Structural color in Myxomycetes

Structural color in Myxomycetes Marina Inchaussandague,1,∗ Diana Skigin,1 Cecilia Carmaran,2 and Sonia Rosenfeldt2 1 Grupo

de Electromagnetismo Aplicado, Departamento de F´ısica, FCEN, Universidad de Buenos Aires, and IFIBA, CONICET Ciudad Universitaria, Pabell´on I, C1428EHA Buenos Aires, Argentina 2 Departamento de Biodiversidad y Biolog´ıa Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabell´on II, C1428EHA Buenos Aires, Argentina *[email protected]

Abstract: In this paper we report evidence of structural color in Myxomycetes, a group of eukaryotic microorganisms with an uncertain taxonomic position. We investigated the Diachea leucopoda, which belongs to the Physarales order, Myxomycetes class, and found that its peridium -protective layer that encloses the mass of spores- is basically a corrugated layer of a transparent material, which produces a multicolored pointillistic effect, characteristic of this species. Scanning (SEM) and transmission (TEM) electron microspcopy techniques have been employed to characterize the samples. A simple optical model of a planar slab is proposed to calculate the reflectance. The chromaticity coordinates are obtained, and the results confirm that the color observed is a result of an interference effect. © 2010 Optical Society of America OCIS codes: (000.1430) Biology and medicine; (240.0310) Thin films; (260.3160) Interference; (310.6860) Thin films, optical properties; (330.1690) Color.

References and links 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

S. Berthier, Iridescences, the physical colours of insects, (Springer Science+Business Media, LLC, France, 2007). S. Kinoshita, Structural colors in the realm of nature, (World Scientific Publishing Co., Singapore, 2008). A. Parker, “515 million years of structural colour,” J. Opt. A, Pure Appl. Opt. 2, R15-R28 (2000). M. Srinivasarao, “Nano-optics in the biological world: beetles, butterflies, birds, and moths,” Chem. Rev. 99, 1935-1961 (1999). P. Vukusic and J. R. Sambles, “Photonic structures in biology,” Nature 424, 852-855 (2003). P. Vukusic and D. G. Stavenga, “Physical methods for investigating structural colours in biological systems,” J. R. Soc. Interface 6, S133–S148 (2009). S. M. Doucet and M. G. Meadows, “Iridescence: a functional perspective,” J. R. Soc. Interface 6, S115-S132 (2009). S. Yoshioka and S. Kinoshita, “Single-scale spectroscopy of structurally colored butterflies: measurements of quantified reflectance and transmittance,” J. Opt. Soc. Am. A 23, 134–141 (2006). W. Zhang, D. Zhang, T. Fan, J. Ding, J. Gu, Q. Guo, and H. Ogawa, “Biomimetic zinc oxide replica with structural color using butterfly (Ideopsis similis) wings as templates,” Bioinsp. Biomim. 1, 89-95 (2006). R. J. Mart´ın-Palma, C. G. Pantano, and A. Lakhtakia, “Biomimetization of butterfly wings by the conformalevaporated-film-by rotation technique for photonics,” Appl. Phys. Lett. 93, 083901 (2008). “Biomimetics and bioinspiration,” Proceedings of SPIE - The International Society for Optical Engineering Volume 7401, 183 (2009). S. Stephenson and H. Stempen, “Myxomycetes. A handbook of slime molds,” Timber Press, Hong Kong, pp. 183 (2000). H. W. Keller, M. Skrabal, U. Eliasson, and T. Gaither, “Tree canopy biodiversity in the Great Smoky Mountains National Park: Ecological and developmental observations of a new Myxomycete species of Diachea,” Mycologia 96, 537–547 (2004).

#130057 - $15.00 USD

(C) 2010 OSA

Received 14 Jun 2010; revised 28 Jun 2010; accepted 29 Jun 2010; published 14 Jul 2010

19 July 2010 / Vol. 18, No. 15 / OPTICS EXPRESS 16055

14. J. D. Schoknecht and H. W. Keller, “Peridial composition of white fructifications in the trichiales (Perichaena and Dianema),” Can. J. Bot. 55, 1807–1819 (1977). 15. H. C. Aldrich, “Influence of inorganic ions on color of lime in the myxomycetes,” Mycologia 74, 404–411 (1982). 16. T. W. Gaither and H. W. Keller, “Taxonomic comparison of Diachea subsessilis and D. Deviata (Myxomycetes, Didymiaceae) using scanning electron microscopy,” Syst. Geogr. Pl. 74, 217–230 (2004). 17. T. P. O’Brien and M. E. McCully, “The study of plant structure. Principles and selected methods,” Termarcarphi Pty. Ltd., Melbourne, Australia (1981). 18. U. Eliasson, “Ultrastructure of Lycogala and Reticularia,” Trans. Br. Mycol. Soc. 77, 243–249 (1981). 19. E. F. Haskins and M. D. McGuiness, “Sporophore ultrastructure of Echinostelium arboreum,” Mycologia 81, 303–307 (1989). 20. R. McHugh, and C. Reid, “Sporangial ultrastructure of Hemitrichia minor (Myxomycetes: Trichiales),” Mycological Research 94, 1144–1146 (1990). 21. E. Hecht, Optica, (Addison Wesley Iberoamericana ed., Madrid, 2000). 22. R. Lozano, El color y su medici´on, (Americalee Ed., Argentina, 1978). 23. B. Gralak, G. Tayeb and S. Enoch, “Morpho butterflies wings color modeled with lamellar grating theory,” Opt. Express 9, 567 (2001). 24. The website EasyRGB http://www.easyrgb.com has the application Color calculator which converts color data to different color standards.

1.

Introduction

Structural color in the biological world has recently attracted the attention of biologists and physicists [1, 2]. The study of iridescent coloration provides insight into the fundamentals of optics [3–6], and also contributes to biological sciences by identifying their behavioural functions such as communication, thermoregulation, camouflage, and predator deterrence [7]. Besides, natural structures inspire biomimetic technologies for applications in different industries related to color [8–11]. Iridescent colors are found in a broad diversity of animals and plants, and they are produced by the selective reflectance of incident light by the microscopic structures present in their cover tissues. The hue often changes with viewing angle, and the color is often very intense and highly saturated. Optical mechanisms such as interference, diffraction and scattering are involved to achieve colorful patterns and metallic colors. These effects usually appear considerably brighter than those of pigments, although they often result from completely transparent materials. The Myxomycetes are a group of organisms that exhibit characteristics of both fungi and animals, and are considered to be more closely related to the protozoans [12]. These organisms show very particular morphologies, presenting plasmodia that eventually sporulate developing different types of fruiting body. There are some genera which exhibit bright colors. One of these genera is Diachea, which belongs to the Physarales order. Species of this genus are found on ground habitats such as leaf litter, little pieces of wood, among others. Diachea leucopoda (Bull.) Rostaf. is characterized by a cylindrical stalked fruiting body (sporangia), with a thin, external membranous layer (peridium), that contains very small dark brown spores. The stalk is typically calcareous. The peridium is a thin layer that covers the mass of spores and a structure called capillitium, consisting of branched threads, sometimes with cross connections [13]. The Myxomycetes present a great variety of colors that have been studied in connection to their utility as a taxonomic tool. A few works have given details about the nature of color in Myxomycetes [14–16]. Aldrich used energy dispersive X-ray spectroscopy combined with scanning electron microscopy to examine several species of Myxomycetes to determine whether the presence of specific inorganic ions correlated with particular colors in the peridium. He suggested that inorganic elements contribute to the bright colors characteristic of several members of the order Physarales [15]. However, Diachea leucopoda has not been included in this investigation. Gaither and Keller studied specimens of Diachea subsessilis and D. Deviata and found that the peridium of D. subsessilis displays beautiful bronze iridescent colors, sometimes tinged with blue, whereas the peridium of D. Deviata lacks iridescent colors [16]. They mentioned for

#130057 - $15.00 USD

(C) 2010 OSA

Received 14 Jun 2010; revised 28 Jun 2010; accepted 29 Jun 2010; published 14 Jul 2010

19 July 2010 / Vol. 18, No. 15 / OPTICS EXPRESS 16056

the first time that iridescent color in Diachea could be related to structural characteristics. They observed that the membranous peridium is colorless in water mounts, and this suggests that pigments are not involved in the color production. To the best of our knowledge, no further efforts have been made to elucidate the origin of the bright colors present in members of this group of organisms. In this paper we investigate the color present in Diachea leucopoda, a species of Myxomycetes. Fresh samples were collected and observed by different microscopy techniques. We found that the multicolored puntillistic effect is the result of the interference of light within the structure of the peridium, i.e., the thin transparent layer that covers the sporangium. The peridium is a multilayer structure and its surface exhibits a periodic distribution of bumps. An electromagnetic model was developed to calculate the reflectance and the color of the system, and the numerical results confirm the existence of structural color in Myxomycetes. 2.

Materials and Methods

The material of Diachea leucopoda was collected in Santa Catalina, Buenos Aires province, Argentina. Also, herbarium material from Buenos Aires (Argentina) on bark of Melia azederach and Fragaria species, and from Maryland (USA) was used. The peridium was observed by an Olympus SZ6045 stereoscopic microscope, and images were captured with a digital camera. The samples were also observed by an Olympus BX60M Brightfield reflected light metallurgical microscope, and in this case the images were captured by a Photometrics CoolSnapc f camera. The microstructure of the peridium was characterized by a scanning electron microscope Zeiss Supra 40 FESEM, previous an Au sputtering treatment of 5 - 10 nm. For scanning electron microscopy (SEM) studies, herbarium material was used. Also, scanning micrographs were taken with a Philips SEM 505 microscope; the sputtering treatment was made with goldpalladium for 3 minutes. For transmission electron microscopy (TEM) studies, the material was pre-fixed in 2,5% glutaraldehyde in phosphate buffer (pH 7,2) for 2 hours and then post-fixed in OsO4 at 2◦ C in the same buffer for 3 hours, was dehydrated in ethanol series and embedded in Spurr’s resin. Fine sections were made on a Sorvall ultramicrotome, stained with uranyl acetate and lead citrate [17]. The sections were observed and photographed in a JEOL - JEM 1200 EX II TEM at 85.0 Kv. 3. 3.1.

Results Color observation

In Fig. 1 we show images of Diachea leucopoda observed under the microscope with different magnifications. A dehiscent peridium, typical of mature sporangia, is shown. The peridium breaks at the apex with portions remaining intact and attached to the capillitium in the lower half. When fresh samples are observed under an optical microscope, the peridium exhibits pixels of bright colors mounted on a dark background. The optical microscope images of the peridium are shown in Fig. 1. The observed colors and their distribution over the peridium surface depend strongly on the samples examined. Some of the species present a mix of many colors along the surface of the whole peridium. Conversely, in others, areas with different hues can be distinguished, typically orange, blue and purple (Fig. 1). Due to the fragility of the peridium, it is extremely difficult to separate it from the sporangium for better observation. However, several images obtained from small fragments of peridium that became detached from the sporangium in the sample preparation procedure, evidence that the

#130057 - $15.00 USD

(C) 2010 OSA

Received 14 Jun 2010; revised 28 Jun 2010; accepted 29 Jun 2010; published 14 Jul 2010

19 July 2010 / Vol. 18, No. 15 / OPTICS EXPRESS 16057

(a)

(b)

Fig. 1. Diachea leucopoda observed under the optical microscope with different magnifications.

(a)

(b)

(c)

Fig. 2. Scanning electron microscope images of the peridium with different magnifications.

peridium is a transparent film, as already noticed in [16]. This interesting observation suggests that the bright colors observed in the peridium are not related to pigments but rather they are a result from interference effects in a completely transparent material. 3.2.

Structural characterization

Scanning electron microscope (SEM) images of the peridium are shown in Fig. 2. A typical sporangium of Diachea leucopoda, with the multiple branches of the capillitium and the mass of spores, is observed in Fig. 2(a). The peridium is the thin and dehiscent layer that surrounds

#130057 - $15.00 USD

(C) 2010 OSA

Received 14 Jun 2010; revised 28 Jun 2010; accepted 29 Jun 2010; published 14 Jul 2010

19 July 2010 / Vol. 18, No. 15 / OPTICS EXPRESS 16058

(a)

(b)

(c)

(d)

Fig. 3. Peridium cross section observed under SEM [(a), (b) and (c)] and TEM (d).

the sporangium. In the figure, peridium appears broken at the apex with portions remaining intact and attached to the capillitium in the lower half. As the peridium is supported by the spores, its surface takes the form of a fairly regular array of protuberances or bumps of heights ≈ 5μ , smoothly separated a distance of 10 μ m, approximately [Figs. 2(b) and 2(c)]. In Fig. 2(b), some spores that have fallen out of their branches can be observed. Figures 3(a)–3(c) show SEM images of the peridium cross section. Detailed observations on different samples and on different parts of the same specimen reveal that the peridium thickness is not uniform. For example, in Fig. 3(a) the thickness of the peridium exhibits variations between 300 and 700 nm, whereas in Figs. 3(b) and 3(c) the thickness is approximately 200 nm and does not vary significantly along the different parts of the fragment studied. Although the fractures of the peridium are very irregular, some interesting features of its cross section structure can be appreciated. We observe a dense material which presents very thin layers of air of thicknesses smaller than ≈ 10 nm in localized areas. The images show that there are areas with several layers of air (5, 6 or more) and areas in which no layers are observed. The external part of the peridium presents a kind of shell with rather periodic protuberances of height and period smaller than 100 nm. Its material is labile and fragile, and in some images [as in Fig. 3(c)] it appears folded upon itself as a consequence of cutting. In Fig. 3(d) a TEM image of the peridium cross section is shown. Since the roughness of its topography reduces the optical density of the outer regions of the peridium, these zones appear more traslucid than the central part.

#130057 - $15.00 USD

(C) 2010 OSA

Received 14 Jun 2010; revised 28 Jun 2010; accepted 29 Jun 2010; published 14 Jul 2010

19 July 2010 / Vol. 18, No. 15 / OPTICS EXPRESS 16059

incident light

θ0 n1 d

n2

peridium

n3

sporangium

(a)

(b)

Fig. 4. Simplified model for the scattering process within the peridium. (a) For normal incidence, the light impinges upon the sample with different local angles; (b) the system is locally represented by a planar slab with varying incidence angle.

3.3.

Model and color calculation

As stated above, the peridium is a multilayer structure with air layers of thickness ≈ 10 nm, and its topography exhibits periodic bumps of period around 10 μ m. Since this period is larger than the visible wavelengths, no diffraction effects are expected to influence the observed colors. On the other hand, the thickness of the air layers is much smaller than the visible wavelengths, and then their effects can be accounted for by means of an effective refraction index of the peridium. Therefore, a simple model is proposed to account for the color effect observed in the Diachea leucopoda, which consists in representing the peridium as a dielectric slab. For a fixed incidence, light impinges upon the sample with different local angles, depending on the local curvature of the peridium, as schematically shown in Fig. 4(a). The significant parameters of our model are the layer thickness d, its dielectric permittivity ε2 , and the local angle of incidence θ0 . There are only a few works that report information about the peridium thickness in Myxomycetes [18–20], and the available data suggest that the thickness is very variable. According to these works and to our observations in the SEM and TEM images for several samples, at different parts of the same individual the peridium thickness is not uniform, and ranges from 50 to 500 nm. Due to the size and geometry of the microorganism under study, it is extremely difficult to optically characterize the peridium. Moreover, no measurements of its refraction index have been reported in the literature. Therefore, in our model we consider ε ranging from 1.79 to 3.34, taking into account that refraction indices that are widespread in nature span from 1.34 for cytoplasm to 1.83 for guanine crystals [1]. The system is schematized in Fig. 4(b). The reflectance of a planar slab between two media is given by [2]: q q q q (1) rq = r12 + t12 r23 t21 eiφ κ q , q q iφ r21 e ), φ = 4π n2 d cos θ2 /λ , λ is the incident wavelength, θ2 is the rewhere κ q = 1/(1 − r23 fraction angle in medium 2, the superscript q = s, p denotes the polarization state (s corresponds to the electric field perpendicular to the plane of incidence and p corresponds to the electric field

#130057 - $15.00 USD

(C) 2010 OSA

Received 14 Jun 2010; revised 28 Jun 2010; accepted 29 Jun 2010; published 14 Jul 2010

19 July 2010 / Vol. 18, No. 15 / OPTICS EXPRESS 16060

parallel to the plane of incidence), and rijq and tijq are the reflection and transmission coefficients at an interface for the light propagating from medium i to j, and their expressions for each polarization state can be found in textbooks [21]. If the refraction indices of the media involved are real quantities, the difference of the reflectivity between s and p polarizations affects the amplitude of the reflected light. Since the sample is illuminated by unpolarized light, we consider that the incident field has two components, s and p. In this work we assume equal amplitudes of both components, and calculate the average reflectance R = (rs + r p )/2. This reflectance is used to calculate the observed colors. In 1931, the International Commission on Illumination (CIE) defined three standard primaries, the CIE X, Y, and Z tristimulus values. The corresponding functions x, y, and z are called color-matching functions. The y color-matching function is defined to match the eye’s sensitivity to brightness; the other two do not correlate with any perceptual attibutes. X, Y and Z represent the weights of the respective color-matching functions needed to approximate a particular spectrum [22]. Let us consider that the body under study is illuminated by an illuminant characterized by its energy distribution D(λ ). If the body has a reflectivity R(λ ), the tristimulus values can be computed by the formulae [23] 

1 D(λ ) R(λ ) x(λ ) d λ , k  1 D(λ ) R(λ ) y(λ ) d λ , k  1 D(λ ) R(λ ) z(λ ) d λ , k

X = Y

=

Z

=

(2)

where k is a normalization factor defined in such a way that an object with a uniform reflectivity R(λ ) = 1 gives a luminance component Y equal to 1. Since the observation of color in the Diachea leucopoda samples is done through an optical microscope, in this paper we use the CIE standard illuminant A, which is intended to represent typical, domestic, tungsten-filament lighting. This illuminant is used in all applications of colorimetry involving the use of incandescent lighting [22]. To analyze the color observed by the human eye, it is enough to retain in the integrals of Eq. (2) only the wavelengths within the range 380 - 780 nm. To visualize the colors in the screen, the XYZ components are converted into RGB components through a linear transformation [24]. Since the human eye has three types of color sensors that respond to different ranges of wavelengths, a full plot of all visible colors is a three-dimensional figure. However, the concept of color can be divided into two parts: brightness and chromaticity. The CIE XYZ color space was deliberately designed so that the Y parameter was a measure of the brightness of a color. The chromaticity of a color was then specified by the two derived parameters x and y, two of the three normalized values which are functions of all three tristimulus values X, Y, and Z: x

=

y = z

=

X , X+Y+Z Y , X+Y+Z Z . X+Y+Z

(3)

The chromaticity diagram is then a 2D plot, where the chromaticity of a color can be represented. In this paper we use this kind of diagrams to illustrate the color variation with the relevant parameters of the model. #130057 - $15.00 USD

(C) 2010 OSA

Received 14 Jun 2010; revised 28 Jun 2010; accepted 29 Jun 2010; published 14 Jul 2010

19 July 2010 / Vol. 18, No. 15 / OPTICS EXPRESS 16061

(a)

(c)

(b)

θ0=75° θ0=75° θ0=0

θ0=0

Fig. 5. Chromaticity coordinates of a homogeneous slab. (a) Normal incidence, for varying n2 d; (b) d = 200 nm, n2 = 1.48, for varying incidence angle; (c) d = 500 nm, n2 = 1.58, for varying incidence angle. The arrows indicate the direction of increasing n2 d (a) or θ0 (b and c).

As it is well known, for normal incidence the condition for constructive interference depends on n2 d. Therefore, we analyze the dependence of the color with this parameter. In Fig. 5(a) we show the chromaticity coordinates calculated using the reflectance of a homogeneous slab, for varying n2 d, with n2 being its refraction index, 1.34 < n2 < 1.83 and 200 nm < d < 500 nm. For the smallest values of n2 d considered, the chromaticity coordinates are located in the orange-red zone of the diagram, and as this parameter is increased, the coordinates move to the blue region, to come back to the orange region through the green-yellow zone. As n2 d is further increased, the points move to the green region to finally end in the orange-pink zone. The behaviour of the chromaticity coordinates evidences that the resulting color is highly dependent on n2 d. Therefore, even if the material of the peridium is considered homogeneous and uniform all along the sample, it is to expect that variations of the thickness would produce significant changes in the observed color. This result confirms that the peridium thickness plays an important role in the color generation. To analyze the iridescent effect, in Fig. 5(b) we show the chromaticity coordinates for a fixed thickness d = 200 nm and for n2 = 1.48, for several values of the incidence angle θ0 . For this particular set of parameters, the color for normal incidence is mainly blue, and as the incidence angle is increased it moves towars the orange region. However, it is important to remark that for other pairs of parameters d and n2 , this dependence can vary significantly and the color coordinates cover a completely different path while the incidence angle is changed, as can be observed in Fig. 5(c) for d = 500 nm and n2 = 1.58. In this case, for normal incidence we get a color in the red region, which turns to the yellow-green zone as the incidence angle is increased. Consequently, the proposed model accounts for the multiple colors observed in the samples (Fig. 1). In this simplified approach, the hues depend on several parameters such as the refraction index, the peridium thickness and the incidence angle. 4.

Discussion

The origin of the bright colors and the pointillistic effect exhibited by Diachea leucopoda was investigated. The peridium was identified as the layer responsible for the color generation in this species. Its topography and internal structure were characterized using SEM and TEM techniques. The peridium topography presents bumps of diameters around 10 μ m. Light reflected #130057 - $15.00 USD

(C) 2010 OSA

Received 14 Jun 2010; revised 28 Jun 2010; accepted 29 Jun 2010; published 14 Jul 2010

19 July 2010 / Vol. 18, No. 15 / OPTICS EXPRESS 16062

by these protuberances produces the pointillistic effect, since only the light that impinges in the vicinity of the top parts is collected back and observed. It was found that the peridium is an inhomogeneous multilayer structure, with air layers of thicknesses of a few nanometers. The total thickness of the peridium varies significantly along the analyzed samples (between 200 and 700 nm), and in different samples. The peridium was modeled by a planar slab, and its reflectance was calculated for different incidence angles. The chromaticity coordinates have been obtained using the calculated reflectance, and it was found that the different hues exhibited by this species can be explained in terms of light interference in the peridium. In conclusion, this study reveals that structural color is found not only in minerals, animals, and plants, but also in Myxomycetes. The bright and multicolored effect is produced by intereference within the peridium, which is a transparent material with varying thickness along each specimen. According to our model, the color also depends on its refraction index and on the local incidence angle. Acknowledgments D. S. and M. I. acknowledge financial support from CONICET (Grant PIP 112-200801-01880), ANPCyT (ANPCYT-BID Grant No. 1728/OC-AR06-01785), and UBA (Grant X208).

#130057 - $15.00 USD

(C) 2010 OSA

Received 14 Jun 2010; revised 28 Jun 2010; accepted 29 Jun 2010; published 14 Jul 2010

19 July 2010 / Vol. 18, No. 15 / OPTICS EXPRESS 16063