biological systems Physical methods for investigating structural colours in

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Physical methods for investigating structural colours in
biological systems

P Vukusic and D.G Stavenga

J. R. Soc. Interface 2009 6, S133-S148 first published online 21 January 2009
doi: 10.1098/rsif.2008.0386.focus
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J.R.Soc.Interface(2009)6,S133–S148
doi:10.1098/rsif.2008.0386.focus
Publishedonline21January2009
Physical methods for investigating
structural colours in biological systems
P. Vukusic1,* and D. G. Stavenga2
1School ofPhysics, University of Exeter, Exeter EX44QL,UK
2Department ofNeurobiophysics, University of Groningen, Nijenborgh4,
9747 AGGroningen, The Netherlands
Many biological systems are known to use structural colour effects to generate aspects of
their appearance and visibility. The study of these phenomena has informed an eclectic
group offields ranging, for example, from evolutionary processes in behavioural biology to
micro-optical devices in technologically engineered systems. However, biological photonic
systems are invariably structurally and often compositionally more elaborate than most
syntheticallyfabricatedphotonicsystems.Forthisreason,anappropriategamutofphysical
methodsandinvestigativetechniquesmustbeappliedcorrectlysothatthesystems’photonic
behaviour may be appropriately understood. Here, we survey a broad range of the most
commonly implemented, successfully used and recently innovated physical methods. We
discuss the costs and benefits of various spectrometric methods and instruments, namely
scatterometers, microspectrophotometers, fibre-optic-connected photodiode array spec-
trometers and integrating spheres. We then discuss the role of the materials’ refractive
index and several of the more commonly used theoretical approaches. Finally, we describe
therecentdevelopmentsintheresearchfieldofphotoniccrystalsandtheimplicationsforthe
further study of structuralcoloration inanimals.
Keywords:structuralcolour;photonics;iridescence;multilayers;spectrometry;microscopy
1.INTRODUCTION refractive index (RI); its value only has a real
component when it comprises no optical absorption,
Thevisualappearancesofbiologicalsystemshavebeen
and it has both real and imaginary components when
investigated and characterized using numerous
light is absorbed. The spatial distribution of the
different methods. The results, invariably collected
sample’s constituent dielectric material, and hence its
using arange of microscopic, optical interrogation and
RI, will largely determine the flow of light through or
modelling techniques, have been wide reaching. They
from it. It is this light scattering, in reflection and
have offered insight into a variety of biological topics
transmission, which subsequently determines the
suchas:species’behaviourandcommunication;crypsis
object’s visual appearance.
strategies and pressure-driven adaptation methods;
It is now well known that, generally, biological
and morphological, developmental and evolutionary
objects with inhomogeneous irregular RI distributions
processes(e.g.Fox&Vevers1960;Silberglied&Taylor
are colourless or diffusely white when there is no
1973;Durrer1977;Prum2006).Furthermore,manyof
absorption.However,whentheirconstituentRIdistri-
the investigations have revealed ingenious optical and
butions have ordered or quasi-ordered sub-micrometre
photonic system designs that are appropriate for
domains,thenwavelength-dependentcoherentscatter-
biomimeticsynthetic replication (Forbes 2006).
ing can occur. This can give rise to bright structural
Particular recent advances have been made in
colours (Mason 1926, 1927; Ghiradella et al. 1972;
the study and understanding of animals that exhibit
Schultz & Rankin 1985; Vukusic et al. 2000, 2001;
structural colour. Structural colour comprises the
Vukusic & Sambles 2003; Kinoshita & Yoshioka 2005;
opticaleffectsproducedwhenincidentelectromagnetic
Prum 2006; Kinoshita et al.2008).
radiation(suchasvisible,ultravioletandnear-infrared
The requisite ordered or quasi-ordered variation in
light) encounters ordered spatial variations in a
the RI for the production of structural colour effects
sample’s constituent dielectric material that are on
may occur in one, two or three spatial dimensions.
thesamelengthscaleasthewavelengthoftheincident
Generally, one-dimensional RI modulations create
light. A dielectric material is characterized by its
multilayers or surface monogratings; RI variations in
two dimensions form analogues of photonic crystal
*Authorforcorrespondence([email protected]). fibres or surface bigratings; and three-dimensional
spatial RI modulations are designated as full photonic
One contribution of 13 to a Theme Supplement ‘Iridescence: more
thanmeetstheeye’. crystals. In some animals, such as hummingbirds,
Received28October2008
Accepted8December2008 S133 Thisjournalisq2009TheRoyalSociety
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S134 Structurallycoloured biological systems P.Vukusic and D. G. Stavenga
(a) (b) (c)
Figure1. (a) The upper wings ofthe tropical butterfly M.didius have a strikingbluecolour owingto multilayerinterference
reflection.(b)Uponobliqueillumination,outsidetheangleofinterferencereflection,alightbrownpigmentarycolourinthewing
scalesbecomesvisible.Notetheappearanceof‘eyespots’fromthewings’ventralsurfaces,whichareoftenquitemarkedinthe
dorsalwingsofotherbutterflies.Withwide-angleillumination,theeyespotsarecompletelyunnoticedowingtothehighintensity
oftheblueinterferencereflection.(c)TEMimageofacrosssectionthroughthebilayerofthedorsalwingscalesonM.didius,
showingthehighlyreflectivescalepresentbasally,whichiscoveredbythehighlydiffractivescalepresentsuperficially(Vukusic
etal.1999).Scalebars:(a,b)1cmand(c)1.5mm.
Morpho butterflies and jewel beetles, the systems
(a)
creating structural colours dominate the animals’
visualappearances. Inmany other examples,however,
for instance in male pigeons, brimstone butterflies and
tiger beetles, the animals’ structural colours may be
convolvedwithoneormorepigments,whichcontribute
additional colour or related optical effects due to local
and spectrally selective light absorption (Mason 1926,
1927; Vukusic et al. 2004; Hariyama et al. 2005;
Stavenga et al. 2006a; Yoshioka & Kinoshita 2006a;
Morehouseet al. 2007).
A striking example is one of the icons of structural
coloration, the Morpho butterfly family. In Morpho
didius (figure 1), for instance, the dorsal (upper) wing
surfaces display a brilliant blue reflection when illumi-
nated from directly overhead (figure 1a). This colour
(b)
effect vanishes upon oblique illumination. Phenomena
suchasthesearecontrolledbythescalesthatimbricate
Morpho wings, as occurs in virtually all lepidoptera.
In M. didius and other brightly coloured Morphos,
multilayered structures in the scale ridges reflect blue
light that can be strongly dependent on the angle of
illumination (Ghiradella 1998; Vukusic et al. 1999;
Kinoshitaetal.2002;Vukusic&Sambles2003;Kinoshita
etal.2008).Forinstance,withobliqueillumination,the
dorsalwingscalesbecomelargelytransparent,andthen
themoreheavilypigmentedscalesontheventral(lower)
sideofthewingsbecomevisible(figure1b).
Structurally coloured objects are often iridescent;
thatis,theircoloursappeartochangewiththeangleof
light incidence (figure 2) or with the observer’s Figure 2. Iridescent feather of a peacock, illuminated from
perspective. Depending on animal species, this may directionsdifferingbyapproximately908.
have a functional role in, for instance, intraspecific
communication. Therefore, one of the typical aims of In all biological systems, it is a complex set of
investigating structurally coloured biological systems variables that controls or contributes to a specific
that exhibit this form of effect would be to develop an optical signature and the resulting overall visual
understandingoftheanimal’sangle-dependentspectral appearance. To understand the significance of each of
phenomena from the perspective of their anatomical these variables both qualitatively and quantitatively,
structureandtheirconstituentmaterial.Thechemical and to appreciate the way in which they create an
properties and associated characterization methods animal’s complete colour appearance, it is essential to
of the materials comprising structurally coloured measure the spectral and spatial characteristics of the
biologicalsystemsaresummarizedelsewhere(Shawkey animal’s body reflectance. It is with this in mind that
et al.2009). we therefore first introduce the central concept of the
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Structurally coloured biologicalsystems P.Vukusic and D. G. Stavenga S135
bidirectionalreflectancedistributionfunction(BRDF).
We subsequently apply this to the experimental
methods that are available, and that have been
successfully used for investigating structural colours
in biological systems. We aim to assess the reliability
andthenatureoftheinformationtowhicheachmethod
may lead.
2.THE BIDIRECTIONAL REFLECTANCE
DISTRIBUTIONFUNCTION
From a measurement perspective, biological structural
colour effects can have a very broad set of associated
experimental variables. These are related to four
aspects: the illumination conditions; the measurement
orlightdetectionconditions;thestructuralgeometryof
the sample; and, lastly, the compositional materials
ofthesample.Formally,therelationthatconnectsallof
Figure 3. The BRDF relates the reflected light I(q,4) in a
these is the BRDF (dimension srK1; Nicodemus 1965; r r r
small solid angle du to the incident light flux I(q,4) in
r i i i
Nicodemus et al. 1977), which is defined as the ratio asmallsolidangledu (seeequation(2.1)).
betweenthereflectedradiance(dimensionWmK2srK1) i
andtheincidentirradiance(dimensionWmK2;figure3), elucidatetherangesofexperimentalmethodsthathave
been successfully used, and which have recently been
R ðq;4;q ;4 ÞZ Irðqr;4rÞdur : ð2:1Þ developed, for investigating structural colours in
l i i r r Iðq;4Þcosqdu biological systems.
i i i i i
The BRDF assumes light scattering from only one
surfacepoint.This,however,maynotalwaysbevalidfor 3.MEASURINGSTRUCTURALCOLORATION
manymacroscopicobjectswheresubstantialsubsurface
3.1. Spatial spectrophotometry
scattering occurs. The more general function that
and scatterometry
incorporates non-negligible subsurface scattering is the
bidirectional scattering surface reflectance distribution A straightforward and direct method for measuring
function(Marschneretal.2001).It is thefunctionthat lightscatteringfrombiologicalsamplesisschematically
completely describes the interaction of the incident represented in figure 4a. It is a method that has been
light with a material. For most structurally coloured widelyusedforsamplesofvarioussizesfromarangeof
biological systems, however, strong subsurface scatter- different animals (for a review, see Kinoshita et al.
ing(i.e.associatedwithlightthatentersthematerialat 2008).Lightfromanappropriatenarroworbroadband
one location and emerges at a spatially significantly source is focused onto a sample from which it is
different location) is negligible, and the limiting case subsequentlyscattered.Thespatialdistributionofthis
of the more straightforward BRDF is therefore fully scatteredlight(bothinthereflectionandtransmission
adequate (Shimada & Kawaguchi 2005). It should be hemispheres)canthenbeinvestigatedbyitscollection
noted from equation (2.1) that a single BRDF only using a spatially filtered detector or light guide
appliesforelasticscatteringataspecificwavelength(i.e. designed to rotate around the sample and deliver it to
fluorescenceisnegligible).TheformalBRDFcharacter- aspectrometer.Absolutereflectanceandtransmittance
izationofasample,therefore,requiresaseparateBRDF measurements, using a range of monochromatic laser
ateachwavelengthofinterest. light sources, have been performed on single Morpho
Generally, biological systems are extended objects butterfly scales (Vukusic et al. 1999) and single pierid
with large-area surfaces. Accordingly, the task of butterflyscales(Morehouseetal.2007).Otherdetailed
characterizing a biological sample’s visual appearance spectral analyses were performed on isolated scales of
viaitsBRDF,bothacrosstheentiretyofitssurfaceand various butterflies (Yoshioka & Kinoshita 2006b;
at all wavelengths of interest, is an extremely challen- Giraldo et al. 2008), using a white light source and a
gingundertaking.MeasurementoftheBRDFevenata diode arrayspectrometer.
singlelocationof,say,aMorphobutterfly(figure1)ora By rotation of the specimen, the BRDF can be
peacockfeather(figure2)isextremelyburdensomeifit measured as a function of the angle of illumination.
is done via sampling the light scattered into numerous Alternatively,thelightsourceoffigure4acanbereplaced
directions (q,4) of the hemisphere above the surface byaflexiblelightguidefittedwithasuitablelens,which
r r
bothateachangleofincidence(q,4)inthehemisphere is rotated around the sample. Spectrophotometry can
i i
andatdifferentincidentwavelengths(figure3).Realis- then be carried out with an experimental arrangement
tically, therefore, to provide experimental insight into similar to that offigure 4a on areas minimally of a few
the scattering processes of a sample, experimental millimetresindiameter(Rutowskietal.2007).
approachesmustbeappropriatelysimplifiedwhilestill An elegant and effective method for visualizing the
generatingusefulinformationaboutthesample’svisual spatial light scattering by a small- to medium-sized
appearance. The principal purpose of this paper is to object, such as a butterfly scale or a bird feather
J.R.Soc.Interface(2009)
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S136 Structurallycoloured biological systems P.Vukusic and D. G. Stavenga
(a) light guide spectrometer
rotating stage
(b) sample
light
source pinhole screen camera
Figure4.Diagramofanopticalsystemformeasuringtheangulardistributionofthelightscatteringfromasample.Lightfroma
lightsourceisfocusedonapinhole,whichisimagedonthesample.(a)Thelightscatteredbythesampleiscollectedatarangeof
polaranglesbyalightguidemountedonarotatingstageanddeliveredtoaspectrometer.(b)Awhitescreenwithasmallhole
placed in between the imaging lens and the scale allows visualization of the spatial distribution of the light reflected by the
butterflyscale;thismayalsobedoneintransmission(modifiedfromGiraldoetal.2008).
S
2
D
2
L
6
D
3
L
7
D
4
L
3
S D L C L M F H F L I L C
1 1 1 1 2 1 2 4 5 2
Figure5.Imagingscatterometer.S ,lightsources;L ,lenses;D ,diaphragms;C ,digitalcameras;M,ellipsoidalmirror;
1,2 1–6 1–4 1,2
F ,focalpointsofthemirror;H,half-mirror.LightfromS createsanarrowapertureaxialbeam.CameraC visualizesthe
1,2 1 1
sampleandcameraC visualizesplaneI,theback-focalplaneoflensL ,wherethescatteringpatternofthesamplelocatedinF
2 4 1
(black speck) is imaged and a spatial filter is placed to interrupt the first-order transmitted light. Light from S passes the
2
movable diaphragm D in the front-focal plane of lens L , which thus creates a light beam with a variable incident angle.
3 7
DiaphragmD isintheback-focalplaneofL ,andisconjugatedtoadiaphragminplaneF byhalf-mirrorH.
4 7 2
barbule, is shown in figure 4b. A white screen with a has associated limitations (see Kinoshita et al. 2008).
small hole, placed in front of the light scattering As discussed in the following, alternative methods are
sample, acting as a secondary scatterer, yields an fortunatelyavailable.
immediate image of the spatial distribution of
light reflected by the sample (Kinoshita et al. 2002;
Yoshioka&Kinoshita2006a;Giraldoetal.2008).The 3.2. Imaging scatterometry
pattern on the screen can be photographed, and then Generally, it is very difficult to capture experimentally
the spatial properties can be quantitatively analysed, the data associated with the numerator in the BRDF
for instance by correcting for the angular distortion of formula shown in equation (2.1). This is in fact the
the flat screen. However, the camera captures only a hemispherical light intensity distribution of scattered
minor fraction of the light scattered by the sample lightperunitsurfaceareaasafunctionofthepolarand
(figure4b),andthereforethemethodisinsensitive.To azimuthalangle,I(q,4).Whileitisnotsomethingthat
r r r
measure the scattering pattern as a function of the canbeachievedwithnormalphotographicalmethods,it
angle of incidence, the scale has to be rotated and is something that can be captured by the recently
additionalscreenshavetobeplaced,butthisapproach developed imaging scatterometer of figure 5 (see also
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Structurally coloured biologicalsystems P.Vukusic and D. G. Stavenga S137
Stavenga et al. 2009; Wilts et al. 2009). Its central back-focal plane of lens L in figure 7) can then be
5
element is an ellipsoidal mirror, where the sample is photographed. Obviously, the objective aperture
mountedinthefirstfocalpoint.Lightfromtheprincipal determines the spatial extent of both the illumination
source is focused at the sample via a small hole in the and scattering, but for objectives with numerical
ellipsoid.Themirrorfoldshemisphericallyscatteredlight apertures as large as 0.85 or more, angles of approxi-
into a narrow cone that is accepted by a photographic mately 608 can be achieved, allowing the imaging of
lens.Thefar-fieldscatteringpatternemergesintheback- scatteringoveralargespatialangle.
focal plane of that lens, and that plane is imaged by a The dependence of the scattering pattern on
secondlensonadigitalcamera.Asecondarylightpath wavelengthcanbestudiedwiththemicroscatterometer
allows measurement of the scattering pattern as a usingthemethodofmultispectralimaging,asexplained
function of the angle of incidence. Light from the in §3.2, that is, by taking images with a series of
secondarysource passes a diaphragm, inthe front-focal illuminations at different wavelengths. Alternatively,
planeofalens,whichis,viaabeamsplitter,confocalwith thetipofalightguide,connectedtoaspectrometer,can
the second focal point of the ellipsoid. Varying the beplacedinplaneI(figure7a),sothatthen,usingwhite
positionofthediaphragmthusresultsinavaryingangle light, the reflectance spectrum can be measured as a
ofincidenceofthelightbeamreachingthescale. function ofscattering direction.
Figure 6 presents measurements performed with Thelatterapproachisanalogoustoepi-illumination
the imaging scatterometer on a single blue scale of a microspectrophotometry. In this method, light from
Morpho aega butterfly. Confirming previous measure- the reflecting sample reaches the image plane, where
mentswiththeset-upoffigure4b(Kinoshitaetal.2002; the tip of the spectrometer-connected light guide or,
Giraldoetal.2008),uponperpendicularillumination,a alternatively, an adjustable diaphragm in front of a
Morphoscalereflectsbluelightintoanarrowspatialstrip photomultiplier tube is positioned. The measured
(figure6f).Thisintensitydistributionpattern,whichis reflectance spectra then are the integrated spectra
matched by much earlier work ona large areaofwhole over the objective aperture, so that the directional
Morphowing(Wright1963),canbedirectlyunderstood informationis lost.
from the structuring of the scale ridges into slender Reflectance spectra are usually measured with
multilayers (Vukusic et al. 1999; Kinoshita et al. 2002, respect to a reference standard, so that the convolving
2008).Rotationofthescaleresultsinascatteringpattern effect of the characteristic wavelength dependence of
shiftedtowardsshorterwavelengths(cf.figure6fwithb the illuminant may be removed. This reference
andi).Thisisentirelyexpectedfromamultilayersystem. standard is usually a spectralon diffuse reflectance
The iridescence, the illumination-dependent colour, is standard.Itis,however,importanttonotethatthiscan
clearly observable on the reflecting wings of intact frequently lead to confusing and adverse results for
Morphobutterflies(Berthier2003). structurally coloured objects. This is because the
To determine the scattering patterns as a function reflectance standard may scatter the incident light
of the wavelength, the light source can be filtered spatially very differently from the sample under
with interference filters or a monochromator, and a investigation. Microspectrophotometrically measured
black-and-white cameracan be used instead ofa colour reflectance spectra from natural samples may then
digital camera. From images taken at a series of often show reflectance values exceeding the value
wavelengths(multispectralimaging),reflectancespectra 100 per cent, unless special consideration is given to
foreachpixel,orspatialdirection(q,4),canbederived, the nature of the sample. Quantified scattered
r r
for instance using IMAGEJ (http://rsbweb.nih.gov/ij/). light intensity measurements that appear to exceed
When the scale is rotated by an angle of G458, the the incident light intensity in this way reveal the
scatteredlightescapesthehemisphereofthesolidangle potentially ambiguous nature of the measurements
captured by the ellipsoid. For this reason, the scatte- carried out.
rometeroffigure5isequippedwithasecondarybeamto
allowstudyingthescatteringforallanglesofincidence.
3.4. Integrating sphere
Although the scatterometer arrangement offigure 5
providesthebenefitofaccesstoquantifyingasample’s An integrating sphere is used for integrating the
BRDF, it is not without its costs. The ellipsoidal radiant flux scattered from or through a sample over
mirror,forinstance,hastobeofextremelyhighquality an entire hemisphere. The instrument is formed of a
and the imaging is very sensitive to an accurate hollow sphere, which has its internal surface prepared
positioning of the sample in the ellipsoid’s focal point. with a diffusely reflective coating. For applications
In many cases, a simpler approach based on epi- associated with investigating coloured samples, it
illuminationmicroscopy is attractive. generally features three openings or ports that are
usedforintroducinglightfromtheilluminant,present-
ingsamplesurfacestothatlightandasanoutletforthe
3.3. Microscatterometryand
light that is subsequently scattered (e.g. Andersson &
microspectrophotometry
Prager2006).A fibre-optic-fed spectrometer is used to
In the microscatterometer of figure 7, the sample is analyse this scattered light if the illuminant is a
illuminatedviaahalf-mirrorandamicroscopeobjective. broadband source. Generally, the intensity of light
The beam size and direction are determined by dia- inside an integrating sphere may be considered nearly
phragmsundervisualcontrolviaaphotomicroscope,and uniform, because the multiple diffuse reflections from
the scattering pattern at infinity (i.e. projected in the the sphere’s interior wall reduce the introduced light’s
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S138 Structurallycoloured biological systems P.Vukusic and D. G. Stavenga
(a) (b) (c) (d) (e)
(f) (g) (h) (i) (j)
Figure6.ScatteringdiagramsofasingleMorphoaegawingscalecreatedbytheprincipalbeamoftheimagingscatterometerof
figure5.Thescalewasrotatedinstepsof108aroundahorizontalaxis,aboutperpendiculartotheridgesrunninglongitudinallyin
the scale surface. The black strip at 2708 in (f) is the shadow of a thin glass micropipette carrying the scale. The red circles
indicatescatteringanglesof308,608and908.
(a) (b)
P
I
L
5
D D
3 3
L (c)
4
H
S D L D L
1 1 2 2 L
3
O
Figure7.(a)Microscatterometerbasedonanepi-illuminationmicroscope.Alllenses,L ,areconfocal,thusformingtelescopic
1–5
systems.D ,diaphragms;S,lightsource;H,half-mirror;O,sample;P,photomicroscope.Anarrowlightbeam,restrictedby
1–3
diaphragms D and D , reaches the sample O under a certain angle. The illumination spot is observable by focusing the
1 2
photomicroscope at the plane of diaphragm D . By inserting lens L , the far-field (infinity) scattering pattern is observed in
3 5
planeI,theback-focalplaneofL (modifiedfromStavenga2002).(b)PartofaM.aegascalephotographedwiththeright-hand
5
arrangementof(a).(c)Theassociatedfar-fieldpatternphotographedwiththeleft-handarrangementof(a).
angular dependence. As a result, every point on the ports in the sphere also reduces the incident and
sphere wall produces an equal light-field density per scattered light fields, which further complicate any
unit solid angle. Further effects of the multiple subsequent quantitative analysis. The same caution in
reflections are the removal of any short-time-scale theuseofwhitereferencesurfacesforthecalibrationof
temporal and polarization information associated with the incident light, described previously, should be
theincidentor scattered light. applied to the use of a white reference when using
It is important to note that the light which is an integrating sphere. Despite these limiting factors,
scattered fromthesamplewillitselfformacomponent however, reflectance spectra measured with an
ofthelightfieldwithinthesphereandwillsubsequently integrating sphere can provide valuable information,
alsointeractwiththesample.Thisleadstoascattered especially for diffusively scattering materials (e.g.
field measurement, which may be rather convolved. Ghiradella et al. 1972; Yoshioka & Kinoshita 2006b;
Similarly, the presence of two or three (or more) Giraldo &Stavenga 2007).
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Structurally coloured biologicalsystems P.Vukusic and D. G. Stavenga S139
(a)
(b)
Figure8.(a)Largereplicamodelmadeofpolymerandbasedexplicitlyon(b)theTEMcrosssectionthroughaMorphorhetenor
butterflywingscale.Scalebars:(a)1.5cmand(b)800nm.
3.5. Bifurcated flexiblefibre-optic probe firstrequirethoseindividualscalestobeappropriately
separated from the wing substrate and mounted by
Averyusefulandhighlyversatile,commerciallyavailable
their proximal tips onto adapted mechanical supports,
deviceisthebifurcated,flexiblefibre-opticprobe(foran
such as ground-down needle tips or micropipettes
extensive description, see Andersson & Prager (2006)).
(Vukusic et al. 1999; Giraldo et al. 2008; Kinoshita
Thefibre-probecomprisessixlightguidesthatcandeliver
etal.2008).Thereisthefurthercomplexityofoptically
the incident light to a surface and which collectively
probing and characterizing these small area samples
surroundacentralfibrethatcollectsthescatteredlight
(typical butterfly scales are approximately 150mm!
anddeliversittoaspectrometer.Thenumericalaperture
75mm). These procedures are not without difficulties
ofthedeviceassociatedwiththelightitcollectsdepends
and can come at a substantial cost in terms of
ontheproximityofthetiptothesurface.Foratypical
limitations in the way in which the experimental
working distance of a few millimetres, the numerical
variables, such as sample orientation and interrogated
aperture is then equivalent to a few degrees. The
surface areas, may beselected.
prevailingdisadvantagesofthistechnique,however,are
An attractive alternative route is available by using
associatedwiththe poorresolution ofspatial scattering
high-magnification transmission electron microscopy
characteristics. However, in situations where spectral
(TEM) images of sample cross sections and subsequent
reflectance information is at a premium, large datasets
laser-sinteringorrapid-prototypingmanufacturingpro-
canbeassembledquicklyandatlittlecost.
cessesasthebasisofthereplicationoflarge-scalemodels
Polarization characteristics are fully lost when using
(approx. 20000 times larger). These models reproduce
common optical fibres. The polarization state of
the natural sample nano- and micro-structures at the
the incident light has receivedlittle attentioninall but
millimetre and centimetre scale out of polymer
a few studies on structural coloration. However, when
materials. They permit the exploration of the samples’
polarizationsignallingisanimportantcomponentof,say,
optical properties using microwave wavelengths due to
ananimal’scommunicationchannel,thenmeasurement
thescalabilityofelectromagneticscatteringphenomena
of the reflectance spectra and scattering profiles for
inthepresenceofanalogousmaterialproperties.Afirst
different polarizations becomes necessary (Vukusic &
study, using this approach on the silver scales of the
Sambles2001, 2003; Sweeney et al. 2003; Douglasetal.
butterfly Argyrophorus argenteus, has been completed
2007;Yoshioka&Kinoshita2007).
(Vukusic etal.2009),and otherscale modelssimilarto
that of the butterfly Morpho rhetenor (figure 8) are
presentlyunderinvestigation.
3.6. Scaled-up modelsfor interrogation at
microwave wavelengths
The experimental methods described so far often
3.7. Lightsources
require the skilful physical manipulation and optical
interrogation of micro- and nano-scale animal ultra- For spectrometric investigations, the chosen light
structures. For instance, the techniques for optically source should be suitably broadband and comprise
interrogating the single isolated scales of butterflies appropriate intensity across the entire wavelength
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S140 Structurallycoloured biological systems P.Vukusic and D. G. Stavenga
range of interest. This intensity should either not
fluctuatewithtimeafterasuitablewarm-upperiod,or (a)
its fluctuation should be accounted for in data proces-
sing.Combinedwithaphotodiodearrayspectrometer,
a halogen–deuterium lamp may be used as a light
source (Stavenga et al. 2006a). Alternatively, a
(pulsed) Xe lamp may be used for spectrophotometry
(Prum 2006; Yoshioka & Kinoshita 2006b; Rutowski
etal.2007).Whitelightsuper-continuumsourceshave
also been used to characterize aspects of Morpho
butterfly appearance (Plattner 2004). However, while
theyprovideexcellent flat-band intensityfrom500nm
upwards,theirpooreroutputbelow450nmcanhinder
effective investigation of samples for which shorter
wavelength characterization is a vital component.
(b)
For all broadband sources, the effect of their
characteristic spectral profiles should be carefully
removed by appropriately ratioing the spectra associ-
ated with the data by that of the light source. As has
been mentioned previously, where a spectrum from a
whitereferencesurfaceisusedinplaceofthespectrum
associated directly with the light source itself, great
care should be taken to account rigorously for
discrepancies associated with wavelength-dependent
absorption, polarization and intensity-related angle
dependence of the reference. These variables may be
tosomedegreemanipulatedbythereferencestandard.
Figure 9. (a) A triangular hole cut into a cover and ground
For non-spectrometric investigations, the chosen
wing scale of the common grass blue (Zizina labradus
light source may be a laser or a high-quality mono- labradus) with a focused-ion-beam scanning electron
chromator, the intensities and polarizations of which microscope (courtesy of Dr Sally Stowe, ANU, Canberra).
may be controlled with appropriate filters. Lasers (b) The area indicated by the dashed rectangle in (a). The
have the advantage of delivering a considerable light cover scale lumen appears to consist of a four-tiered,
flux, but they are spectrally limited to a discrete perforated multilayer that scatters blue light (see Wilts
number of wavelengths. etal.2009).Scalebars:(a)3mmand(b)0.5mm.
either commercial cryosectioning or freeze–fracture
4.STRUCTURALMETHODS
facilities. A more controlled preparation method is
Physicalunderstandingofthespatialandspectraldata offered by focused-ion-beam SEM (FIBSEM).
measuredfromstructurallycolouredbiologicalsamples With this facility, the directed ion beam incises the
inherently demands detailed knowledge of both the materialalongachosendirectionandatachosenrate,
ultrastructure and the RI of the samples. Light so that it becomes possible to observe the exposed
microscopy is generally not suitable for gathering this section profile (figure 9).
structureinformationsincetheassociatedperiodicities The FIBSEM technique has been used for the
are generally in the range of 50–500nm. Electron examinationofscatteringbeadstructuresonthescales
microscopy is therefore an indispensable tool, since ofpieridbutterflies(Stavengaetal.2004;Smentkowski
with appropriate sample preparation, it enables et al. 2006). More recently, Galusha et al. (2008) have
imaging of the crucial structures that determine the used FIBSEM in an elegant, alternative procedure.
structuralcolours. In their study on the photonics of the weevil Lampro-
cyphus augustus, they took a series of SEM pictures
fromthecuticle,aftersectioningconsecutivethinlayers
4.1. Scanning electron microscopy
and resputtering each newly exposed surface for SEM.
A direct and generally straightforward way to gain This enabled reconstruction of the diamond-like
some insight into structures that create structural structure that causes the weevil’s intense green
colour in biological systems with micrometre and iridescence. This method is somewhat analogous to
nanometre dimensions is through use of scanning theprocedureappliedinTEM,oneofthekeymethods
electron microscopy (SEM). However, while SEM forstudyingthe anatomyof a biological system.
generallyshowssurfaceprofilesandfeatures,itsuseful-
ness may be limited with certain samples, because the
ultrastructure responsible for biological structural
4.2. Transmission electron microscopy
colour is very often subsurface. Therefore, when
using SEM, it is necessary to induce or expose Imaging using TEM methods can be of fundamental
mechanically a cross section in the sample. This may importance for studying samples, the subsurface fine
be carried out with sharp razor blades or by using structure of which creates their structural colour
J.R.Soc.Interface(2009)
Downloaded from rsif.royalsocietypublishing.org on 2 June 2009
Structurally coloured biologicalsystems P.Vukusic and D. G. Stavenga S141
properties. As a method, however, it requires consider- (a)
ableeffortandexpertisewhencomparedwithSEM.The
material must be fixed, stained and embedded in a
suitableresinthatallowsextremelythin(typically70nm
thick) sectioning. Insight into the three-dimensional
structure of the material may then be deduced from
imagesofaseriesofsections.Onoccasion,thecomplete
three-dimensional architecture of a sample may be
inferred using appropriate computer modelling and a
series of high-quality good TEM images, even from
sectionswhosethicknessmaybelessthantheperiodicity
ofthestructure(Michielsen&Stavenga2008).
An alternative approach can also lead to virtual
reconstruction of the three-dimensional model. TEM
tomography is a widely applied technique that can
image relatively large structures of various types;
periodic or aperiodic. A series of TEM images that is
tiltedsequentiallyovermanydifferenttiltanglesabout
one axis can be combined tomographically to recon-
struct a virtual three-dimensional model that is based
explicitlyontheoriginalthree-dimensionalstructureof
(b)
thesample.Thismethodwastrialledonthewingscales
oftheKaiser-I-Hindbutterfly(Teinopalpusimperialis)
structure(Argyros et al.2002).
Inthecaseofsimplermultilayerstructures,likethose
of damselfly wing-cell membranes (figure 10), straight-
forward TEM imaging may yield the dimensions of a
sample’sconstituent layers. These thenmay be usedto
predictthewingreflectancespectra(Vukusicetal.2004).
4.3. Atomicforcemicroscopy
The three-dimensionalprofile oftheintact surface ofa
sample can be quantitatively determined by atomic
forcemicroscopy(AFM).Thismethodhasbeenapplied
(c)
to the corneal nipple array of moth eyes, where the
surface structure counteracts reflections (Stavenga
etal.2006b).Theapplicabilitytostructurallycoloured
biologicalsystemsislimited,however,becauseAFMis
unabletoprobestructuresbeneaththeoutersurfaceof
asample.
4.4. Refractive index measurements
In addition to the structure, the RI of the material
determines its structural colour. Although the classical
methodsofrefractometryarenotapplicabletomaterials
Figure 10. (a) The damselfly Neurobasis chinensis exhibits
with structural colours, there are alternatives. The
brightandhighlysaturatediridescentgreenhindwingsasa
simplest one, applying suitable immersion fluids with
result of the melanin-backed multilayer in its (b) wing
known refractive indices, can be applied when the
membrane. (c) TEM images resolve the periodic spatial
materialhasanopenstructure and the immersion fluid
variation in the constituent material. Scale bars: (a) 1cm,
can permeate through its air-filled spaces. This is the (b)5mmand(c)0.5mm.
case for many butterfly wing scales. The reflection
vanisheswhentheimmersionfluidhasaRIapproaching
thatofthematerial(Nijhout1991;Vukusic etal.1999; Interestingly, the RI changes in a subtle way when
Berthier 2003; Stavenga et al. 2004). Of course, the vapours adhere to Morpho wing scales, causing a
immersion method only works when the RI is constant change in reflectance. This phenomenon can be
throughout the sample.Fortunately, this seems tohold exploited in extremely sensitive RI measurements
formanybutterflywingscales,whichhavechitinastheir (Portyrailoet al. 2007;Biro´ et al.2008).
mainconstituent,withaRIof1.56(Vukusicetal.1999). A complicating factor is that structurally coloured
Damselflywingshavelayerswithlower(1.47)aswellas biologicalsystems almostuniversallycontain absorbing
higher (1.68 and 1.74) real refractive indices (Vukusic pigments, notably the melanins (Fox & Vevers 1960).
etal.2004). The presence of melanin in a sample increases
J.R.Soc.Interface(2009)