestive that they may play a role in aerodynamically easy flight (MatèjkováPlskova et al., 2009). The periodicity of photonic crystals in butterfly wing scales has also been studied using three-dimensional electron tomography, a computer-based visualization (Argyros et al., 2002). The periodic air filled nano-voids described in these studies are a common feature in manmade photonic crystals.
∗ Corresponding author. Tel.: +55 19 3521 6123; fax: +55 19 3521 6185. E-mail address: [email protected] 0968-4328/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2011.04.006
The atomic layer deposition of Al2 O3 has been used to replicate the butterfly wing structure and to study the wave guide and beam splitter properties of the wing (Huang et al., 2006). ZrO2 replicas of natural butterfly wings revealed that the periodic dielectric structures of the wings can be observed even at the visible wavelength range; butterfly wings are thus examples of naturally occurring photonic structures (Chen et al., 2009). Each of the aforementioned replicated samples of butterfly wings exhibited the same photonic colors as the natural samples. These studies have demonstrated that supra-organizations having photonic properties are structured in a periodic arrangement of nano-units and display dielectric properties. Form birefringence and nonlinear optical properties are thus expected to be also displayed by butterfly wing components. Because of the physical properties of butterfly scales, investigation on optical anisotropic properties to better understand butterfly scales is reasonable. 1.1. Form birefringence The principles and properties of form birefringence (F ) have been researched systematically by biophysicists and physicists for academic and utilitarian purposes. The definition of Bêche and Gaviot (2003) “In optics, an anisotropy property on the permittivity tensor, called form birefringence, occurs in sundry multiple quantum wells consisting of two constituents” exemplifies the possible relation of scale structures to F .
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F can be mathematically expressed as a two component body (e.g., quantum wells consisting of two constituents, one of which is rod like) F = (n21 − n22 ) = [f1 f2 (n21 − n22 )]/[(1 + f1 )n22 + n21 f2 ] (permittivity ε = n2 ), where f1 is defined as the fraction of the component with refraction index n1 and f2 is defined as the fraction of the component with n2 , such that (f1 + f2 ) = 1. This equation is valid for rod-like molecules such as chitin and collagen (Wiener, 1912; Schmidt and Keil, 1958; Cassim and Taylor, 1965). The use of this equation can be applied to birefringent porous silica and dichroic Bragg reflectors (DBR), which have been proposed as sensors in various applications (Diener et al., 2001; Vukusic and van Hooper, 2005). Bodies that display F must be composed of a periodic nanocomposition and at least one of the components must be smaller than the wavelength of the electromagnetic incident light (Wiener, 1912; Schmidt and Keil, 1958; Cassim and Taylor, 1965). 1.2. Form birefringence (F ) and photonics No study establishing a correlation between the reported photonic properties of butterflies with the anisotropic optical properties as linear dichroism (LD), intrinsic (I ) and form (F) birefringence could be found in our survey of the literature. Currently, the physical principles of F are applied to construct fibers endowed with birefringence, and photonic crystal that exhibit higher nonlinearity, chirality and F . The reports on this subject are abundant. Harris et al. (2008), for example, have produced transparent films exhibiting F that are electrically conductive and capable of aligning liquid crystals. These products have many potential applications. Birefringence in optical fibers helps them to maintain polarization guide modes, which permits the construction of various optical devices as well as highly birefringent optical fibers and DBR (Yokohama et al., 1985; Diener et al., 2001; O et al., 2004; Kotynski et al., 2005; Pena et al., 2005; Golovan et al., 2006; Mitrofanov et al., 2006). An extreme instance of F principles application is the construction of a nano-engine for the all-optical control of a microfluidic component (Neale et al., 2005). Birefringent image analysis yields informative images that reveal morphological as well as topographic properties that allow measurements of the molecular order and degrees of ordered aggregation of the periodic dielectric structures involved (Vidal, 2010). To date, no detection of birefringence and linear dichroism has been reported in butterfly wing scales where photonic properties have been simultaneously observed. In the present study, individual whole scales from butterfly wings were studied under conditions where the influence of alternating layers of ordered scale components on the optical anisotropy can be detected and birefringence can be measured. 1.3. Aims This work has the objective of studying fluorescence properties as well as anisotropic optical properties (LD at visible and IR light, and I and F ) of butterfly wing scales, contributing to the knowledge of morphological details of the scale structure. The findings were interpreted in terms of the state of the art in the field of photonic properties, periodic constructed structures, nonlinearity and birefringence for several other materials. 2. Material and methods 2.1. Wings and scales of butterflies Wings of the butterflies Eryphanis reevesi (Doubleday, 1849) and Morpho aega (Hübner, 1822) (Nymphalidæ) were used. Isolated whole scales were obtained by touching the blue iridescent sur-
face of the wings to the surface of histological slides, applying a light pressure to promote adherence of the scales on the slides, and drying the preparations at room temperature. 2.2. Fluorescence Fluorescence of the wing scales was observed by epiillumination in a Zeiss Axiophot-2 microscope (Eching/Munich, Germany) using the filters UV–G363/FT-395/LP-420 and blue excitation 485/20, FT-500/600. 2.3. Birefringence and linear dichroism Polarized light microscopy was used to search for linear dichroism (LD) and birefringence using an Olympus BX51 polarized light microscope. LD was studied after removal of the microscope analyzer and after the long axis of the scales was oriented successively parallel and perpendicular to the polarizer plane of polarized light (PPL). To determine if the birefringence was positive or negative, and to detect the orientation of the fibrillar components of the wing scales, Sénarmont’s and Bräce-Köhler’s compensators were used. Form birefringence was measured after immersing the scales in Cargille oils of different refractive indexes (n = 1.33–1.70) according to previously described procedures (Vidal, 1965, 1980, 1986, 2010).The slides were covered by coverslips and the imbibition of the preparations lasted for 10–24 h. Measurements of optical path differences due to the birefringence were performed with a Sénarmont (, 1/4) and/or a Brace-Köhler compensator depending on the birefringence intensity. 2.4. FT-IR FT-IR investigation was done using the Illuminat IR IITM microspectrometer (Smiths Detection, Danbury, USA) with a liquid nitrogen-cooled mercury–cadmium–telluride (MCT) detector and Grams/AI 8.0 spectroscopy software (Thermo Electron Corporation, Waltham, USA). An attenuated total reflection (ATR) diamond objective, magnification 36× was employed. The diamond objective ATR 36× generally avoids the dispersion and Mie scattering that are more common using all reflecting objectives (ARO). 3. Results and discussion 3.1. General aspects The wings of both butterfly species exhibit a strong iridescent glow depending on the incident light angle. The entire surface of the wing in M. aega exhibited iridescence (Fig. 1), whereas only a particular region of the wing in E. reevesi revealed an iridescent glow (Fig. 2). Even the scales isolated on slides showed iridescence when illuminated by light from different directions (data not shown). The scales are ordered and fixed by peg-and-socked attachments on the surface of a matrix such that the scales of all species appear arranged in parallel rows like shingles on a house, as was previously reported (Prum et al., 2006). In E. reevesi as well as in M. aega, the ordered scales are separated into two layers. The scales, from one layer to another, are distributed out of phase. The more superficial layer of the scales is darker and likely to be responsible for the iridescent colors. Beneath this layer, there is another layer, composed of clear scales that fit the description of glass scales (Fig. 4A and B) (Prum et al., 2006). The brown color of these scales has been reported to be due to melanin (Matèjková-Plskova et al., 2009). Taking in consideration that the scales are composed by chitins, and that in arthropods chitin is darkened by tanning phenolic processes (Neville, 1975), it is proposed that the dark appearance
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Fig. 1. Iridescent image of half wings of M. aega under orthogonal illumination. The iridescence covers the entire surface of the wings.
of the scales in the present case may also be caused by a similar process. 3.2. Fluorescence The dark scales of M. aega and E. reevesi, when excited by UV light ( = 363 nm), and provided that the long-pass filter LP-420 is used, show a blue fluorescence ( in the range of 420–460 nm, obeying the Stokes law) (Fig. 3A and B). They show a green fluorescence when excited by blue light (data not shown). No difference in fluorescence intensity or hues was detected when comparing M. aega to E. reevesi.
Fig. 2. Iridescent image of half wings of M. reevesi under orthogonal illumination. The iridescence is limited to certain areas of the wings.
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Vukusic and van Hooper (2005) have described fluorescence in the butterfly Princeps nireus, uttering that the periodic photonic crystal in the wing scales is infused with a highly fluorescent pigment; they tested whether there was an “out of plane emission”, by studying the specimen with and without immersion in an index-matching fluid. They have also described that the intense fluorescence they described was directionally enhanced by a Dichroic Brag Reflector (DBR). Diener et al. (2001) reported construction of a DBR based on birefringence, which is in the context of the anisotropic findings described in the present work. Vukusic and van Hooper (2005) concluded that immersion of the butterfly wing effectively removes the fluorescence due to a nanostructure’s photonic influence. It must be emphasized that this method is similar to the method used here to determine the intrinsic birefringence in the F curve. A 3-D fluorescence study of wings of the butterfly Morphoa sulkowskyi has been reported by Kumazawa and Tabata (2001), relating a peak emission near = 410 nm to a mixture of three pteridin pigments; they also attributed the emission peak at 470 nm (blue fluorescence) in Papilio xuthus wings to the papiliochrom II pigment. Considering the results obtained by Vukusic and van Hooper (2005), it is concluded that the observed fluorescence in the scales of the butterflies M. aega and E. reevesi is due to a photonic periodic structure. 3.3. Anisotropic optical properties: linear dichroism (LD)and birefringence The images observed by optical anisotropy in the wing scales allowed the detection of their morphological characteristics. 3.3.1. Linear dichroism (LD) The scales of M. aega and E. reevesi when attached to the wing matrix or when isolated on glass slides displayed LD visually. LD is recognized by selective absorptions of polarized light with respect to the plane of polarized light (PPL). In this case, when the long axis of the scales was parallel to the PPL the scales appear darker than when the scales long axis was perpendicular to the PPL. A second layer of scales (clear scales) out of focus may appear underneath (Fig. 4A and B). It is important to consider the influence of the matrix wing on the whole wing thickness in the total structure focus on the differences of absorption. The LD observed in the wing is dependent on the periodic ordered distribution of the ribs, columns and their dimensions of scales. LD observed in isolated scales is more evident. When the long axis of a scale is parallel to the PPL, there is a predominance of orange-yellow colors; when this axis is perpendicular to the PPL, a green color is predominant. The scale tip is very dark, and a small area of the scale, at its insertion region, is yellow. Variation of LD colors is detected in all other orientations of the scales as regards the PPL (Fig. 4C). Actually, in this case the LD can be designed as a pleochroism, and it is based on DBR (Diener et al., 2001; Vukusic and van Hooper, 2005). The LD shown by air-dried scales was more intense than that observed after imbibing the scales in Cargille oils (data not shown). This finding is in agreement with previous reports on dichroism properties (Fig. 7 in Schmidt, 1937). 3.3.2. Birefringence The wing scales of the butterflies studied here revealed positive intrinsic (I ) and form birefringence (F ) after these structures were imbibed in a series of oils of different refractive indices. As the observed images of anisotropical anisotropy of M. aega and E. reevesi were very similar, all the figures shown are from E. reevesi. The scale column’s birefringence decreases at the point of their contact with the scale ribs (Fig. 5). Such a finding allowed the
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Fig. 3. Blue fluorescence image of scales from M. aega (A), and from E. reevesi (B). Bar = 30 m.
inference that the rib fibrilar structure could insert into the column in a subtraction way; in terms of chitin fibrilar and molecular orientation, this induces an internal compensation of the column birefringence. Birefringence of the scales causes an optical retardation or optical path difference (OR) visible in the field of observation as brilliant images that allowed detection of various scale characteristics. Emphasis should be given to the fact that the birefringent image has a 3D-dimensional information which results from a bidimensional information provided by the topographic relationship of the object optical axis with the PPL, plus the intensity of brilliance that directly depends on the object thickness and the concentration of the birefringent material (details in Vidal, 2010, Figs. 1 and 2; Vidal and Mello, 2010). The birefringent object appears brilliant
against a dark field; the brilliant ribs are thus observed perpendicular to the columns and ordered among them. Compensation of the column birefringence using a Bräce-Köhler’s or a Sénarmont’s compensator provides a better visualization of the column morphology. It is important to consider that the morphological information presented here is generated at the level of molecular orientation (nanometric dimension) of the structures. When performing OR measurements, the birefringence corresponding to the long axis of the columns compensates perpendicular to the ribs (Fig. 6). Compensation of the ribs’ birefringence occurs when they are positioned with an orientation of 90◦ in relation to the orientation of the columns. After this compensation, the ribs appear dark, whereas the columns continue to show a brilliant birefringence (Fig. 7).
Fig. 4. Whole mount of a wing fragment of M. aega. The images were obtained for part of the blue iridescent wing illuminated by polarized light. (A) The long axis of the scales was oriented parallel to the PPL, revealing higher absorptions than when the scales are oriented perpendicular to the PPL (B). Differences in absorption result in linear dichroism (LD). The lower layer with clear scales appears out of the focus plane. (C) Isolated scales illuminated with polychromatic polarized light such that the direction of the PPL was east to west. When the long axis of the scales (pad-socket to apical direction) was parallel to the PPL, the wing shows colors from orange to orange-green; in the 1/3 apical region, the scales were clear. When the long axis of the scales was perpendicular to the PPL, the colors were predominantly green, and they were darker than under parallel orientation. The color varied with the orientation of the scale relative to the PPL, and is dependent on each wavelength (coherent light scattering). Bar = 30 m.
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Fig. 8. Birefringence curve for scales of the butterfly wings represented by a scatter diagram of optical retardation (OR) with respect to refractive index values. Dashed line, E. reevesi; solid line, M. aega. Fig. 5. Birefringence image of a E. reevesi wing scale. The long axis of the scale was oriented at 45◦ with respect to the crossed polarizers of the PPL. The birefringence of the ribs appears more brilliant than that of the columns. The interference colors observed here are due to the dispersion of the birefringence. Bar = 20 m.
Fig. 6. Image of the compensated birefringence on the columns of the same scale depicted in Fig. 5. The columns now appear black and the ribs show a strong brilliance. Bar = 20 m.
Fig. 7. Same scale as in Fig. 5 after compensation of the ribs’ birefringence. The columns appear brilliant. Bar = 20 m.
A scatter diagram dispersion of form birefringence was constructed for F and I comparison of the wings’ scales of M. aega and E. reevesi. The minimal OR value, corresponding to I , was found at n = 1.55 for both M. aega and E. reevesi (Fig. 8). Generally, a F curve is considered to be a U-shaped parabola-like curve (Wiener, 1912; Schmidt, 1937; Frey-Wyssling, 1948; Schmidt and Keil, 1958; Bennet, 1967). Actually, the geometry of the curves varies as a function of the composition and relative concentration of each oriented component that plays a role in the supramolecular architecture under study. In the present case, the shape of the scatter diagram indicates a tendency to a hyperbolic shape; the line from n = 1.00 to n = 1.55 gives the sharp feature to the scatter diagram, supporting the conclusion that, for both butterfly wing scales, the major component causing form birefringent is the same as for cuticles, i.e., chitin. Similar results have been reported for the chitinous tendon of the grasshopper and the pen of Loligo brasiliensis (Vidal and Carvalho, 1986; Carvalho and Vidal, 1991). The major difference between species in the scatter diagram for scales was found at n = 1.50, n = 1.60 and n = 1.70, which could be caused by variations in the ordered arrangements of their periodic constructions. A hypothesis could be raised that where the ribs of M. reevesi are in contact with a column they penetrate the column with different directions of its chitin fibrilar entanglement, originating differences at the level of the apparent birefringence intensity. Reports from electron scanning microscopy give support to this interpretation (see figures in Huang et al., 2006; Matèjková-Plskova et al., 2009). Because the birefringence measurements were performed for entire scales and the polarized photons of light travel perpendicular to the three dimensional lattice of the scale’s periodic construction, the sum of these interactions with the nano-units of the scale causes the anisotropic optical phenomena reported here. This information is not obtained with electron tomography, which is limited by the thickness of the specimen, although Shawkey et al. (2009) have interpreted that thickness can have enough repeating units to allow Fourier analysis to be meaningful. In the case of the optical anisotropies the wave of photons traveling through the scale can give a better statistical approach to the statistical meaning of its structural orientation. Biophotonic properties are not restrained to butterflies. These properties have been reported in non-chitinous quasi-periodic nanostructures of keratinous bird feathers and King Penguin beaks (Dresp et al., 2005; Shawkey et al., 2009), and in collagen fibers (Prum and Torres, 2003). In this context, it is pertinent citing that collagen fibers induce nanometric silver particles to link in their nanostructure, causing LD and abnormal dispersion of birefrin-
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chitin exists in the scales as a macromolecule making nanometric aggregate ordered units with a periodic supramolecular structure. The FT-IR data obtained with polarized IR light revealed a positive LD and provided results which are in agreement with birefringence and visible light LD observations. Acknowledgments The support of Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants no. 2003/04597-0 and 2007/058251 is gratefully acknowledged. The author is indebted to Dr. Maria Luiza S. Mello and Dr. André Victor Lucci Freitas for providing him with the butterfly specimens. References Fig. 9. FT-IR spectrum for LD exhibited by butterfly wing scales. Measurements were carried out using polarized infrared light. The red and blue lines refer to absorptions when the long axis of the scales was parallel and perpendicular, respectively, to the plane of polarization of the IR light.
gence phenomena recognized to be photonic (Vidal et al., 1975; Vidal and Joazeiro, 2002; Vidal, 2003). An interesting and inspiring example of practical application of these concepts is the constructed nanomotor that moves by a photonic moment (Neale et al., 2005). A question that may arise from this example is “Could a photonic moment be generated in the wings of, for instance, M. aega that makes flying easier, especially considering that their wings present a large area of iridescence?” Furthermore, what is the biophotonic role in the evolutionary process of the butterflies? 3.4. FT-IR analysis and LD Data were obtained only for scales of M. aega because of the similarity in the above-mentioned LD birefringence data between the two butterfly species. The results obtained for FT-IR of M. aega scales are similar, but not identical, to those already reported for chitin and chitosan (Neville, 1975; Brunner et al., 2009; Skorig et al., 2010; Sudheesh Kumar et al., 2010). However, the present reports are considered to be the first approach to pave future developments in the LD explored by FT-IR. Vibrational absorption peaks were found at the wavenumbers: 3287 cm−1 ,corresponding to (–N–H) symmetric and asymmetric stretching, 2923 and 2850 cm−1 , corresponding to (–C–H) symmetric stretching, 1644 cm−1 , corresponding to amide I peak (–C O) stretching, 1550–1530 cm−1 , corresponding to amide II (–N–H), and 895–902 cm−1 , corresponding to -linking. Examination of the scales with polarized IR demonstrated a positive LD, i.e., when the long axis of the scales was parallel to the PPL of the IR light, higher vibrational absorptions were detected (Fig. 9). The differences found in the LD characteristics when comparing the scales of M. aega and other chitin-containing materials (Neville, 1975), may be due to differences in their nanostructure supraorganizations. 4. Conclusions The wing scales of the butterflies M. aega and E. reevesi exhibit fluorescence, LD, and F and I properties when examined with fluorescence, polarization and IR microscopy. The LD displayed by the scales at the visible light is due to the scale periodic structure and its Dichroic Brag Reflector properties. F represents 91.44% of the total birefringence of the scales, whereas I represents only 8.56% of the total birefringence. The higher participation of F in the total birefringence confirms that
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B.d.C. Vidal / Micron 42 (2011) 801–807 Schmidt, W.J., Keil, A., 1958. Die Gesungen und die Erkrankten Zahngeweve des Menschen und der Wierbeltiere im Polarizationmikroskop. Catl. Hanser Verlag, München, pp. 1–11. Skorig, Y.A., Pestov, A.V., Yatluk, Y., 2010. Evaluation of various chitin-glucan derivatives from Aspergillus niger as transition metal adsorbents. Bioresour. Technol. 101, 1769–1775. Sudheesh Kumar, P.T., Abhilash, S., Manzoor, K., Nair, S.V., Tamura, H., Jayakumar, R., 2010. Preparation and characterization of novel -chitin/nanosilver composite for wound dressing applications. Carbohydr. Polym. 80, 761–767. Vidal, B.C., 1965. The part played by the mucopolysaccharides in the form birefringence of collagen. Protoplasma 59, 472–479. Vidal, B.C., 1980. The part played by proteoglycans and structural glycoproteins in the macromolecular orientation of collagen bundles. Cell. Mol. Biol. 26, 415–421. Vidal, B.C., 1986. Evaluation of carbohydrate role in the molecular order of collagen bundles: microphotometric measurements of textural birefringence. Cell Mol. Biol. 32, 527–535. Vidal, B.C., 2003. Image analysis of linear dichroism in collagen-nano-silver complexes. Micr. Anal. 97, 21–23. Vidal, B.C., 2010. Form birefringence as applied to biopolymer and Inorganic material supraorganization. Biotechnol. Histochem. 85, 365–378.
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Butterfly scale form birefringence related to photonics Benedicto de Campos Vidal ∗ Departamento de Anatomia, Biologia Celular e Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas, CEP, 13083-863 Campinas, SP, Brazil
a r t i c l e
i n f o
Article history: Received 17 February 2011 Received in revised form 28 April 2011 Accepted 29 April 2011 Keywords: Butterfly Wing scale Optical anisotropy Photonics FT-IR
a b s t r a c t Wings of the butterflies Morpho aega and Eryphanis reevesi were investigated in the present study by fluorescence, polarization and infra-red (IR) spectroscopic microscopy with the aim of identifying the oriented organization of their components and morphological details of their substructures. These wings were found to exhibit a strong iridescent glow depending on the angle of the incident light; their isolated scales exhibited blue fluorescence. Parallel columns or ridges extend from the pad and sockets to the dented apical scale’s region, and they are perpendicular to the ribs that connect the columnar ridges. The scales reveal linear dichroism (LD) visually, when attached on the wing matrix or isolated on slides. The LD was inferred to be textural and positive and was also demonstrated with IR microscopy. The scale columns and ribs are birefringent structures. Images obtained before and after birefringence compensation allowed a detailed study of the scale morphology. Form and intrinsic birefringence findings here estimated and discussed in the context of nonlinear optical properties, bring to the level of morphology the state of molecular order and periodicity of the wing structure. FT-IR absorption peaks were found at wavenumbers which correspond to symmetric and asymmetric (–N–H) stretching, symmetric (–C–H) stretching, amide I (–C O) stretching, amide II(–N–H), and -linking. Based on LD results obtained with polarized IR the molecular vibrations of the wing scales of M. aega and E. reevesi are assumed to be oriented with respect to the long axis of these structures. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Butterfly wings are composed of scales of photonic properties of which are well characterized. These structures are often used as templates to achieve manmade photonic structures. An examination of four families and twelve species of lepidopterans concluded that all the butterfly scales observed exhibit coherent scattered visible wavelengths (Prum et al., 2006). The scales of swallowtail Papilionidae butterflies exhibit a nanostructure characterized by a 2D photonic crystal cylinder of hollow air (Vukusic and van Hooper, 2005). The air cylinders are arranged in the photonic crystal quasiperiodically and are infused with a highly fluorescent pigment (Vukusic and van Hooper, 2005). Scanning electron microscopy aspects of the photonic scales of Sasakia charonda revealed the detailed morphology of these structures and were suggestive that they may play a role in aerodynamically easy flight (MatèjkováPlskova et al., 2009). The periodicity of photonic crystals in butterfly wing scales has also been studied using three-dimensional electron tomography, a computer-based visualization (Argyros et al., 2002). The periodic air filled nano-voids described in these studies are a common feature in manmade photonic crystals.
∗ Corresponding author. Tel.: +55 19 3521 6123; fax: +55 19 3521 6185. E-mail address: [email protected] 0968-4328/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2011.04.006
The atomic layer deposition of Al2 O3 has been used to replicate the butterfly wing structure and to study the wave guide and beam splitter properties of the wing (Huang et al., 2006). ZrO2 replicas of natural butterfly wings revealed that the periodic dielectric structures of the wings can be observed even at the visible wavelength range; butterfly wings are thus examples of naturally occurring photonic structures (Chen et al., 2009). Each of the aforementioned replicated samples of butterfly wings exhibited the same photonic colors as the natural samples. These studies have demonstrated that supra-organizations having photonic properties are structured in a periodic arrangement of nano-units and display dielectric properties. Form birefringence and nonlinear optical properties are thus expected to be also displayed by butterfly wing components. Because of the physical properties of butterfly scales, investigation on optical anisotropic properties to better understand butterfly scales is reasonable. 1.1. Form birefringence The principles and properties of form birefringence (F ) have been researched systematically by biophysicists and physicists for academic and utilitarian purposes. The definition of Bêche and Gaviot (2003) “In optics, an anisotropy property on the permittivity tensor, called form birefringence, occurs in sundry multiple quantum wells consisting of two constituents” exemplifies the possible relation of scale structures to F .
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F can be mathematically expressed as a two component body (e.g., quantum wells consisting of two constituents, one of which is rod like) F = (n21 − n22 ) = [f1 f2 (n21 − n22 )]/[(1 + f1 )n22 + n21 f2 ] (permittivity ε = n2 ), where f1 is defined as the fraction of the component with refraction index n1 and f2 is defined as the fraction of the component with n2 , such that (f1 + f2 ) = 1. This equation is valid for rod-like molecules such as chitin and collagen (Wiener, 1912; Schmidt and Keil, 1958; Cassim and Taylor, 1965). The use of this equation can be applied to birefringent porous silica and dichroic Bragg reflectors (DBR), which have been proposed as sensors in various applications (Diener et al., 2001; Vukusic and van Hooper, 2005). Bodies that display F must be composed of a periodic nanocomposition and at least one of the components must be smaller than the wavelength of the electromagnetic incident light (Wiener, 1912; Schmidt and Keil, 1958; Cassim and Taylor, 1965). 1.2. Form birefringence (F ) and photonics No study establishing a correlation between the reported photonic properties of butterflies with the anisotropic optical properties as linear dichroism (LD), intrinsic (I ) and form (F) birefringence could be found in our survey of the literature. Currently, the physical principles of F are applied to construct fibers endowed with birefringence, and photonic crystal that exhibit higher nonlinearity, chirality and F . The reports on this subject are abundant. Harris et al. (2008), for example, have produced transparent films exhibiting F that are electrically conductive and capable of aligning liquid crystals. These products have many potential applications. Birefringence in optical fibers helps them to maintain polarization guide modes, which permits the construction of various optical devices as well as highly birefringent optical fibers and DBR (Yokohama et al., 1985; Diener et al., 2001; O et al., 2004; Kotynski et al., 2005; Pena et al., 2005; Golovan et al., 2006; Mitrofanov et al., 2006). An extreme instance of F principles application is the construction of a nano-engine for the all-optical control of a microfluidic component (Neale et al., 2005). Birefringent image analysis yields informative images that reveal morphological as well as topographic properties that allow measurements of the molecular order and degrees of ordered aggregation of the periodic dielectric structures involved (Vidal, 2010). To date, no detection of birefringence and linear dichroism has been reported in butterfly wing scales where photonic properties have been simultaneously observed. In the present study, individual whole scales from butterfly wings were studied under conditions where the influence of alternating layers of ordered scale components on the optical anisotropy can be detected and birefringence can be measured. 1.3. Aims This work has the objective of studying fluorescence properties as well as anisotropic optical properties (LD at visible and IR light, and I and F ) of butterfly wing scales, contributing to the knowledge of morphological details of the scale structure. The findings were interpreted in terms of the state of the art in the field of photonic properties, periodic constructed structures, nonlinearity and birefringence for several other materials. 2. Material and methods 2.1. Wings and scales of butterflies Wings of the butterflies Eryphanis reevesi (Doubleday, 1849) and Morpho aega (Hübner, 1822) (Nymphalidæ) were used. Isolated whole scales were obtained by touching the blue iridescent sur-
face of the wings to the surface of histological slides, applying a light pressure to promote adherence of the scales on the slides, and drying the preparations at room temperature. 2.2. Fluorescence Fluorescence of the wing scales was observed by epiillumination in a Zeiss Axiophot-2 microscope (Eching/Munich, Germany) using the filters UV–G363/FT-395/LP-420 and blue excitation 485/20, FT-500/600. 2.3. Birefringence and linear dichroism Polarized light microscopy was used to search for linear dichroism (LD) and birefringence using an Olympus BX51 polarized light microscope. LD was studied after removal of the microscope analyzer and after the long axis of the scales was oriented successively parallel and perpendicular to the polarizer plane of polarized light (PPL). To determine if the birefringence was positive or negative, and to detect the orientation of the fibrillar components of the wing scales, Sénarmont’s and Bräce-Köhler’s compensators were used. Form birefringence was measured after immersing the scales in Cargille oils of different refractive indexes (n = 1.33–1.70) according to previously described procedures (Vidal, 1965, 1980, 1986, 2010).The slides were covered by coverslips and the imbibition of the preparations lasted for 10–24 h. Measurements of optical path differences due to the birefringence were performed with a Sénarmont (, 1/4) and/or a Brace-Köhler compensator depending on the birefringence intensity. 2.4. FT-IR FT-IR investigation was done using the Illuminat IR IITM microspectrometer (Smiths Detection, Danbury, USA) with a liquid nitrogen-cooled mercury–cadmium–telluride (MCT) detector and Grams/AI 8.0 spectroscopy software (Thermo Electron Corporation, Waltham, USA). An attenuated total reflection (ATR) diamond objective, magnification 36× was employed. The diamond objective ATR 36× generally avoids the dispersion and Mie scattering that are more common using all reflecting objectives (ARO). 3. Results and discussion 3.1. General aspects The wings of both butterfly species exhibit a strong iridescent glow depending on the incident light angle. The entire surface of the wing in M. aega exhibited iridescence (Fig. 1), whereas only a particular region of the wing in E. reevesi revealed an iridescent glow (Fig. 2). Even the scales isolated on slides showed iridescence when illuminated by light from different directions (data not shown). The scales are ordered and fixed by peg-and-socked attachments on the surface of a matrix such that the scales of all species appear arranged in parallel rows like shingles on a house, as was previously reported (Prum et al., 2006). In E. reevesi as well as in M. aega, the ordered scales are separated into two layers. The scales, from one layer to another, are distributed out of phase. The more superficial layer of the scales is darker and likely to be responsible for the iridescent colors. Beneath this layer, there is another layer, composed of clear scales that fit the description of glass scales (Fig. 4A and B) (Prum et al., 2006). The brown color of these scales has been reported to be due to melanin (Matèjková-Plskova et al., 2009). Taking in consideration that the scales are composed by chitins, and that in arthropods chitin is darkened by tanning phenolic processes (Neville, 1975), it is proposed that the dark appearance
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Fig. 1. Iridescent image of half wings of M. aega under orthogonal illumination. The iridescence covers the entire surface of the wings.
of the scales in the present case may also be caused by a similar process. 3.2. Fluorescence The dark scales of M. aega and E. reevesi, when excited by UV light ( = 363 nm), and provided that the long-pass filter LP-420 is used, show a blue fluorescence ( in the range of 420–460 nm, obeying the Stokes law) (Fig. 3A and B). They show a green fluorescence when excited by blue light (data not shown). No difference in fluorescence intensity or hues was detected when comparing M. aega to E. reevesi.
Fig. 2. Iridescent image of half wings of M. reevesi under orthogonal illumination. The iridescence is limited to certain areas of the wings.
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Vukusic and van Hooper (2005) have described fluorescence in the butterfly Princeps nireus, uttering that the periodic photonic crystal in the wing scales is infused with a highly fluorescent pigment; they tested whether there was an “out of plane emission”, by studying the specimen with and without immersion in an index-matching fluid. They have also described that the intense fluorescence they described was directionally enhanced by a Dichroic Brag Reflector (DBR). Diener et al. (2001) reported construction of a DBR based on birefringence, which is in the context of the anisotropic findings described in the present work. Vukusic and van Hooper (2005) concluded that immersion of the butterfly wing effectively removes the fluorescence due to a nanostructure’s photonic influence. It must be emphasized that this method is similar to the method used here to determine the intrinsic birefringence in the F curve. A 3-D fluorescence study of wings of the butterfly Morphoa sulkowskyi has been reported by Kumazawa and Tabata (2001), relating a peak emission near = 410 nm to a mixture of three pteridin pigments; they also attributed the emission peak at 470 nm (blue fluorescence) in Papilio xuthus wings to the papiliochrom II pigment. Considering the results obtained by Vukusic and van Hooper (2005), it is concluded that the observed fluorescence in the scales of the butterflies M. aega and E. reevesi is due to a photonic periodic structure. 3.3. Anisotropic optical properties: linear dichroism (LD)and birefringence The images observed by optical anisotropy in the wing scales allowed the detection of their morphological characteristics. 3.3.1. Linear dichroism (LD) The scales of M. aega and E. reevesi when attached to the wing matrix or when isolated on glass slides displayed LD visually. LD is recognized by selective absorptions of polarized light with respect to the plane of polarized light (PPL). In this case, when the long axis of the scales was parallel to the PPL the scales appear darker than when the scales long axis was perpendicular to the PPL. A second layer of scales (clear scales) out of focus may appear underneath (Fig. 4A and B). It is important to consider the influence of the matrix wing on the whole wing thickness in the total structure focus on the differences of absorption. The LD observed in the wing is dependent on the periodic ordered distribution of the ribs, columns and their dimensions of scales. LD observed in isolated scales is more evident. When the long axis of a scale is parallel to the PPL, there is a predominance of orange-yellow colors; when this axis is perpendicular to the PPL, a green color is predominant. The scale tip is very dark, and a small area of the scale, at its insertion region, is yellow. Variation of LD colors is detected in all other orientations of the scales as regards the PPL (Fig. 4C). Actually, in this case the LD can be designed as a pleochroism, and it is based on DBR (Diener et al., 2001; Vukusic and van Hooper, 2005). The LD shown by air-dried scales was more intense than that observed after imbibing the scales in Cargille oils (data not shown). This finding is in agreement with previous reports on dichroism properties (Fig. 7 in Schmidt, 1937). 3.3.2. Birefringence The wing scales of the butterflies studied here revealed positive intrinsic (I ) and form birefringence (F ) after these structures were imbibed in a series of oils of different refractive indices. As the observed images of anisotropical anisotropy of M. aega and E. reevesi were very similar, all the figures shown are from E. reevesi. The scale column’s birefringence decreases at the point of their contact with the scale ribs (Fig. 5). Such a finding allowed the
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Fig. 3. Blue fluorescence image of scales from M. aega (A), and from E. reevesi (B). Bar = 30 m.
inference that the rib fibrilar structure could insert into the column in a subtraction way; in terms of chitin fibrilar and molecular orientation, this induces an internal compensation of the column birefringence. Birefringence of the scales causes an optical retardation or optical path difference (OR) visible in the field of observation as brilliant images that allowed detection of various scale characteristics. Emphasis should be given to the fact that the birefringent image has a 3D-dimensional information which results from a bidimensional information provided by the topographic relationship of the object optical axis with the PPL, plus the intensity of brilliance that directly depends on the object thickness and the concentration of the birefringent material (details in Vidal, 2010, Figs. 1 and 2; Vidal and Mello, 2010). The birefringent object appears brilliant
against a dark field; the brilliant ribs are thus observed perpendicular to the columns and ordered among them. Compensation of the column birefringence using a Bräce-Köhler’s or a Sénarmont’s compensator provides a better visualization of the column morphology. It is important to consider that the morphological information presented here is generated at the level of molecular orientation (nanometric dimension) of the structures. When performing OR measurements, the birefringence corresponding to the long axis of the columns compensates perpendicular to the ribs (Fig. 6). Compensation of the ribs’ birefringence occurs when they are positioned with an orientation of 90◦ in relation to the orientation of the columns. After this compensation, the ribs appear dark, whereas the columns continue to show a brilliant birefringence (Fig. 7).
Fig. 4. Whole mount of a wing fragment of M. aega. The images were obtained for part of the blue iridescent wing illuminated by polarized light. (A) The long axis of the scales was oriented parallel to the PPL, revealing higher absorptions than when the scales are oriented perpendicular to the PPL (B). Differences in absorption result in linear dichroism (LD). The lower layer with clear scales appears out of the focus plane. (C) Isolated scales illuminated with polychromatic polarized light such that the direction of the PPL was east to west. When the long axis of the scales (pad-socket to apical direction) was parallel to the PPL, the wing shows colors from orange to orange-green; in the 1/3 apical region, the scales were clear. When the long axis of the scales was perpendicular to the PPL, the colors were predominantly green, and they were darker than under parallel orientation. The color varied with the orientation of the scale relative to the PPL, and is dependent on each wavelength (coherent light scattering). Bar = 30 m.
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Fig. 8. Birefringence curve for scales of the butterfly wings represented by a scatter diagram of optical retardation (OR) with respect to refractive index values. Dashed line, E. reevesi; solid line, M. aega. Fig. 5. Birefringence image of a E. reevesi wing scale. The long axis of the scale was oriented at 45◦ with respect to the crossed polarizers of the PPL. The birefringence of the ribs appears more brilliant than that of the columns. The interference colors observed here are due to the dispersion of the birefringence. Bar = 20 m.
Fig. 6. Image of the compensated birefringence on the columns of the same scale depicted in Fig. 5. The columns now appear black and the ribs show a strong brilliance. Bar = 20 m.
Fig. 7. Same scale as in Fig. 5 after compensation of the ribs’ birefringence. The columns appear brilliant. Bar = 20 m.
A scatter diagram dispersion of form birefringence was constructed for F and I comparison of the wings’ scales of M. aega and E. reevesi. The minimal OR value, corresponding to I , was found at n = 1.55 for both M. aega and E. reevesi (Fig. 8). Generally, a F curve is considered to be a U-shaped parabola-like curve (Wiener, 1912; Schmidt, 1937; Frey-Wyssling, 1948; Schmidt and Keil, 1958; Bennet, 1967). Actually, the geometry of the curves varies as a function of the composition and relative concentration of each oriented component that plays a role in the supramolecular architecture under study. In the present case, the shape of the scatter diagram indicates a tendency to a hyperbolic shape; the line from n = 1.00 to n = 1.55 gives the sharp feature to the scatter diagram, supporting the conclusion that, for both butterfly wing scales, the major component causing form birefringent is the same as for cuticles, i.e., chitin. Similar results have been reported for the chitinous tendon of the grasshopper and the pen of Loligo brasiliensis (Vidal and Carvalho, 1986; Carvalho and Vidal, 1991). The major difference between species in the scatter diagram for scales was found at n = 1.50, n = 1.60 and n = 1.70, which could be caused by variations in the ordered arrangements of their periodic constructions. A hypothesis could be raised that where the ribs of M. reevesi are in contact with a column they penetrate the column with different directions of its chitin fibrilar entanglement, originating differences at the level of the apparent birefringence intensity. Reports from electron scanning microscopy give support to this interpretation (see figures in Huang et al., 2006; Matèjková-Plskova et al., 2009). Because the birefringence measurements were performed for entire scales and the polarized photons of light travel perpendicular to the three dimensional lattice of the scale’s periodic construction, the sum of these interactions with the nano-units of the scale causes the anisotropic optical phenomena reported here. This information is not obtained with electron tomography, which is limited by the thickness of the specimen, although Shawkey et al. (2009) have interpreted that thickness can have enough repeating units to allow Fourier analysis to be meaningful. In the case of the optical anisotropies the wave of photons traveling through the scale can give a better statistical approach to the statistical meaning of its structural orientation. Biophotonic properties are not restrained to butterflies. These properties have been reported in non-chitinous quasi-periodic nanostructures of keratinous bird feathers and King Penguin beaks (Dresp et al., 2005; Shawkey et al., 2009), and in collagen fibers (Prum and Torres, 2003). In this context, it is pertinent citing that collagen fibers induce nanometric silver particles to link in their nanostructure, causing LD and abnormal dispersion of birefrin-
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chitin exists in the scales as a macromolecule making nanometric aggregate ordered units with a periodic supramolecular structure. The FT-IR data obtained with polarized IR light revealed a positive LD and provided results which are in agreement with birefringence and visible light LD observations. Acknowledgments The support of Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants no. 2003/04597-0 and 2007/058251 is gratefully acknowledged. The author is indebted to Dr. Maria Luiza S. Mello and Dr. André Victor Lucci Freitas for providing him with the butterfly specimens. References Fig. 9. FT-IR spectrum for LD exhibited by butterfly wing scales. Measurements were carried out using polarized infrared light. The red and blue lines refer to absorptions when the long axis of the scales was parallel and perpendicular, respectively, to the plane of polarization of the IR light.
gence phenomena recognized to be photonic (Vidal et al., 1975; Vidal and Joazeiro, 2002; Vidal, 2003). An interesting and inspiring example of practical application of these concepts is the constructed nanomotor that moves by a photonic moment (Neale et al., 2005). A question that may arise from this example is “Could a photonic moment be generated in the wings of, for instance, M. aega that makes flying easier, especially considering that their wings present a large area of iridescence?” Furthermore, what is the biophotonic role in the evolutionary process of the butterflies? 3.4. FT-IR analysis and LD Data were obtained only for scales of M. aega because of the similarity in the above-mentioned LD birefringence data between the two butterfly species. The results obtained for FT-IR of M. aega scales are similar, but not identical, to those already reported for chitin and chitosan (Neville, 1975; Brunner et al., 2009; Skorig et al., 2010; Sudheesh Kumar et al., 2010). However, the present reports are considered to be the first approach to pave future developments in the LD explored by FT-IR. Vibrational absorption peaks were found at the wavenumbers: 3287 cm−1 ,corresponding to (–N–H) symmetric and asymmetric stretching, 2923 and 2850 cm−1 , corresponding to (–C–H) symmetric stretching, 1644 cm−1 , corresponding to amide I peak (–C O) stretching, 1550–1530 cm−1 , corresponding to amide II (–N–H), and 895–902 cm−1 , corresponding to -linking. Examination of the scales with polarized IR demonstrated a positive LD, i.e., when the long axis of the scales was parallel to the PPL of the IR light, higher vibrational absorptions were detected (Fig. 9). The differences found in the LD characteristics when comparing the scales of M. aega and other chitin-containing materials (Neville, 1975), may be due to differences in their nanostructure supraorganizations. 4. Conclusions The wing scales of the butterflies M. aega and E. reevesi exhibit fluorescence, LD, and F and I properties when examined with fluorescence, polarization and IR microscopy. The LD displayed by the scales at the visible light is due to the scale periodic structure and its Dichroic Brag Reflector properties. F represents 91.44% of the total birefringence of the scales, whereas I represents only 8.56% of the total birefringence. The higher participation of F in the total birefringence confirms that
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