structural color in myxomycetes -...

Structural color in Myxomycetes

Marina Inchaussandague,1,∗ Diana Skigin,1 Cecilia Carmaran,2 andSonia Rosenfeldt2

1Grupo de Electromagnetismo Aplicado, Departamento de Fısica,FCEN, Universidad de Buenos Aires, and IFIBA, CONICET

Ciudad Universitaria, Pabellon I, C1428EHA Buenos Aires, Argentina2Departamento de Biodiversidad y Biologıa Experimental,

Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,Ciudad Universitaria, Pabellon II, C1428EHA Buenos Aires, Argentina

*[email protected]

Abstract: In this paper we report evidence of structural color inMyxomycetes, a group of eukaryotic microorganisms with an uncertaintaxonomic position. We investigated the Diachea leucopoda, which belongsto the Physarales order, Myxomycetes class, and found that its peridium-protective layer that encloses the mass of spores- is basically a corrugatedlayer of a transparent material, which produces a multicolored pointillisticeffect, characteristic of this species. Scanning (SEM) and transmission(TEM) electron microspcopy techniques have been employed to charac-terize the samples. A simple optical model of a planar slab is proposed tocalculate the reflectance. The chromaticity coordinates are obtained, and theresults 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) Interfer-ence; (310.6860) Thin films, optical properties; (330.1690) Color.

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(2009).8. 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).9. 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).10. R. J. Martın-Palma, C. G. Pantano, and A. Lakhtakia, “Biomimetization of butterfly wings by the conformal-

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ume 7401, 183 (2009).12. S. Stephenson and H. Stempen, “Myxomycetes. A handbook of slime molds,” Timber Press, Hong Kong, pp.

183 (2000).13. 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,” Mycologia96, 537–547 (2004).

#130057 - $15.00 USD Received 14 Jun 2010; revised 28 Jun 2010; accepted 29 Jun 2010; published 14 Jul 2010(C) 2010 OSA 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 (Perichaenaand 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),” Myco-

logical Research 94, 1144–1146 (1990).21. E. Hecht, Optica, (Addison Wesley Iberoamericana ed., Madrid, 2000).22. R. Lozano, El color y su medicion, (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 andphysicists [1, 2]. The study of iridescent coloration provides insight into the fundamentals ofoptics [3–6], and also contributes to biological sciences by identifying their behavioural func-tions such as communication, thermoregulation, camouflage, and predator deterrence [7]. Be-sides, natural structures inspire biomimetic technologies for applications in different industriesrelated to color [8–11].

Iridescent colors are found in a broad diversity of animals and plants, and they are producedby the selective reflectance of incident light by the microscopic structures present in their covertissues. The hue often changes with viewing angle, and the color is often very intense and highlysaturated. Optical mechanisms such as interference, diffraction and scattering are involved toachieve colorful patterns and metallic colors. These effects usually appear considerably brighterthan those of pigments, although they often result from completely transparent materials.

The Myxomycetes are a group of organisms that exhibit characteristics of both fungi andanimals, and are considered to be more closely related to the protozoans [12]. These organismsshow very particular morphologies, presenting plasmodia that eventually sporulate developingdifferent types of fruiting body. There are some genera which exhibit bright colors. One ofthese genera is Diachea, which belongs to the Physarales order. Species of this genus are foundon 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 istypically calcareous. The peridium is a thin layer that covers the mass of spores and a structurecalled capillitium, consisting of branched threads, sometimes with cross connections [13].

The Myxomycetes present a great variety of colors that have been studied in connection totheir utility as a taxonomic tool. A few works have given details about the nature of color inMyxomycetes [14–16]. Aldrich used energy dispersive X-ray spectroscopy combined with scan-ning electron microscopy to examine several species of Myxomycetes to determine whether thepresence of specific inorganic ions correlated with particular colors in the peridium. He sug-gested that inorganic elements contribute to the bright colors characteristic of several membersof the order Physarales [15]. However, Diachea leucopoda has not been included in this investi-gation. Gaither and Keller studied specimens of Diachea subsessilis and D. Deviata and foundthat the peridium of D. subsessilis displays beautiful bronze iridescent colors, sometimes tingedwith blue, whereas the peridium of D. Deviata lacks iridescent colors [16]. They mentioned for


the first time that iridescent color in Diachea could be related to structural characteristics. Theyobserved that the membranous peridium is colorless in water mounts, and this suggests thatpigments are not involved in the color production. To the best of our knowledge, no furtherefforts have been made to elucidate the origin of the bright colors present in members of thisgroup of organisms.

In this paper we investigate the color present in Diachea leucopoda, a species of Myx-omycetes. Fresh samples were collected and observed by different microscopy techniques. Wefound that the multicolored puntillistic effect is the result of the interference of light withinthe structure of the peridium, i.e., the thin transparent layer that covers the sporangium. Theperidium is a multilayer structure and its surface exhibits a periodic distribution of bumps. Anelectromagnetic 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, Ar-gentina. Also, herbarium material from Buenos Aires (Argentina) on bark of Melia azederachand Fragaria species, and from Maryland (USA) was used.

The peridium was observed by an Olympus SZ6045 stereoscopic microscope, and imageswere captured with a digital camera. The samples were also observed by an Olympus BX60MBrightfield reflected light metallurgical microscope, and in this case the images were capturedby a Photometrics CoolSnapc f camera.

The microstructure of the peridium was characterized by a scanning electron microscopeZeiss Supra 40 FESEM, previous an Au sputtering treatment of 5 - 10 nm. For scanning elec-tron microscopy (SEM) studies, herbarium material was used. Also, scanning micrographswere taken with a Philips SEM 505 microscope; the sputtering treatment was made with gold-palladium 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 inthe 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 leadcitrate [17]. The sections were observed and photographed in a JEOL - JEM 1200 EX II TEMat 85.0 Kv.

3. Results

3.1. Color observation

In Fig. 1 we show images of Diachea leucopoda observed under the microscope with differentmagnifications. A dehiscent peridium, typical of mature sporangia, is shown. The peridiumbreaks at the apex with portions remaining intact and attached to the capillitium in the lowerhalf.

When fresh samples are observed under an optical microscope, the peridium exhibits pixelsof bright colors mounted on a dark background. The optical microscope images of the perid-ium are shown in Fig. 1. The observed colors and their distribution over the peridium surfacedepend strongly on the samples examined. Some of the species present a mix of many colorsalong the surface of the whole peridium. Conversely, in others, areas with different hues can bedistinguished, typically orange, blue and purple (Fig. 1).

Due to the fragility of the peridium, it is extremely difficult to separate it from the sporangiumfor better observation. However, several images obtained from small fragments of peridium thatbecame detached from the sporangium in the sample preparation procedure, evidence that the


(a) (b)

Fig. 1. Diachea leucopoda observed under the optical microscope with different magnifi-cations.

(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 suggeststhat the bright colors observed in the peridium are not related to pigments but rather they are aresult 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 typicalsporangium of Diachea leucopoda, with the multiple branches of the capillitium and the massof spores, is observed in Fig. 2(a). The peridium is the thin and dehiscent layer that surrounds


(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 remainingintact and attached to the capillitium in the lower half. As the peridium is supported by thespores, its surface takes the form of a fairly regular array of protuberances or bumps of heights≈ 5μ , smoothly separated a distance of 10 μm, 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 ondifferent samples and on different parts of the same specimen reveal that the peridium thick-ness is not uniform. For example, in Fig. 3(a) the thickness of the peridium exhibits variationsbetween 300 and 700 nm, whereas in Figs. 3(b) and 3(c) the thickness is approximately 200 nmand does not vary significantly along the different parts of the fragment studied. Although thefractures of the peridium are very irregular, some interesting features of its cross section struc-ture can be appreciated. We observe a dense material which presents very thin layers of air ofthicknesses smaller than ≈ 10 nm in localized areas. The images show that there are areas withseveral layers of air (5, 6 or more) and areas in which no layers are observed. The external partof the peridium presents a kind of shell with rather periodic protuberances of height and periodsmaller than 100 nm. Its material is labile and fragile, and in some images [as in Fig. 3(c)] itappears 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 itstopography reduces the optical density of the outer regions of the peridium, these zones appearmore traslucid than the central part.


incident light

sporangium

peridium

θ0

n1

n3

n2d

(a) (b)

Fig. 4. Simplified model for the scattering process within the peridium. (a) For normalincidence, the light impinges upon the sample with different local angles; (b) the system islocally 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, andits topography exhibits periodic bumps of period around 10 μm. Since this period is larger thanthe 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 theperidium. Therefore, a simple model is proposed to account for the color effect observed in theDiachea leucopoda, which consists in representing the peridium as a dielectric slab. For a fixedincidence, light impinges upon the sample with different local angles, depending on the localcurvature of the peridium, as schematically shown in Fig. 4(a).

The significant parameters of our model are the layer thickness d, its dielectric permittivityε2, and the local angle of incidence θ0. There are only a few works that report information aboutthe peridium thickness in Myxomycetes [18–20], and the available data suggest that the thick-ness is very variable. According to these works and to our observations in the SEM and TEMimages for several samples, at different parts of the same individual the peridium thicknessis not uniform, and ranges from 50 to 500 nm. Due to the size and geometry of the microor-ganism 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 ourmodel we consider ε ranging from 1.79 to 3.34, taking into account that refraction indices thatare 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 mediais given by [2]:

rq = rq12 + tq

12 rq23 tq

21 eiφ κq , (1)

where κq = 1/(1− rq23rq

21eiφ ), φ = 4πn2d cosθ2/λ , λ is the incident wavelength, θ2 is the re-fraction angle in medium 2, the superscript q = s, p denotes the polarization state (s correspondsto the electric field perpendicular to the plane of incidence and p corresponds to the electric field


parallel to the plane of incidence), and rqij and tq

ij are the reflection and transmission coefficientsat an interface for the light propagating from medium i to j, and their expressions for eachpolarization state can be found in textbooks [21].

If the refraction indices of the media involved are real quantities, the difference of the reflec-tivity between s and p polarizations affects the amplitude of the reflected light. Since the sampleis illuminated by unpolarized light, we consider that the incident field has two components, sand p. In this work we assume equal amplitudes of both components, and calculate the averagereflectance R = (rs + rp)/2. This reflectance is used to calculate the observed colors.

In 1931, the International Commission on Illumination (CIE) defined three standard pri-maries, the CIE X, Y, and Z tristimulus values. The corresponding functions x, y, and z arecalled color-matching functions. The y color-matching function is defined to match the eye’ssensitivity to brightness; the other two do not correlate with any perceptual attibutes. X, Y andZ represent the weights of the respective color-matching functions needed to approximate aparticular spectrum [22].

Let us consider that the body under study is illuminated by an illuminant characterized byits energy distribution D(λ ). If the body has a reflectivity R(λ ), the tristimulus values can becomputed by the formulae [23]

X =1k

∫D(λ )R(λ )x(λ )dλ ,

Y =1k

∫D(λ )R(λ )y(λ )dλ , (2)

Z =1k

∫D(λ )R(λ )z(λ )dλ ,

where k is a normalization factor defined in such a way that an object with a uniform reflectivityR(λ ) = 1 gives a luminance component Y equal to 1.

Since the observation of color in the Diachea leucopoda samples is done through an opticalmicroscope, in this paper we use the CIE standard illuminant A, which is intended to repre-sent typical, domestic, tungsten-filament lighting. This illuminant is used in all applications ofcolorimetry involving the use of incandescent lighting [22]. To analyze the color observed bythe human eye, it is enough to retain in the integrals of Eq. (2) only the wavelengths within therange 380 - 780 nm. To visualize the colors in the screen, the XYZ components are convertedinto RGB components through a linear transformation [24].

Since the human eye has three types of color sensors that respond to different ranges ofwavelengths, a full plot of all visible colors is a three-dimensional figure. However, the conceptof color can be divided into two parts: brightness and chromaticity. The CIE XYZ color spacewas 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 ofthe three normalized values which are functions of all three tristimulus values X, Y, and Z:

x =X

X+Y+Z,

y =Y

X+Y+Z, (3)

z =Z

X+Y+Z.

The chromaticity diagram is then a 2D plot, where the chromaticity of a color can be rep-resented. In this paper we use this kind of diagrams to illustrate the color variation with therelevant parameters of the model.


(c)(b)(a)

θ0=0

θ0=75°

θ0=0

θ0=75°

Fig. 5. Chromaticity coordinates of a homogeneous slab. (a) Normal incidence, for varyingn2 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 dependson 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 < 500nm. For the smallest values of n2 d considered, the chromaticity coordinates are located in theorange-red zone of the diagram, and as this parameter is increased, the coordinates move tothe blue region, to come back to the orange region through the green-yellow zone. As n2 d isfurther 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 de-pendent on n2 d. Therefore, even if the material of the peridium is considered homogeneousand uniform all along the sample, it is to expect that variations of the thickness would producesignificant changes in the observed color. This result confirms that the peridium thickness playsan important role in the color generation. To analyze the iridescent effect, in Fig. 5(b) we showthe chromaticity coordinates for a fixed thickness d = 200 nm and for n2 = 1.48, for severalvalues of the incidence angle θ0. For this particular set of parameters, the color for normal inci-dence 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 dependencecan vary significantly and the color coordinates cover a completely different path while theincidence angle is changed, as can be observed in Fig. 5(c) for d = 500 nm and n2 = 1.58. Inthis case, for normal incidence we get a color in the red region, which turns to the yellow-greenzone as the incidence angle is increased. Consequently, the proposed model accounts for themultiple colors observed in the samples (Fig. 1). In this simplified approach, the hues dependon several parameters such as the refraction index, the peridium thickness and the incidenceangle.

4. Discussion

The origin of the bright colors and the pointillistic effect exhibited by Diachea leucopoda wasinvestigated. The peridium was identified as the layer responsible for the color generation inthis species. Its topography and internal structure were characterized using SEM and TEM tech-niques. The peridium topography presents bumps of diameters around 10 μm. Light reflected


by these protuberances produces the pointillistic effect, since only the light that impinges in thevicinity of the top parts is collected back and observed. It was found that the peridium is aninhomogeneous multilayer structure, with air layers of thicknesses of a few nanometers. Thetotal thickness of the peridium varies significantly along the analyzed samples (between 200and 700 nm), and in different samples. The peridium was modeled by a planar slab, and its re-flectance was calculated for different incidence angles. The chromaticity coordinates have beenobtained using the calculated reflectance, and it was found that the different hues exhibited bythis 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 interef-erence within the peridium, which is a transparent material with varying thickness along eachspecimen. According to our model, the color also depends on its refraction index and on thelocal 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).


structural color in myxomycetes -...

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