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Guanine-Based Biogenic Photonic-Crystal Arrays in Fishand Spiders

By Avital Levy-Lior, Eyal Shimoni, Osip Schwartz, Efrat Gavish-Regev,

Dan Oron, Geoff Oxford, Steve Weiner, and Lia Addadi*

Biological photonic systems composed of anhydrous guanine crystals evolved

separately in several taxonomic groups. Here, two such systems found in fish

and spiders, both of which make use of anhydrous guanine crystal plates to

produce structural colors, are examined. Measurements of the photonic-

crystal structures using cryo-SEM show that the crystal plates in both fish skin

and spider integument are �20-nm thick. The reflective unit in the fish

comprises stacks of single plates alternating with �230-nm-thick cytoplasm

layers. In the spiders the plates are formed as doublet crystals, cemented by

30-nm layers of amorphous guanine, and are stacked with �200 nm of

cytoplasm between crystal doublets. They achieve light reflective properties

through the control of crystal morphology and stack dimensions, reaching

similar efficiencies of light reflectivity in both fish skin and spider integument.

The structure of guanine plates in spiders are compared with the more

common situation in which guanine occurs in the form of relatively

unorganized prismatic crystals, yielding a matt white coloration.

1. Introduction

Structural colors have captivated the human mind for over threecenturies. The first attempts to understand structural colorswere made by Robert Hooke in his book Micrographia (1665),[1]

where he described observations of peacock feathers, as well as

[*] Prof. L. Addadi, Dr. A. Levy-Lior, Prof. S. WeinerDept of Structural BiologyWeizmann Institute of ScienceRehovot 76100 (Israel)E-mail: Lia.Addadi@weizmann.ac.il

Dr. E. ShimoniElectron Microscopy UnitWeizmann Institute of ScienceRehovot 76100 (Israel)

O. Schwartz, Dr. D. OronPhysics of Complex SystemsWeizmann Institute of ScienceRehovot 76100 (Israel)

Dr. E. Gavish-RegevDepartment of ZoologyTel-Aviv University, Tel Aviv (Israel)

Dr. G. OxfordDepartment of BiologyUniversity of YorkYork YO10 5YW (UK)

DOI: 10.1002/adfm.200901437

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silverfish scales. Anderson and Richards(1942)were the first to investigate structuralcolors using electron microscopy. Theydescribed the structure of an iridescentwing-cover of the Serica sericea beetle and awing-scale of the blue Morpho butterfly.[2]

Since then, electron microscopy has beenone of the major tools used to investigatestructural colors and their spatial arrange-ments.[3–7]

Spiders, beetles, mollusks, and manyspecies of fish[8–10] and other aquaticanimals produce two main types of light-interaction effects: light-scattering andspectral (mirror-like) reflection. The latteris also known as thin-film interference.[10]

Light scattering occurs as a result of lightinteracting with non-oriented particles,while spectral reflection occurs when lightinteracts with multilayer arrays of thinfilms. Animal reflective systems are used as

camouflage, as light filters and to enhance sensitivity in eyes, andin light emitting organs (photophores) to increase the lightemitting efficiency.[10–12] The organism must produce high andlow refractive index layers to form multilayer reflector systems.One such system is composed of guanine crystals (refractive indexn¼ 1.83 in one direction) interspersed with cytoplasm layers(n� 1.36), that together form multilayer arrays [10,13].

A well-studied guanine-crystal-based reflector system is in theskin of fish. Themetallic color observed in fish skinwas first notedbyReaumur in1760 (cited inRef. [14]), but itwasnotuntil 1861 thatguanine was identified by Barreswil and Voit[14–16] as theresponsible structural component. Other purines such as uricacid and hypoxanthine are also present as minor compo-nents.[14,17–20] Guanine, uric acid, xanthine, and hypoxanthineare components of cellular metabolic processes and are thusreadily available. Guanine is by far the most commonly usedpurine in biological structural color systems.[8,21–23]

Fish coloration is believed to play an important role ascamouflage for protection against predators and in communica-tion within schools.[24] Much work has been conducted on thedistributionof theguanine crystals and their orientation in relationto the body contours of the fish. These studies showed how controlover crystal orientation in three dimensions gives rise to nearlyperfect camouflage of the fish over a wide range of angles.[8,25–28]

The crystals are intracellular and form in specialized cells,[19]

referred to in the literature as iridophores, iridocytes, leucophores,guanocytes, or guanophores. Morphological studies of fish skin

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structure have identified a number of iridophore-type cells. Someare globular, while others show dendritic morphology, dependingon the location of the cell in the skin.[9,29] Iridophores are located intwo main areas, underneath the scales[15] and adjacent to themuscular layer of the fish (Pouchet, cited in Ref. [15]). Iridophoreswith disordered guanine crystals have also been observed.[9] Eachcrystal is located in a crystal chamber where crystallization mostlikely occurs.[14] The developing crystal platelet is enveloped by anendoplasmic double membrane which eventually meshes into asingle membrane layer.[30]

Optically, it has been widely accepted that the crystals arearranged in quarter wavelength (‘‘ideal’’) stacks. This implies thatthe optical thickness (nd) of the crystals and the cytoplasmicspacing are approximately the same, and the maximumwavelength reflected is 4nd. Here, n is the refractive index ofthematerial and d is the thickness of the layer. The basic reflectingunit was described as a stack of 4–5 single crystals separated bycytoplasm.[8] By observing detached scales, Denton and Nicolconcluded that the stacks in any given scale do not have the samereflectiveproperties,which iswhatmakes it possible for thescale toreflect light over a wide range of incidence angles andwavelengths.[26] The concept of an ideal reflector present in fishskin was challenged by McKenzie et al., who argued that thecrystals are randomly spaced. Using transmission electronmicroscopy (TEM) they observed that crystals from the skin ofTrichiurus lepturus and Lepidopus caudatus are oriented parallel toone another but lack any periodic stack arrangement.[31]

We identified that the crystals from Koi fish are composed ofanhydrous guanine (and not guanine monohydrate) and solvedtheir crystal morphology.[7] The crystal structure of anhydrousguanine is monoclinic, with space group P21/c and celldimensions a¼ 3.55, b¼ 9.69, c¼ 16.35 A, b¼ 95.88.[32] Thebiogenic crystals in fish have an intriguing structure–morphologyrelation: they form as elongated semi-hexagonal (102) thin platesgrowingperpendicular to the shortest crystallographic axis. As thisaxis is generally predicted to be the fastest growth direction, the(102) crystal face should be very small, if developed at all. The (102)plate morphology enhances light reflectivity of the multilayerstacks in fish by exposing light to the high refractive index plane.[7]

Some inconsistencies exist in the literature regarding guaninecrystal dimensions and spacings in fish skin. The crystals werereported to bebetween6–100-nm thick andonaverage 20-mmlongand 3-mm wide,[9,33,34] based on TEM images. Different specieshave been reported to have different crystal thicknesses.

Spiders are also known to produce a variety of colors, includingstructural colors.[21,35–37] Bright markings on spiders are thoughtto facilitate camouflage, attraction of prey, intimidation ofpredators, or UV protection.[21,38,39] The colors are ofteninfluenced by guanine crystal formations, usually in combinationwith subcutaneous pigments.[21,40] Guanine is the principalnitrogenous excretory product in spiders, but in many species aportion is diverted to specialized cells of the intestinal divertic-ula,[36] referred to as guanocytes, where it acts as a colorant.Crystal formation occurs in intracellular vesicles, which aftermaturation form crystal sacs. Crystallization starts at the sac walls,where fibrous structures develop into coils located in the center ofthe sac, and crystalline platelets subsequently form parallel to thefibers.[41] Seitz (1972) also documented prismatic crystalsencapsulated within vesicles[42] in Araneus diadematus. In some

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spiders the guanine layer produces a matt-white effect, while inothers it generates a highly reflective silver color.[21] Millot(1926)[43] noted that guanine crystals producing amatt-white colorare small and cuboid, whereas those forming a silver color are thinplates.[21] Oxford (1997)[44] confirmed this observation usingscanning electronmicroscopy (SEM). These crystals are describedas guanine althoughno reference ismade to the hydrous state or totheir crystal structure.

Here we used powder X-ray diffraction to solve the crystalstructure of guanine crystals from different spiders and comparethe structure to that of guanine crystals in fish skin. We used cryo-SEM to obtain reliable high-resolution structural information oncrystal organization in fish and spider systems displayingstructural colors. A comparison of the two systems providesinsights into the mechanism of color production. Finally, weevaluate the reflective efficiency of these systems using measure-ments of the photonic crystal dimensions derived from cryo-SEMimages.

2. Results

2.1. Biogenic Guanine Crystal Organization and Morphology

Isolated guanine crystals from the scales of Koi fish (Fig. 1A) arethin (102) plates with defined crystal facets.[7] Cryo-SEM was usedto study freeze-fractured cross-sections of the fish scale, whichincluded the epidermal and iridophore cellular layers beneath thescale. The cellular layer includes two distinct areas; a layer of largeiridophore cells adjacent to the scale and a few layers of smallerepidermal cells. The guanine crystals are arranged in orderedarrays nearly parallel to one another (Fig. 1B). The space betweenthe crystals is most likely filled with cytoplasm (as described inRef. [8]), as some vesicles are observed between the crystals(Fig. 1C).

The crystal thicknesses and the spacing between the crystalswere measured from cryo-SEM images (Fig. 1D). Cryo-SEMsample preparation and imaging allows the samples to remainhydrated, thus avoiding the drying effects of conventional samplepreparation techniques. The average crystal thicknessmeasured is19.1� 5.5 nm (standard deviation (SD), n¼ 119) and the averagespacing between the crystals is 230.8� 94 nm (SD, n¼ 138;Supporting Information Fig. S1). Due to sample preparationconstraints, the crystals observed in the cryo-SEM imagesmay notbe oriented perfectly edge-on to the fracture, that is, to the surfaceobserved. This causes the measurements to be biased towardssmaller crystal thicknesses.

Conventional SEMwasused to studydispersedguanine crystalsfrom three species of spiders:Tetragnathamontana (silver colored)(Fig. 2A), Latrodectus pallidus (matt-white colored), and Argiopelobata (matt-white colored). The crystals of Tetragnatha montanaare thin and plate-shaped (Fig. 2A), while crystals of Latrodectuspallidus (c.f. Fig. 5A) and Argiope lobata are prismatic. This isconsistent with the observations ofG. S. Oxford, who documentedprismatic crystals in matt-white colored Nephila komaci(Nephilidae) and Misumena vatia (Thomisidae), and plateletcrystals in silver-colored Tetragnatha polychromata.[44]

Crystals extracted from the silver Tetragnatha montana areirregular platelets of generally homogeneous dimensions

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Figure 1. A) Single isolated guanine crystals extracted from the skin of a Koi fish. B–D) Cryo-SEM

micrographs of fish-scale cross-sections prepared using high pressure freezing and freeze

fracture techniques. B) A single layer of large, highly oriented iridophore cells (Ir) lined up

underneath the fish scale (Sc). C) A single iridophore cell. Note the crystal (Cr) between the

cytoplasm layers and also vesicles (V) embedded within cytoplasm layers. D) A magnified image

of an iridophore cell. Note the regular layered organization of the crystals as well as the

cytoplasmic layers (Cy) between the crystals.

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(Fig. 2A). Their facets are jagged and less distinct compared tocrystals extracted fromtheKoi fish (Fig. 1A).Cryo-SEMwasused tostudy crystal organization within the tissues of several spiders. InZygiella x-notata, Tetragnatha extensa (silver-colored spiders withplate-shaped guanine crystals) (Fig. 2B), and Latrodectus pallidus(matt-white-colored spider), the crystals are confirmed to beintracellular. In Zygiella x-notata and Tetragnatha extensa theguanocytes are densely packed with plate-shaped guanine crystals(Fig. 2C) and form discrete areas or ‘‘lobes’’ that are distinguish-able from the surrounding area, as previously described.[41,42,44]

The cells adjacent to the crystal-filled guanocytes contain sphericalvesicles. These vesicles appear to be membrane bound and tocontain material other than cytoplasm because they do not freeze-etch in the samemanner as the rest of the surrounding cell (Fig. 3).

The crystal plate organization appears to have long-rangedisorder, although some local order is observed (Fig. 2B). Thecrystals are arranged in doublets, that is, two single guanine crystalplates are separated by a material that is clearly distinguishablefrom them (Fig. 4A, arrow). The presence of these doublets isstrikingly different from the crystal stacks observed in the fish. Thecrystal thicknesses and spacings were measured from cryo-SEMmicrographs. In Tetragnatha extensa the average thickness of asingle crystal is 19.1� 6 nm (SD, n¼ 56), while the average

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spacing between crystals within a doublet isapproximately 30.0� 14 nm (SD, n¼ 22). Thespacing between crystal doublets is203� 59 nm (SD, n¼ 4).

Less etching occurs between the crystalswithin a doublet than in the surroundingcytoplasm. It is therefore reasonable to assumethat the material separating the crystal pairs isnot cytoplasm. This is further supported byobserving dispersed crystals of Tetragnathamontana in conventional SEM at a 308 tilt.The crystal doublets, although dried, are still ofsignificant thickness, measured as 59.2� 9 nm(SD, n¼ 39; Fig. 4B). Because cytoplasm ismostly water, if this was thematerial separatingthe crystals the shrinkage of the doublets wouldhave been much greater. On the other hand,amorphous material typically contains somewater, which would account for the smallamount of shrinkage observed. We note thatthe thickness of the doublets of anotherTetragnatha species, T. polychromata, observedin conventional SEM[44] appears to be the sameas those reported here. We therefore assumethat the crystal doublets in the Tetragnathaspecies all have the same overall dimensions.

The dispersed biogenic guanine crystalsextracted from the matt-white area beneaththe integument of Latrodectus pallidus andArgiope lobata were examined by SEM(Fig. 5A). Crystals isolated from the two speciesare similar in size and morphology: an overallprismatic habit can be identified, although thesmall limiting crystal faces are heterogeneousin size andhabit. The crystals are often twinned.The dihedral angle between the larger crystal

faces is 69.9� 78 (SD, n¼ 46). The calculated dihedral anglebetween the (012) and (102) planes in anhydrous guanine is 68.78.Based on the known crystal structure of anhydrous guanine, themorphology of biogenic guanine crystals from the skin of fish, andtheoretical morphology predictions,[7] we conclude that the largeface expressed is (102).

Using cryo-SEM, Latrodectus pallidus guanocytes are seen to bepacked with prismatic crystals (Fig. 5B, D, E). The cells adjacent tothe densely packed guanocytes are characterized by having fewprismatic crystals, but many spherical vesicles filled with a solidmaterial other than cytoplasm, possibly amorphous guanine(Fig. 5C, C1, C2). In Figure 5C and its magnification C1 one ofthese vesicles fractured by chance through the membrane. Thefracture exposes a prismatic crystal, similar to those previouslyshown in TEM sections.[41,42]

2.2. X-Ray Powder Diffraction from Spider Crystals

X-Ray powder diffraction data were collected at ambienttemperature from guanine deposits extracted from severalspider species (Fig. 6A). The diffraction patterns from the isolated

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Figure 2. A) Dispersed guanine crystals extracted from the tissue underneath the integument of

the silver-colored spider Tetragnatha montana. B,C) Cryo-SEMmicrographs of the tissue beneath

the integument (top) of silver-colored Tetragnatha extensa. B) Guanine platelets in situ, viewed

almost edge-on to the crystals. Some local order exists. C) In the center of the micrograph is a

basin-like shape composed of crystal-filled guanocytes.

plate-shaped crystals of the silver-colored Tetragnatha extensa,Tetragnatha montana, and Zygiella-x-notata show three reflectionpeaks belonging to the (012), (110), and (102) planes. The absenceofmany of the reflections is due to the preferred orientation of theplate-like crystals. The (204) reflection, clearly visible in the powderdiffraction pattern of the fish crystals, is not detected in the silver-colored spider diffraction patterns as it is too weak (2.75% of the(102) reflection). The presence of the (012) and (110) reflectionsmay be due to incomplete dispersion of the crystals. Prismaticcrystals from four additional species of matt-white spiders wereexamined. Enoplognatha ovata, Nephila komaci, Latrodectuspallidus, and Argiope lobata all have similar diffraction patternsand closely resemble patterns obtained for in vitro grownanhydrous guanine and the calculated diffraction patternpredicted from the crystal structure of anhydrous guanine. Allshow the same reflections observed in the silver spiders as well asadditional reflections of the (011), (002), (121), and (003) planes.The shift in the peak positions relative to the calculated diffraction(measured at 120K) is due to the difference in temperature of themeasurements.

In all the spider X-ray diffraction patterns a broad peak appearsin the low angle range, centered around 2u¼ 108. This is indicativeof the presence of amorphousmaterial in the sample and concurswith the suggestion of amorphous material-filled vesicles in the

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guanocytes. Interestingly, the powder diffrac-tion spectrum from Tetragnatha extensa, whichhas crystal-plate doublets, shows the highestproportion of amorphous material, and ingeneral the crystal-plate samples all have moreprominent amorphous peaks compared to theprismatic crystal samples. This observationsupports the hypothesis that the materialsandwiched between the crystal doublets isamorphous guanine.

A pole-figure measurement was conductedon the plate-shaped crystals of Tetragnathamontana at a constant 2u angle correspondingto that measured for the (102) and (012) planes(Fig. 6B). The (102) plane is detected in a rangeof 90–508 tilt angle. This indicates that there ispreferred orientation of the crystals along the(102) plane, that is,most of the crystal plates arelying on the (102) plane. In contrast, in the pole-figure measurement of the (012) plane thereflection is detected in a range of 20–908 tiltangle, clearly indicating a lack of preferredorientation of the crystals along this plane. Weconclude that the crystals of Tetragnathamontana are (102) plates, similar to thoseobserved in the fish crystal plates.

2.3. Electron Diffraction Patterns from

Spider Crystals

Electron diffraction patterns were obtainedfrom guanine plate-shaped crystals fromTetragnatha montana and compared to plate-shaped crystals from Koi fish (Fig. 7). The

diffraction patterns from the Koi crystals closely correspond to the[100] zone axis.[7] The diffractionpatterns obtained fromcrystals ofTetragnatha montana generally indicate the presence of two orthree superimposed single crystals. The presence of super-imposed crystals is consistent with the SEM micrographs, wherethe crystals are observed to exist as doublets. The zone axis is [100],as in the Koi fish. Themultiple crystals have the samemorphologyand do not detach during sample extraction, cleaning, orpreparation, including vortexing, shaking, and sonication of thesample, all of which would be expected to separate any aggregatesnot held together by strong adhesion forces. This is consistentwiththe two crystals of a doublet having grown in a single crystalchamber and being held together by a material other thancytoplasm.Aftermeasuring the angle fromnumerous diffractionswe concluded that the angle between superimposed single crystaldiffractions is arbitrary.

2.4. Percent-Reflectivity of Biogenic Guanine Photonic-Crystal

Arrangements

A prediction of the percent reflectivity at normal incidence can becalculated using the mean measured values and standarddeviations for crystal thicknesses and the spacing between them

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Figure 3. Cryo-SEM images of vesicle-filled cells adjacent to the guanocytes in the silver colored

Tetragnatha extensa. A) The distinctly different areas of crystal filled guanocytes (right) and vesicle

filled cells (left) is shown. Some of the vesicles have been fractured. B) The vesicles are less

etched than the surrounding cytoplasm-filled cellular area. C) A vesicle showing the surrounding

membrane.

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(see Experimental Section). This was performed for photoniccrystal arrays both from the Koi and the silver-colored spider,Tetragnatha montana.

2.4.1. Predicted Reflectivity of Photonic Crystal Arrays in Koi Fish

Assuming that light enters the plate-shaped crystals in a directionperpendicular to the large face, we estimate that on average aperiodic stack of 30 crystal layers, corresponding to3 cellswith�10crystals in each, is exposed to light (Fig. 1). The photonic-crystalstack thicknesses (�20 nmþ 230 nm) differ significantly from therequirements of a quarter-wave stack (as assumed in Ref. [8]),because even the highest possible index of refraction n, the opticalthickness nd¼ 20 nm, cannot reach a value close to thewavelengthof visible light (400 nm).

The first calculation was performed using the thickness andspacing parameters from the Koi, namely 19.1� 5.5 nm and230.8� 94 nm, respectively. This calculation was performed byaveraging 500 random values of crystal thicknesses and spacings,taken within the measured standard deviations of both. Thepredicted reflectivity curve shown in curve (a) of Figure 8A wasobtained.

Experimentally, we find a correlation between the spacing ofadjacent crystals that can be describedmathematically as a nearest-neighbor correlation of 60% (See Experimental Section). To takethis into account we performed a new calculation inwhich the firstvalue of the spacingbetween thefirst and second crystalswas takenat random as above, but the next value was taken as 60% correlatedto the first value. The applied correlation roughly corresponds toten crystals, which is the average number of crystals in a single cell.Experimentally we observe that indeed the correlation between

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crystals within one cell is higher than thatbetween crystals in different cells. The percentreflectivity was calculated by averaging 500 runsof 30 layers of crystals. The resulting reflectivitygraph (curve (b), Fig. 8A), is different from thatin curve (a) of Figure 8A, in that it shows a widereflectivity over the whole spectrum with amaximum reflectivity of 40–50% in the green–red spectral region. The percent reflectivitycalculated is very sensitive to changes in thecorrelation of the crystal spacing; a highercorrelation would result in substantially higherpercent reflectivity.

2.4.2. Predicted Reflectivity of Photonic Crystal

Arrays in the Spider Tetragnatha extensa

The SEM images show that there is no long-distance organization of the guanine crystalscomparable to that observed in the fish. Theshort-distance organization is restricted to asingle cell, which encapsulates between six andten crystal doublets. The percent reflectivity wascalculated using ten crystal doublets, assumingno correlation and the same averaging condi-tions for the dimer spacings as for the fish. Thiswas performed for two cases: the first considersthe reflectivity from a stack of crystals in which

the single crystalswithin a doublet are separated by cytoplasmwitha refractive index of n¼ 1.33 (curve (a), Fig. 8B), while the secondcalculates the reflectivity if the crystals are separated by a higherrefractive indexmaterialn¼ 1.6 (curve (b), Fig. 8B). This refractionindex is taken as a weighted average of the indices of guaninecrystals in the direction [102] (n¼ 1.83) and perpendicular to it(n¼ 1.48).[45] In the first case, the predicted percent reflectivity isonly around 25% back reflectance, while in the second case it isapproximately 40%, similar to the percent reflectivity calculatedfrom a 30 crystal array from the fish.

3. Discussion

Several animal taxonomic groups independently utilize guaninecrystals in order to generatewhite, silver and iridescence structuralcolors. We have endeavored to study the detailed in vivoarrangements of anhydrous guanine crystals in the iridescentscales of Koi fish and in matt-white and silver spiders using cryo-SEM imaging. This allowed reliable measurements of the crystalthicknesses and spacings, crucial elements of any photonic crystalsystem. Comparing and contrasting the arrangement of thecrystals provides useful insight into the interplay between crystalorganization in vivo, crystal structure, crystal morphology, and theresulting optical properties.

3.1 Structure/Morphology–Function Relations

A multilayer reflector system is characterized by the opticalthickness of its constituents, that is, their refractive indices and thethickness of the layers. The fish and silver-colored spiders form

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Figure 4. Guanine plates from silver-colored Tetragnatha spiders. A) Cryo-

SEM micrograph of the crystal doublets of the guanine platelets from T.

extensa. A typical architecture is observed, consisting of pairs of crystals

(Cr) with a layer of different material sandwiched between them (arrow). B)

Conventional SEM image of dry guanine doublets from T. montana at a 308angle.

photonic crystals that are multi-layered arrays of two media withdifferent optical properties: theguanine crystals represent thehighrefractive index medium, and the cytoplasm represents the lowerrefractive index medium.

The crystal plate morphology and its relation to the crystalstructure are fundamental to the reflective properties of singlecrystals and of the crystal arrays. The predicted morphology ofanhydrous guanine, calculated from its crystal structure, isprismatic. The crystals are elongated along the shortest crystal-lographic axis and perpendicular to the planar guanine mole-cules.[7] The prismatic crystal morphology observed from thematt-white spiders closely resembles this predicted morphology.Fish and silver-colored spiders have evolved a mechanism toinhibit crystal growth in the molecular stacking direction, as thebiogenic crystals are very thin plates, with the shortest dimensionof the crystals in the direction that is expected to exhibit the highestgrowth rate. This results in the formation of crystal plates wherethe main crystal face exposed to light is parallel to the planarH-bonded network of guanine molecules. The refractive index ofthe (102) crystal face is significantly higher (n¼ 1.83) than in thedirection perpendicular to this face (n¼ 1.48).[45] Thus, throughcontrol ofmorphology and lattice orientation, both species are ableto optimize light reflectivity at the level of single crystals.

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Studies of guanine crystals in different fish species havereported varying crystal thicknesses. Denton and Land measuredcrystal thicknesses from a wide variety of fish using interferencemicroscopy, and found that they range from 34–100 nm.[25]

Crystals of much lower thickness, in the range of 5–20 nm, havebeen documented using transmission electronmicroscopy (TEM)in Neon tetra,[33] Harengula zunasi,[34] and Salmo solar.[9] Samplepreparation for conventional TEM includes sample dehydrationand the use of materials that frequently dissolve guanine crystals.This inevitably influences measurements of crystal thickness andspacing, as noted by Lythgoe and Shand.[46]

Using cryo-SEM we confirmed the multilayer arrangement ofguanine platelet crystals arranged in the epidermal layer of skinbeneath the collagenous fish scale. We determined that thebiogenic crystals of Koi average 19.1� 5.5 nm in thickness, withspacing between two neighboring crystals of 230� 94 nm.

It is evident from these measurements that the guanine platesare too thin to function as ‘‘ideal’’ reflectors in the visible range(400–800 nm). Ideal reflectors would require that for alternatingmedia a and bwith refraction indexes na and nb spaced at distancesda and db, nada¼ nbdb, and the maximum reflectance wavelengthl¼ 4nd. Denton and Land[25] described the photonic crystalarrangement in fish as ‘‘ideal’’ andmade up of 4–5 single guaninecrystals interspaced with cytoplasm. They also addressed thepossibility of ‘‘non-ideal’’mechanisms of broadband reflectance insilvery fish, based either on a randomdistribution of the thicknessof the layers or to systematic changes in layer thickness.[13,25] Anon-idealmultilayer system is one inwhich the optical thicknessesof the layers are not equal, that is, nada 6¼ nbdb, and the maximumreflectance wavelength is l¼ 2(nadaþ nbdb). The idea of approxi-mately ideal stacks was challenged by McKenzie et al.,[31] whoproposed that the silvery broadband reflectance of fish skin resultsrather from a chaotic arrangement of crystals.

We have found that the crystal thicknesses are relativelyuniform.Each iridophore containsbetween8and12singleplateletcrystals. Although there is variation in crystal spacing this systemdoes not appear chaotic, as proposed by McKenzie et al.[31]

Furthermore, we calculated a nearest neighbor correlation of 60%between adjacent crystal spacings. The correlationdisappears afterapproximately ten crystals, which corresponds to the averagenumber of crystals found in an individual cell. This indicatessystematic regulation of the photonic crystal unit, based on controlof crystal spacing within the confines of a single cell. When thiscorrelation is considered in predicted reflectivity calculations, theresult is a 40–50% broad band reflectance in the green and redspectrum. The number of crystal layers observed by McKenzieet al.[31] and used in their calculations is 100–200 crystals, which isvery different from our own observations, where the highlyreflective layer beneath the scale is composed of a single layer ofaligned iridophores oriented obliquely to the direction of theincident light. This arrangement results in the incident lighttraversing on average through three cells (Fig. 1A). In the Koi fish,an array of approximately 30 crystals is thus sufficient to obtain thereflectivity observed, and our simulations reproduce well theobserved effect.

Spiders produce prismatic and plate-shaped guanine crystalswhich correlate with color.[44] The prismatic crystals produce amatt-white color and the plate-shaped crystals a silver color. Plate-shaped crystals from silver-colored spiders show marked

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Figure 5. A) SEM micrographs of guanine crystals extracted from the matt-white colored spider Latrodectus pallidus. B–D) Cryo-SEM micrographs of the

tissue located beneath the integument of Latrodectus pallidus. B) On the right-hand side of this micrograph are guanocytes (Gy) packed with crystals (Cr).

On the left-hand side are cells filled with few crystals and many spherical vesicles. Note the cellular membrane (Cm) separating the cells and nucleus (N).

C) Amicrograph of the cells adjacent to the crystal-filled guanocytes area. C1) Vesicle containing a crystal. C2) Fractured vesicle (V) filled with solidmaterial.

D) Cellular area filled with prismatic guanine crystals. D1,D2) Two crystals from (D). E) Prismatic guanine crystal in the tissue.

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differences in crystal morphology and arrangement compared toplate-shaped crystals from fish. Interestingly, the crystals arearranged in doublets with a layer separating the two. The averagethickness of a single crystal is 19.1� 6 nm. This is surprisinglysimilar to those of Koi fish. The spacing between two crystalswithin a doublet is approximately 30� 14 nm.

Wesuggest that thematerial separating crystalswithin adoubletis not cytoplasm but amorphous guanine. This is supported by theobservations that during sample preparation thismaterial showedless etching than would be expected from a mostly water-basedmaterial such as cytoplasm. The superimposed single-crystalelectron-diffraction patterns obtained from the doublet crystalsindicate that the crystals did not separate despite rigorous samplehandling. Moreover, X-ray powder diffraction spectra from thespiders in general, and fromthe crystal plates extracted fromsilver-colored spiders in particular, indicate the presence of amorphousmaterial in the samples. Thismay also be thematerial observed inthe vacuoles prior to crystallization, which show little etching inSEM images. The random angle between electron crystaldiffraction patterns of plate doublets indicates that they form byindependent nucleation rather than twinning. The identicalmorphology of the crystals, aswell as their close proximity, suggestthat they grow inside a single chamber, which delineates andrestricts crystal growth.

Might an amorphous guanine phase play a role in theenhancement of light reflectivity? Considering the doublet as

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the high refractive index unit and the cytoplasm as the low indexcomponent of the photonic-crystal array, the system effectivelybecomes a near quarter wave stack in the visible range(4nd¼ 4� 1.7� 70 nm¼ 476 nm). The predicted reflectivity plotcalculated for a single-cell array in the silver-spider system for thecase where the material between the crystals is amorphousguanine, with a refractive index estimated as n¼ 1.6, shows a 40%broad-band reflectivity. Preliminary measurements performed byimmersion in graded liquids with high refractive index indicatethat for the doublet crystals n> 1.61, further confirming thevalidity of the assumption. In contrast, a reflectivity of only�25%would be predicted if the material between the crystals had therefractive index of cytoplasm, namely 1.33. Thus, by producingcrystal doublets, optimization of crystal reflection is achieved,despite the biological constraints. The crystal organization inthe silver-colored spiders shows a local order of crystal stacksconfined to a single cell, including approximately 6–10 crystaldoublets.

Interestingly, the percent reflectivity from one array confined toone cell is nearly that predicted for a 30-crystal array from fish.Spiders are exposed to light, which strikes the integument indifferent directions. The requirement for a multi-directional lightreflector couldbeachieved through theobserved long-range crystaldisorder. These findings indicate that silver-colored spiders haveevolved a solution allowing them to obtain multidirectionalreflectance using minimum crystal order.

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Figure 6. A) X-Ray powder-diffraction patterns of crystals collected from

spiders. Tetragnatha extensa, Tetragnatha montana, and Zygiella-x-notata

spiders are silver in color with thin plate-like crystals. The diffraction pattern

shows predominantly the (102) reflection, with extremely weak (012) and

(110) reflections. The next four diffraction spectra are from spiders all

having a matt-white color and prismatic crystals: Enoplogatha ovata,

Nephila komaci, Latrodectus pallidus, and Argiope lobata. The crystals show

the same diffraction pattern observed in the silver spiders, but (012) and

(110) are much stronger and there are additional reflections (011), (002),

(121), and (033). The diffraction patterns closely resemble those of

synthetic anhydrous guanine and the calculated diffraction pattern of

anhydrous guanine (bottom two spectra). All spectra from spiders show

a large amount of amorphous material (broad peak centered at 2u¼ 108).B) Pole-figure plots of (012) (top) and (102) (bottom) planes from crystals

of Tetragnatha montana. The diffraction angle 2uwas held constant at 14.58and 27.788, respectively, while the orientation angle of the sample (x) was

tilted from 908 to 108. Preferred orientation is detected only around the

(102) plane.

Figure 7. Electron diffraction patterns. A) Electron diffraction from bio-

genic guanine crystals from Tetragnatha montana spider. The diffraction

pattern observed is that of two superimposed single crystals. B) Electron

diffraction from a biogenic guanine crystal from Koi fish. The three

diffraction patterns, that is, two in (A) and one in (B) are essentially the

same and correspond to the [100] zone axis of anhydrous guanine.

Figure 8. A) The percent reflectivity calculated for Koi fish. a) Predicted

percent reflectivity at normal incidence using the average crystal thick-

nesses and spacing and their distribution. b) Predicted percent reflectivity

obtained using the same average crystal thickness and spacing at normal

incidence, taking into consideration the observed distribution as well as the

calculated nearest neighbor correlation of 60%. B) The percent reflectivity

calculated for plates from the silver colored spider Tetragnatha extensa.

a) The reflectivity calculated for crystal doublets spaced with cytoplasm.

b) Percent reflectivity calculated for crystal doublets spaced with a higher

refractive index material n¼ 1.6.

Cryo-SEM images have revealed that prismatic guanine crystalsinmatt-white spider iridophores are generally homogeneous withsharp crystal edges, but the crystals are not organized. The matt-white coloring observed is the result of light scattering off thecrystals, similar to the scattering of light from suspended particlesin milk. Furthermore, cryo-SEM images show that cells not yetfilled with crystals contain many spherical vesicles. This isobserved in both silver and white spiders. The crystallizationprocess in spiders has previously been described by Seitz[41] asoccurring inside such vesicles. It is thus conceivable that thecrystallization of guanine in spiders occurs via an amorphousprecursor phase, as has been demonstrated in other biominer-alization processes.

SEM micrographs of dispersed guanine prismatic crystalsrevealwhat seemtobepairs of closely associated crystals.Althoughwehavenot directly investigated the relationbetween thesepairs ofcrystals, we would like to point out that if the crystals are nottwinned thismaybean indication that theyhave formedwithinone

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vesicle, a feature similar to that observed for the plate crystals.This might indicate that a common mechanism of crystalnucleation evolved in both systems. This is of evolutionaryinterest in that Oxford (1997),[44] using a phylogenetic approach,showed that in spiders silver guanine has evolved from matt atleast eight times. If there is a common mechanism of crystalnucleation these transitions would be more easily explained.Importantly, we show that the silver-guanine genera Tetragnathaand Zygiella both show doublet crystal formations even thoughthey have evolved independently from prismatic crystal (matt-white) ancestors.[44]

4. Conclusions

We have investigated two biological photonic-crystal systems,whichhaveevolved theability togrowanhydrousguanineaswell asto tightly control crystalmorphology and structure to serve specificevolutionary requirements. It is astonishing how, throughevolution, fish and silver-colored spiders have independentlysucceeded in achieving light reflecting structures with similarefficiencies, although differences in mechanism of reflection areapparent. We have shown that silver-colored spiders form crystaldoublets separated by a layer of amorphous guanine, thus takingfull advantage of the increased thickness, and enhanced lightreflection. The use of amorphous guanine results in a higherrefractive index of the double layers and consequently a widerspectrum of wavelengths reflected from a smaller number ofrepeat layers. Furthermore, we have seen that spiders formprismatic crystal morphologies similar to published theoreticalgrowth morphology predictions.[7]

5. Experimental

Materials; Biogenic Guanine Crystals: Biogenic crystals and scales wereextracted from the skins of the Japanese Koi fish (Cyprinus carpio). Onlycompletely silver-colored scales were used. Biogenic guanine crystals wereextracted from seven species of spiders. The silver-colored Tetragnathaextensa (Tetragnathidae), Tetragnatha montana (Tetragnathidae) andZygiella-x-notata (Araneidae), and the matt-white Enoplognatha ovata(Theridiidae), were collected in Britain. The matt-white Nephila komaci(Nephilidae) came from Mombasa, Kenya (a description of the species byM. Kuntner is in preparation). Finally, Latrodectus pallidus (Theridiidae) andArgiope lobata (Araneidae), also matt-white, were collected in Israel.Anhydrous guanine was purchased from Sigma Aldrich (Lot 021K5008).

Methods; Isolation of Biogenic Crystals from Fish: Fish scaleswere mechanically removed and washed in deionized water (DW).Crystals were extracted by placing the scales in a drop of DW and peelingthe skin from the underside of the scale, and dispersing the crystalsinto the water. The crystal suspension was collected and centrifuged(5–10minutes, 10 600 rpm, Eppendorf centrifuge 5417C). The supernatantwas replaced with DW, the pellet re-suspended and the procedure repeatedtwice more.

Isolation of Biogenic Crystals from Spiders: The crystals were extractedby first removing the integument from the abdomen of the spider,thus exposing the iridophores. The iridophores were collected into acentrifuge tube containing 1mL DW, were allowed to settle to the bottomof the vial, and the water volume was reduced to 500mL using a glassPasteur pipette. A solution of proteinase K (35mL,> 600 nAUmL�1 in Tris-Cl, pH 7.5) was added. The sample was incubated at 37 8C for three days ona shaker. The samples were centrifuged (10 600 rpm, Eppendorf centrifuge

� 2010 WILEY-VCH Verlag GmbH &

5417C, 20min) at room temperature. The supernatant was removed andreplaced with 1mL double distilled water (DDW). The pellet was re-suspended and centrifuged using the same conditions. This was repeated3–5 times until the pellet was white. If the pellet was not white and clean, anadditional step of heavy liquid separation was employed. The pellet was re-suspended in a solution of sodium polytungstate (Na6(H2W12O40)�H2O;Sometu-Europa, 1.35 gmL�1) and centrifuged using the same conditionsdescribed above. The pellet was then washed twice with DW, once withabsolute ethanol, and allowed to dry in air.

Synthetic crystals for X-ray analysis of anhydrous guanine were grown inanhydrous dimethyl sulfoxide (DMSO). A saturated solution of guanine inDMSO was prepared and heated to 80 8C. The anhydrous guanineremnants were allowed to settle. The supernatant was transferred into anew vial and stored overnight at 4 8C. The crystal suspension was warmedto room temperature for one hour, and then centrifuged (5min, 1700 rcf).The supernatant was removed and replaced with absolute ethanol. Thepellet was redispersed and centrifuged using the same conditions twice.The crystals were subsequently air dried and stored at ambienttemperature.

Conventional X-Ray Powder Diffraction: X-Ray powder diffractionpatterns were obtained using a powder diffractometer (Ultima III-Rigaku, Japan) with a Cu target. The biogenic powder specimens weredispersed as a thin film on a Si zero-background holder and measured.Alternatively, a powder was prepared from synthetic anhydrous guanine bycrushing in vitro grown crystals with a mortar and pestle before dispersingthem on the holder.

Pole figures were measured in the Bragg–Brentano mode (Schultzmethod) on crystals supported on a Si zero-background holder using thepowder diffractometer (TTRAX III-Rigaku, Japan). The reflections wererecorded at a fixed Bragg angle while performing an in-plane rotation atregularly increasing sample tilts (90–508) relative to the incident/diffractedbeam plane.

The theoretical X-ray diffraction pattern of anhydrous guanine wasgenerated using the known crystal structure of anhydrous guanine and theCerius2 program.

Scanning Electron Microscopy (SEM) was performed with a LEO-Supra55 VP FEG SEM (Zeiss). Samples were prepared by dispersing the crystalsin either ethanol or DW. A drop of the crystal suspension was placed on aglass cover slide and was allowed to dry. The slide was then mounted on analuminum SEM sample holder and coated with gold.

Cryo-SEM: Cryo-SEM was performed with a high resolution LEO-Ultra55 (Zeiss, Germany) at �120 8C. Samples were prepared as follows: Fishscales were removed mechanically and washed in phosphate-bufferedsaline (PBS). Samples of scales were cut into approximately 2-mm squaresand immersed in 10% dextran. The integument of the spider was removedand peeled off. Iridophore-containing tissue was washed in PBS, cut intoapproximately 2mm squares and immersed in 10% dextran. Each samplewas sandwiched between two discs to hold the sample and frozen using thehigh-pressure freezing technique (HPF; Bal-Tec HPM 010). The sampleswere fractured at a temperature of �120 8C using a freeze fracture device(Bal-Tec BAF60). Samples of fish scales used to measure the spacingbetween the crystals were not etched (etching refers to the sublimation ofvitreous water from the fractured sample), while others were etched for 5minutes at�105 8C. Spider samples were etched for 5 minutes at�105 8C.The etching step removes some of the vitreous ice layer from the sample,thus exposing the embedded cellular or crystal material. The samples werecoated with 2-nm Pt/C using double axis rotary shadowing. The sampleswere transferred to the scanning electronmicroscope using a VCT 100 cryo-transfer unit. Note that spider species collected in Britain were preserved in2.5% glutaraldehye and 4% formaldehyde for 36 hours before transfer toDW prior to cryo-sample preparation.

Electron diffraction was performed using a T-12 (Philips) transmissionelectron microscope (TEM) operated at 120 kV. Samples were prepared byplacing a drop of the crystal suspension on a copper carbon-coated TEMgrid, allowing the crystals to settle for 30 seconds, and then removing theremaining suspension.

Predicted percent reflectivity: Every layer is characterized by nj (refractiveindex) and dj (which is the layer thickness randomly picked from the

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experimental distribution). Thus, for each layer we define a 2� 2 matrix

mj ¼cosbj � i

njsinbj

�inj sinbj cos bj

!where bj ¼

2p

lnjdj (1)

The set of ten double layers is characterized by an overall reflectivity2� 2 matrix

Mj ¼Y20j¼1

mj (2)

from which the reflectivity is extracted from the following expression [47]:

R ¼ ðm11 þm12Þ � ðm21 þm22Þðm11 þm12Þ þ ðm21 þm22Þ

��������2

(3)

The refractive index of guanine was taken to be n¼ 1.83 and of thecytoplasm n¼ 1.33. The calculation was performed taking into considera-tion a random distribution of thicknesses d in the range measured.

Acknowledgements

We thank Talmon Arad for assistance in the electron diffractionexperiments, and Dr Issai Feldman for assistance in the powder diffractionspectra. We thank Dr Berta Levavi-Sivan for providing us with the Koi fishand for help in the scale extraction. The electron microscopy studies wereconducted at the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging at the Weizmann Institute of Science.We thank the IsraeliMinistry of Science and the Minerva Foundation for financial support. L.A.is the incumbent of the Dorothy and Patrick Gorman Professorial Chair ofBiological Ultrastructure, and S.W. is the incumbent of the Dr. TrudeBurchardt Professorial Chair of Structural Biology. Supporting Informationis available online at Wiley InterScience or from the authors.

Received: July 31, 2009

Revised: September 29, 2009

Published online: December 15, 2009

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