1
Collaborative Research: Photonic Crystals in Biology: Butterfly Scales
1. Introduction
The beautiful iridescent colors found on the wings of butterflies have attracted the attention
of brilliant minds over the past centuries starting with Newton, who understood that these colors
must be due to the presence of “thin film structures” [1]. Since then, much progress has been
made and it is now recognized that many of these brilliant colors are due to various kinds of
microstructures present on butterfly wing scales [2-6]. In other words, the colors are produced by
periodic structures of cuticle-air that mimic photonic crystals [7-11].
Many butterflies across all five major families (Nymphalidae, Lycaenidae, Riodinidae,
Papilionidae, and Pieridae) display striking colors that result from the nano-morphology on the
surface of their wing scales [4, 5, 12-15]. These structural colors can include deep blacks [16],
reds, oranges and greens [17, 18], as well as the more common blues, violets, ultraviolets, and
whites [19-23]. Typically these colors appear metallic due to the saturation or the purity of the
colors produced [3].
The colors perceived by an observer results from the various types of interaction of the
incident light with the nanostructure on the wings of the butterfly scales. This interaction may
lead to the incident light being scattered in all directions, producing a color that is independent of
the viewing angle [24], or it can have a strong directionality, producing colors that are only seen
at certain angles [3, 6, 25-27]. Biologists and Physicists alike have been drawn to study the nanomorphology of these scales, by randomly sampling a few specimens across the 17,000-specimenrich butterfly superfamily (the Papilionoidea). This initial sampling has uncovered a relatively
broad range of morphological structures that are involved in producing the structural colors.
Because of the relatively sparse number of specimens examined to date, and because many
of these specimens are not closely related, it is still unclear how readily these nano-morphologies
can change to give rise to different colors, and what are the minimal number of morphological
changes that can produce a change in a scale’s color.
In this proposal we will describe and model the structural colors of a group of closely related
species of butterflies with remarkably different colors. We will also apply artificial selection to a
population of live butterflies over several generations to change the color of its wings from brown
to blue in order to describe the causative changes in the scale’s nano-morphology. It is our hope
that the resulting insight into the photonics of butterfly wing scales will stimulate a rapidly
developing field of fundamental physical and biological research. It is also our hope that our
results will inspire technical innovations in the field of applied material science to generate such
structures on other substrates.
2. Specific aims
The aim of the present proposal is to combine descriptive and theoretical approaches coupled
with experimental approaches to understand the structural colors of butterfly wing scales. We will
take into consideration the evolutionary relationships among species, in order to properly evaluate
the direction of morphological modification and the number of independent nano-structural
solutions to similar “colors”, when more than one solution is found. The evolutionary framework
will also help us predict which ancestral scale nanostructures most easily evolve into a diverse
array of different colors. This information will facilitate the future design of synthetic
nanostructures for photonic applications. By probing nature’s diversity, we hope to identify
alternative solutions to the same “color” that may differ in ease of artificial synthesis, or
archetypical solutions that can be easily modified to produce significantly different colors.
2.1. Study of the morphology and optical properties of butterfly wing scales
2
 We will perform a detailed analysis of the purple, violet, blue, white, yellow, orange, and
brown scales on the wings of the members of a single butterfly genus, Bicyclus, and
characterize their scale structural morphology using light microscopy, scanning electron
microscopy (SEM), and transmission electron microscopy (TEM) (see Figs. 1 and 2).
 We will describe their wing scales according to a series of parameters, e.g., scale color, UV
reflectivity, ultra-structure, and iridescence.
 A theoretical-computational analysis of the optics will be performed, using the obtained
structural data. We will determine whether scale color reflectivity, ultra-structure, and other
quantifiable variables of butterfly wing scales are co-varying with each other, i.e., whether a
certain color always appear associated with a particular scale ultra-structure and vice-versa.
 We will perform angle-dependent reflection and transmission microspectrophotometry of
individual wing scales, which will yield the necessary data to test and fine-tune the
theoretical models. We would like to point out that such measurements are nonexistent with
Vukusic’s work being the single exception [24].
 We will determine whether different colors present on the same individual are more likely to
have similar scale ultra-structure, than similar colors found across individuals of the same
species, or across different species.
Figure 2 (left).
Evolution of
transverse band colors
on the dorsal forewing
of females of the
genus Bicyclus.
Estimation of
ancestral states was
carried out using
Figure 1 (above). The diversity
parsimony
of scale coloration on the dorsal
implemented in
surface of the forewing of several
MacClade 4.0.
Bicyclus species. The white pupils
Character states were
of the eyespots of B. anynana (top
assigned to terminal
left), as well as the white, purple,
taxa based on all the
and blue transversal bands are
transverse band color
structural colors that reflect UV
variants found in
light.
Bicyclus.
2.2. Experimental approaches to modifying particular colors on the wing

One of the most exciting things we intend to pursue is to target a change in color of the
scales using artificial selection in replicate lines, and observe which morphological
components of the ultra-structure of the scale change as a result. We will use the “lab-rat”
butterfly, Bicyclus anynana, to experimentally modify wing scale coloration from brown,
found in B. anynana, to blue or purple, found in other Bicyclus species (Fig. 1).
3. Background and significance of project relative to long-term goals
of the investigators
3.1. Monteiro Lab: The long-term goals of the Monteiro Lab are to explore the molecular and
developmental mechanisms of morphological evolution using butterflies as model organisms.
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This lab is the only lab that has established transgenic technology for butterflies [28] and is
currently developing functional genetic tools in order to test the role of several candidate genes in
differentiating specific colors on butterfly wings [29]. This lab has extensive experience with
artificial selection experiments on pigment-based color patterns [30-32] but would like to
continue exploring structural-based colors as well [33]. The traits previously selected were the
rings of colored scales, present in the margin of the wings, which make up an eyespot pattern.
These experiments demonstrated that populations of this species of butterfly carry substantial
amounts of genetic variation for eyespot size, ring color composition, and eyespot shape. In all
cases the mean value of the trait under selection changed to values well beyond those present in
the individuals of the original population, and resembling the phenotypes of different species of
the genus.
It has never been assessed in Bicyclus, or in any other butterfly or moth, whether artificial
selection can be employed to change the nano-structure of the wing scales, i.e., whether there is
sufficient genetic variation underlying morphology of scale nano-structures in individuals of a
population, and whether the alleles of the genes that produce such variation can be recombined in
new ways so as to produce new structural colors on the wing.
Heritability experiments performed on the “brightness” of the
structural scales of another butterfly, Colias eurytheme, showed
that this trait has an heritability of around 50-60% (R. Rutowski,
personal communication), which is comparable to the estimates
obtained in Bicyclus for eyespot traits. It is very likely, therefore,
that selection on the structural component of the brown scales in
B. anynana will also yield a strong response to selection.
The genus Bicyclus contains around 80 species showing
dramatic changes in the color
patterns on their wings [34, 35].
Structural colors in Bicyclus
butterflies are present in scales at
the centers of the eyespot
patterns [36], in brown-purple
colored rings around the
eyespots, and in transversal
white, blue, and purple bands of
scales in the dorsal surface of the
wings (Fig. 1). All of these
Figure 3. a) SEM photograph
scales of different colors are
of the wing area where the large
Figure 4. SEM photographs of the
posterior eyespot on the dorsal
highly reflective in the UV [37].
ultrastructure of a) white, b) black,
surface of B. anynana is located
To date, only the scales of a
and c) gold scales that make up the
(red
rings
indicate
the
rings of the eyespot pattern on the
single Bicyclus species, B.
approximate areas of the
dorsal wings of B. anynana. d)
anynana, have been studied at
differently colored scales), b)
brown background scales on the
the level of their ultra-structure
close-up of the white central
same surface. Darker colors, with
scales, c) black disc scales, d)
using low-resolution scanning
the exception of the white scales,
gold ring scales, and e) brown
have more cross-ridges (vertical
electron microscopy (SEM)
background scales.
lines) per unit area.
(Figs. 3 and 4) [33]. The
experiments outlined here, where
we will attempt to alter scale color in Bicyclus anynana via alteration of the nano-structures at the
surface of wing scales will be the first experiments of its kind. As in the selection experiments for
eyespots traits, however, we expect to attain gradual changes in the structural morphology of the
B. anynana brown background scales that that will mimic the structural colors of other Bicyclus
species. Ultimately, and using the selection experiments described in this project, it will be
possible to begin targeting the genes involved in producing the specific scale nano-structures.
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One of the most well established techniques in Quantitative Genetics to find genes responsible for
specific morphologies, quantitative trait loci (QTL) mapping, involves producing divergent
populations of organisms for the trait under study (e.g. large versus small, blue scales versus
brown scales, etc.), using artificial selection over several generations [38]. Once the populations
have evolved non-overlapping morphologies, a few individuals are crossed and back-crossed
between the two populations, and their offspring are measured with respect to the trait of interest.
An association study is then performed between specific genotypic markers.
3.2. Srinivasarao lab: One of the long-term goals of the Srinivasarao lab is to gain a better
understanding of how light interacts with matter sculptured at the nanometer scale to generate
various optical phenomena. The lab also wants to replicate some of these nano-morphologies and
has already had some success toward these goals (see below). The work detailed in the current
grant will broaden our understanding of the multiple ways that nano-morphologies can produce
color.
4. Aims and results from previous support
4.1 Monteiro has been supported by NSF since 2003. The aims of the first grant, IBN–
0316283 “The role of developmental genes in controlling butterfly eyespot patterns”
(08/01/03-12/31/05, $240,00) were to develop transgenic tools for butterflies. The results from
this work resulted in a highly publicized publication [28] that was featured in BBC News and
several other newspapers and websites around the World. The second grant IOB-0516705
(10/01/05-09/30/08), currently ongoing,, aims to apply these transgenic tools to test the role of
several candidate genes in the development of the wing color patterns. These grants have
supported a total of eleven publications [28, 29, 36, 39-46] , one other [36] highly publicized and
featured in a piece in Science magazine and in the German newspaper, Der Spiegel, among other
media; trained one PhD student who is a woman minority, a member of a Native American tribe,
the Choctaw Nation of Oklahoma; six additional MS and MA students (3 women); and 19
undergraduates (10 women). Also, since 2003, A Monteiro has been invited to speak at 26
venues, including a Gordon Conference on Molecular Evolution, and has co-organized 3
international meetings and/or symposia.
4.2 Srinivasarao currently has 5 active NSF grants, DMR-0312792 (11/15/03-10/31/07) and
DMI-0423619 (07/01/04-06/30/07), DMR-0637233 (08/15/06-07/31/08), CMS-0600600
(07/01/06-06/30/09), DMR-0603026 (07/01/06-06/30/09) and has completed CAREER Award,
DMR-0096240. Currently this has resulted in 22 papers published, 8 submitted, 4 in preparation,
and 55 invited talks presented worldwide. We summarize here only part of our results from the
awards due to the space constraints.
4.2.1 Anchoring Transitions of
Nematic Fluids at a Polymer
Interface: Control by Sidechain
Branching.
We reported a
temperature-driven
anchoring
transition in a polymer/nematic
fluid composite [47], where the
transition temperature is far from
the
bulk
nematic-isotropic
transition temperature. In order to
probe the subtle effects of the side
chain structure of the polymer on
control of the anchoring, a series of
polyacrylates with different side
Figure 5. Left: Polarized microscopy (POM) images under crossed
polarizers, showing only planar anchoring in a wide range of temperature.
Right: POM images under crossed polarizers, showing homeotropic-toplanar anchoring transition.
5
chain structures have been synthesized for the. A polymer dispersed liquid crystal (PDLC) film
made from TL205/1-methyl-heptyl acrylate shows only planar anchoring in the temperature
range of -14o C to TNI (nematic-isotropic transition temperature), while the films made from all
other methylheptyl acrylates are homeotropic at low temperature and show the homeotropic-toplanar anchoring transition at temperatures from 70C to 78C, which are close to that of
TL205/poly(n-heptyl acrylate) system, as shown in Fig. 5. We postulated that this dramatic
difference in the anchoring is due to a tilted conformation of the 1-methylheptyl side chain.
By using two different acrylates in the photopolymerization, it is possible to tune the
anchoring transition temperature at the copolymer interface to be in a wide range of temperatures
between those of the individual homopolymers [48]. When these two acrylates have different
anchoring tendencies and different polymerization rates, photopolymerization through a
photomask produces a spatially varying anchoring that leads to a spatially varying refractive
index, which then behaves as a diffraction grating [49]. We also demonstrated that stable Bloch
walls [50] can be successfully trapped and may be analyzed to provide a very simple method to
determine the anchoring energies. Such walls are not stable in nematic fluids but are in our
system and we have studied the 3-dimensional structure by using a laser scanning confocal
microscope in the polarization mode. We also observed that the LC anchoring is governed by the
control of polymer structures in phase-separated LC-polymer composite structures [51].
4.2.2 Three-Dimensionally Ordered Array
of Air Bubbles in a Polymer Film. We
reported the formation of a three
dimensionally ordered array of air bubbles
of monodisperse pore size in a polymer film
through a templating mechanism based on
thermo-capillary convection [52] (See Fig.
6). We have also demonstrated that both
linear polymers [52] and highly conjugated
semiconducting polymers with stiff
backbones [53, 54] can also generate such
films (Figs. 7 and 8), supporting our model
[52, 55]. This finding is in contrast to the
arguments in the literature that, in order for
such ordered structure formation, one needs
Figure 6. Schematic illustration of a mechanism
to have polymers with a given architecture
proposed for the breath figure process, by which 3dimensionally ordered array of holes are formed in
(like star polymers) or copolymers that have
polymer films. (g)
polystyrene blocks. The dimensions of these
bubbles can be controlled simply by changing the velocity of the airflow across the surface.
Depending on the type of the solvent used, either single layer or multilayer array of hole may be
generated.
When these three dimensionally ordered macroporous materials have pore dimensions
comparable to the wavelength of visible light, they are of interest as photonic band gaps and
optical stop-bands.
We
have
also
shown that one can
fabricate picoliter
beakers in a facile
manner using the
method of self29.7  29.7  3.7 m
Figure 8. Left: Microporous film made of
assembly described
azide substituted
poly(para-phenyleneFigure 7. Macroporous polymer
ethynylene). The 3D image size is 15.2
above.
Organic
3
film showing the interconnected
pores with high degree of order and
uniformity in size.
x15.2x 4.7 (m). Right: Same bubble arrays
after heating to 300oC. The 3D image size is
10.8x10.8x2.2 (m).
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semiconducting polymers have been used to form these picoliter beakers (Fig. 8), which will find
use in analytical chemistry and analytical biochemistry [54].
5. Research Methodology
5.1. Biological material: Butterflies A. Monteiro is already in possession of specimens for
each of 54 species of Bicyclus butterflies and will share wing samples with M. Srinivasarao. A
small travel budget is included to allow additional specimens to be collected for the destructive
SEM and TEM work. For the artificial selection work, we will use a large lab population of B.
anynana butterflies being permanently reared in the Monteiro lab.
5.2. Measurements of scale morphology and correlations of scale color and scale
morphology From the SEM and TEMs performed for individual scales from each species we
will score a variety of parameters such as density of ridges on the surface of the scale, density of
cross ridges, density of trabeculae, thickness of the cuticle at several positions, and number of
cuticle layers. In order to determine whether there is a correlation between a scale’s color and its
nano-morphology, we will perform a correlation analysis using
transformed data sets (phylogenetic independent contrasts), where we
remove the effect of shared ancestry from biasing the value of the
correlation [45, 56, 57]. For these analysis we will use the single
morphological parameters independently or a compound measure of a
scale’s morphology (using principle components analysis), and
correlate it with a scale’s color and/or reflective light spectral
distribution (using the X,Y,Z color system described below).
It is likely that only some aspects of a scale’s morphology will be
involved in producing the observed color. In order to dissect colordependent and color-independent morphologies apart, and avoid biasing
our color-morphology correlation with points from many closely related
species, which are not strictly independent, we will transform our data
to phylogenetic independent contrasts [56], which takes the species
evolutionary relationships into consideration and produces sets of
independent data points for further analysis.
We have started collecting transmission electron microscopy
(TEM) data for the scales of B. anynana and two other species, B.
mandanes and B.sambulos (Fig. 9) that show considerable variation in
ultra-structure. In particular the purple scales of B. medontias (Fig. 9B)
show a considerable thinker cuticle than the brown colored scales from
other species. The brown scales of B. anynana (Fig. 9A) and B.
Figure 9. TEM micromedontias (Fig. 9C) also show considerable structural differences. The
graphs showing cross
brown color is likely produced through the presence of pigments
sections through A)
deposited inside the scale. The variations in ultra-structure found for
Bicyclus anynana brown
brown scales may reflect past instances of selection (in ancestral
scales; B) B. medontias
species) for particular structural colors that are no longer visible, or
purple scales; C) B.
current instances of selection for structural colors in flanking regions of
medontias brown scales;
the wing, that produce correlated changes in other parts of the wing.
D) B. sambulos light
Untangling what characteristics of a scale’s ultra-structure are
violet scales.
actually functionally producing its color, and which characteristics are
due to historical and phylogenetic constraints will be an essential component of this project. By
using a group of closely related species, such as members of the Bicyclus genus, that show
considerable variation in scale coloration and morphology, and which have a well described
evolutionary history, will enable us to dissect these two aspects apart. We will continue to collect
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scale morphology data at larger resolution that the one displayed in Fig. 9 (using SEM and TEM)
and color data for all Bicyclus species
5.3. Artificial selection on a scale’s color
Using a thin fiber optics cable connected to a spectrophotometer, we will obtain spectra from
a constant area in the wings of live virgin male and female specimens of Bicyclus anynana (see
Fig. 10 as an example), and determine the level of color spectral variability for the colony
population. After each measurement the butterfly will be marked with a unique number and kept
in a separate cage with others of its sex, to prevent matings from taking place. Once all the
measurements are processed, we will select a subgroup of butterflies, displaying the largest
deviations from the mean color spectral values for the population, to mate with each other and
provide offspring for the next generation.
We will measure the color spectral value of all offspring from the next generation and
compare it with the mean spectral value from the parental population. From these measurements,
we will determine the heritability for scale coloration for our lab population. This measure will
indicate the proportion of scale color variation in the population that is due to genetic variation
and will indicate the
Figure 10.
Top: Spectrotrait’s
potential
photometer measurements of the
“evolvability”,
or
brown scales of ten individuals of
degree to which it
B. anynana showing variation in
will respond to
wavelength at which reflection isS
artificial selection.
at the highest intensity (red and
For a successful and
white dots). Individuals with the
timely alteration of a
largest wavelength measurements
are marked with red dots. Bottom:
scale’s
coloration
The individuals identified in the
using
artificial
plot above also show variation in
selection, we will
absolute levels of intensity of light
need the heritability
reflection.
The
light
blue
for this trait to be
individual
most
closely
significantly
approximates the color spectrum
different from zero
of the purple scales of B.
and relatively high
mandanes (yellow curve) and
(around 50%) [58].
would be marked as the most
As
already
extreme individual in our selection
experiment.
mentioned
above,
we
predict that
heritabilities of this order of magnitude are likely to be present for structural colors in Bicyclus.
From the measurements already performed in different individuals of the population (Fig. 10)
along with repeatability measurements performed in each individual (not shown), it is clear that
there is substantial amounts of color variation between individuals relative to the variation within
an individual. This indicates that selection for color change is likely to succeed.
We will rear large populations in each generation and will perform two replicate selection
lines, each containing between 300-400 individuals/per generation. The strength of selection
applied in each generation, i.e., the difference in the mean trait value for the individuals selected
as parents for the next generation relative to the population mean will depend on the initial
variance for the trait. We will select the most extreme 30 males and 30 females as parents in each
generation. These numbers have been shown to minimize inbreeding depression and maximize
selection pressure across several generations of selection for this species. Artificial selection
experiments are done routinely in this species and the Monteiro lab has extensive experience in
this area [30-32].
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5.4 Measurement of the optical properties of Individual wing scales
% Reflectance
There have been a number of reports where the “color” of large areas of the butterfly of
interest has been studied. Such measurements are subject to a number of inconsistencies due to
the fact that the precise alignment of the scales for different measurements may be difficult and
that multiple scale types, with different optical qualities, are measured together, among other
things. In order to avoid such complications, one must perform the necessary optical
measurements on single individual wing scales. Such studies have not been carried out on any
species of butterflies, with Vukusic’s study as a single exception in the literature [24]. Therefore
in order to better understand the color generating properties of a collection of wing scales, we
propose to study the optical properties of individual wing scales. We will make measurements of
absolute reflectivity and transmission of various butterfly wing scales as well as angle dependent
reflectivity measurements. To a large extent, optical modeling of the wing scales is hindered by
not having accurate values of the refractive index of the materials that the wing scales are made
of. Hence we will make measurements of refractive index of wings scales of all the butterfly
wings we intend to study.
To this end, we will build a dedicated instrument that will measure the angle dependent
reflectivity of the wing scales. The optical arrangement of the instrument will enable us to
position an individual wing scale at the center of a motorized rotational stage that coincides with
the path of the light source with a detection system mounted on a rotating arm so as to allow us to
make measurements of the absolute intensity both in reflection as a function of angle as well as
the transmitted intensity (intensity at zero angle). The
detection arm will allow for a scan from 0 to 2 around a
horizontal plane about the centrally mounted wing scale.
4
An example of reflectance spectrum of a butterfly wing at a
0
fixed angle is shown in Fig. 11.
2
While data on individual wing scales can be collected
0
over a large enough angles, the above instrument will also
0
allow us to obtain data in the transmission mode, thus
4
5
6
7
8
allowing us to interrogate the scattering as a function of
0
0
0
0
0
Wavelength
(nm)
angle as well, from zero degrees to about 90 degrees or so.
0
0
0
0
0
Such data provides us with information on the structure
Figure 11. Reflectance spectrum of
factor of the object doing the scattering. We will perform
a wing scale of a Morpho butterfly
such measurements on both the cover and ground scales of
From ref. 3.
all the butterfly species we will investigate. Measurements such as the angle dependent scattering
and angle dependent reflection will be done as a function of wavelength of the incident beam as
well with a variety of wavelengths available in Srinivasarao’s lab with the use of an Ar ion laser
(456, 476, 488, 514nm), a Kr ion laser (568nm) and several He-Ne laser (541nm and 633nm)
sources. We will use a Na lamp and a xenon arc lamp to obtain several other wavelengths
covering the entire visible region of the electromagnetic spectrum.
We will also use a micro-spectrophotometer to make measurements of the reflectivity of the
wing scales without having to remove the scales from the wings of the butterfly. The microspectrophotometer is basically a microscope with a spectrometer head with a spectral resolution
of 1nm. The illumination spot size can be adjusted to be in the range of 5-25 m, thus ensuring
that data is collected from a single wing scale. Spot sizes smaller than 5 m can be achieved by
using objectives that have higher numerical apertures. We will add a tilt stage on the rotational
stage of the microscope, thus enabling us to make angle dependent measurements of reflectivity
of the chosen wing scales.
5.5. Modeling the Optical Properties of an Individual Wing Scale
9
5.5.1.Specification of color of the wings The modeling of the colors produced by the wings of
Spectral tristimulus values
butterflies will take three different forms. One is to use the language of color science to convert
the set of complex data of the reflectivities to a simple point on the CIE (Commission
International de l’Eclairage (International Commission on Illumination) xy coordinate system,
and the second is to use the structural details obtained from various techniques (TEM, SEM, and
optical) to model the optical properties of the wing scale – in other words to be able to predict the
color based on the well known optics [59]. As a third avenue for modeling the optical properties,
we will use the language of photonic crystals to compute the band gaps, fully recognizing that the
band gaps may only be pseudogaps due to the weak refractive index contrast of these systems,
since the index contrast arises primarily from the difference in indices between the cuticle and air.
The mathematical description of
2
color by chromaticity diagram relies on
three parameters: an object, an
illuminant, and a detector. An object
1.5
can be described by its Spectral
Reflectance or Spectral Transmission
1
Curve, which maps the reflectance or
transmission of light that interacts with
the object at various wavelengths. An
0.5
illuminant is described by its Spectral
Power Distribution, which denotes the
relative power of the illuminant
0
through its gamut of wavelengths.
350 400 450 500
550 600 650 700 750
Again, the range of wavelengths
Wavelength (nm)
between 400 and 700 nm are of
Figure 12. Color matching functions of 1931 CIE standard
particular interest for the purposes of
2o observer. The filled circles, open circles, and filled
color science. Finally, the sensation of
squares represent the color sensitivity for red ( x ), green
color requires a detector, such as the
( y ), and blue ( z ), respectively. From ref. 3.
human eye. The CIE decided on a
standard set of Color Matching Functions in
1931 so that color could be mathematically
described [60, 61]. This type of function can be
generated by using a split field with one half
illuminated by a single wavelength of light. The
test for the observers is to match the other half of
the field to the standard by adjusting the relative
intensities of red, green, and blue light. In 1931,
a large group of observers with normal color
vision were asked to match every wavelength of
visible light at a 2 viewing angle (so as to
exclude any rod vision participation), and the
CIE 1931 2 Standard Observer was born [60,
61]. The Observer’s perception of red, green, and
blue is denoted by x , y , and z , respectively.
These Color Matching Functions are displayed
graphically in Fig. 12 [62].
By summing the reflectivity curves, R(),
power distribution, P(), and color matching
functions for a given object and light source over
Figure 13. CIE 1931 2 Observer chromaticity
the visible spectrum of light, three so-called
diagram.
10
Tristimulus Values X, Y, and Z are obtained [62]:
X = k  R() P() x () d
(1)
Y = k  R() P() y () d
(2)
Z = k  R() P() z () d
(3)
Here k is a constant determined by normalizing the relationships such that Y = 100 for a perfect
white reflector which reflects 100% of all incident light. After normalization, CIE chromaticity
values x, y, and z are defined as :
x = X / (X + Y + Z)
(4)
y = Y / (X + Y + Z)
(5)
z = Z / (X + Y + Z)
(6)
Since the sum of the three chromaticity coordinates is obviously equal to unity, only two of the
coordinates are needed to describe a color. By convention, x and y are used, and these two values
decide an object’s color point on the CIE 1931 2 Observer chromaticity diagram (Fig. 13). These
two chromaticity coordinates aptly describe two of the three components of color, namely hue
and saturation.
Once the reflectivities of the desired wing scales are measured, it is relatively simple to
convert the reflectivities to their respective tristimulus values (X, Y, Z), from which the
chromaticity coordinates can be calculated. We will make reflectivity measurements of all the
wing scales including those of individual wing scales and map the reflectivity on to the
chromaticity diagram. We fully recognize that the color maps we will obtain are based on the
color perception of human observers. Hence, we will make every attempt to understand the
“color perception” of butterflies by looking at the visual photo-pigments of the butterfly species
involved and attempt to construct a diagram similar to the x, y chromaticity diagram shown in Fig
13. The data necessary to do this will come from the literature where the spectral characteristics
of the photo-pigments have been garnered using “eyeshine spectroscopy”.
Once the tristimulus (X, Y, Z) values are known one can convert these back to RGB values
for the purpose of imaging and displaying the colors.
5.5.2.Modeling of Interference and diffraction
Interference colors are observed in thin films with a periodic variation in refractive index. Such
colors are produced by light waves interfering after reflections from the two or more surfaces of
these films, like soap films seen in sunlight [63], butterfly wings [2-6, 12], or beetles [3, 64]. Here
we are studying colors of butterfly wing scales that are a few microns thick, we can use the theory
of interference developed for coherent sources. The light source under consideration might be
incoherent in comparison to a laser light source, however, the coherence length of these sources
are on the order of a few microns.
One sees interference colors when the film thickness is on the order of the wavelength of
visible light. Therefore, in order to manipulate colors, one changes the film thickness or the
viewing angle. The theoretical foundation for the interference phenomena in thin film structures
is provided by the Fresnel equations [59, 63]. Constructive interference occurs when Fresnel
reflection coefficient is positive for a light beam reflected from the liquid-air interface and is
negative for reflection from the air-liquid interface. A negative reflection coefficient can simply
be thought of as a beam undergoing a 180o phase shift between the incident and reflected light
waves. Therefore the 180o phase shift suffered at the air-liquid interface together with the 180o
phase shift suffered in a round-trip through the quarter-wave plate (the thickness of the film is
/4, where  is the wavelength of the light beam) leads to perfect constructive interference for all
the reflected waves. The optical path difference experienced by the two rays that interfere
constructively is simply equal to the extra distance the light beams have to travel in the medium.
11
When a light beam is incident (at an angle 1) on a thin film of liquid of thickness d, the path
difference turns out to be 2n2dcos2, where n2 is the refractive index of the film and 2 is the
angle of refraction. In addition to this optical path difference,there will be an additional path
difference of ½ due to the additional phase difference  that occurs at the air-film interface
whenever an incident light beam is reflected by a medium of higher refractive index than the
initial medium. Thus the effective path difference between the two rays is 2n2dcos2 + ½.
Consequently if 2n2dcos2 + ½ = m, where m is an integer, the two rays will interfere
constructively and give an intensity maximum. On the other hand, if 2n2dcos2 + ½ = (m + ½),
one has destructive interference resulting in zero intensity. Since by implication we have assumed
the amplitudes, A, of the two beams to be equal, the resulting amplitude of the reflected wave will
be given by, Ar, where Ar = A + Aei with the phase difference, , given by  = (2/) (2n2dcos2
+ ½). The total reflected intensity is Ir = Ar Ar*,
and is equal to 4A2cos2(½). This equation can be
1
rewritten in terms of the reflectivity, R, to have
the following form: 4IiRsin2((2/)n1dcos2),
where Ii is the incident intensity. One can of
0.8
course rather easily eliminate the angle of
refraction from the above formula to show the
dependence of intensity on the incident angle.
0.6
600
An attempt to quantify the above discussion
800
y
400
leads us to the task of deriving expressions for
the reflected and transmitted light intensity.
0.4
Fresnel equations predict the amplitude of the
750
reflected light from thin film structures and can
440
300
be written as [65]:
0.2
n cos 1  n2 cos 2 
rs   1
;
n1 cos 1  n2 cos 2 
500


2n1 cos 1
t s  
250

n1 cos 1  n2 cos  2 
0
where n1, n2 are the refractive indices of the two
0
0.2
0.4
0.6
0.8
media in which light propagates, rs and ts are the
x
amplitudes of reflection and transmission for SFigure 14. Chromaticity diagram calculated
polarization of the incoming light beam. Here Sfor the interference colors. The numbers are
polarization implies the polarization is
the path differences used to calculate the color
perpendicular to the plane of incidence, where
seen in reflection. From ref. 60.
the plane of incidence is defined as the plane
which contains both the incident and reflected light beams. Similar expressions can be derived for
P-polarization.
In order to obtain the angular dependence, one can easily eliminate the angle of refraction
and write the optical path difference in terms of the incident angle. The maximal reflectance can
then be easily shown to be given by:
 4n d 
n2

2
1
 max   2 
1 sin 1 
2n  1 n22

where n is an average refractive index This expression clearly shows that the reflectance is shifted
to shorter wavelengths with increasing angle of incidence, consistent with all the interference
colors, including those due to butterfly wings. In our preliminary study, we calculated the
chromaticity diagram for various interference colors, and Fig. 14 is an example of such a color
seen at a fixed angle [66].
12
We will use the structural data obtained from the SEM micrographs to compute the Fresnel
coefficients and the max for a variety of butterfly wings and compare them to the experimentally
measured values to get an understanding of the color production. We will also convert the
reflectance data into chromaticity coordinates so that a realistic representation of the color
properties of the wings can be made as discussed in section 5.5.1.
5.5.3.Fourier modeling of the wing scale structures As already pointed out, the colors on the
wings of butterflies arises from a complex combination of interactions of light with the structures
on the wing scales, coupled with absorption of light that is not reflected to the observer by the
pigments that exist on the wings. Whenever the colors are structural and angle independent, such
colors have often been attributed to incoherent scattering mechanisms like Rayleigh scattering.
However, when the structures are periodic in nature, it is worthwhile to consider that the colors
produced might be due to coherent scattering. Coherent scattering occurs when the elements
doing the scattering of light are not random, in other words, they are either highly periodic or
quasi-periodic. Consequently, when structures are periodic one can use models that describe
coherent scattering for the production of color. Unlike in incoherent scattering, the phases of the
two scattered beams are the same in coherent scattering. Since coherent scattering occurs only
when there is spatial periodicity in the refractive index, we will analyze all the wing scale
structures and attempt to classify the nature of the periodic structures found on the wing-scales.
By analyzing the real-space images obtained by various microscopy techniques, we will
characterize the extent of order on the individual wing-scales as well as over large enough areas
that pertain to real viewing conditions that produce the color that one sees on the wings of
butterflies. We will also obtain a Fourier transform of the real-space images of the wings and
wing-scales, which will provide us the q-space image. This will of course be compared with the
scattering measurements from individual wing scales as was discussed in the section on optical
measurements, thus providing a direct comparison of the measurement structure factor to those
computed by taking the Fourier transform of the real-space images. In order to predict the color
that a given nanostructure might produce, we will use the 2-D Fourier tool developed by Prof.
Richard Prum at Yale University [67-69]. Such predictions can then be compared with the
measurements that we would make of the color of the individual wing scale as well as the entire
wing of a given butterfly.
5.5.4 Photonic properties of the wings based on
Photonic crystals: Photonic crystals are materials that
possess periodic dielectric structures with different
refractive indices that forbid propagation of
1-D
2-D
3-D
electromagnetic waves in any particular directions and
for any particular polarization, in a certain frequency
Figure 15. Schematic illustration of onerange, the so-called photonic band gap (PBG). Such a
dimensional (1-D), two-dimensional (2-D),
material is schematically shown in Fig. 15 where two
and three-dimensional (3-D) photonic
materials denoted by A and B are stacked alternatively.
crystals.
Such photonic crystals can be classified as onedimensional (1-D), two-dimensional (2-D) and three-dimensional (3-D) based on how the
materials are stacked (Fig. 15). The optical characteristics of such photonic band gap structures
depend on the dielectric contrast between the structures, the symmetry of the structure, and the
filling factor, i.e. the ratio of the volumes occupied by each dielectric with respect to the total
volume of the photonic crystal structure. So far, complete PBGs are found for relatively high
dielectric constant materials. Should the modulation of the contrast be weak, as in the case of the
wings of butterflies, pseudogaps can occur. The propagation of light inside the structure can be
strongly affected by the characteristics of the band structure regardless of the completeness of the
band gaps [7-11]. Hence, these periodic structures can then exhibit unusual optical phenomena,
like the phenomenon of negative refraction [70-74].
13
Many butterflies that are characterized as exhibiting structural colors have structures that are
quite periodic in nature, thus mimicking or behaving as photonic crystals. Some examples of such
wonderfully periodic structures in butterfly wings are shown in Fig. 16 [75]. Hence, knowing the
structure and the refractive indices of the periodic material, one can calculate the band structure
of such a system. The photonic band diagram indicates all possible electromagnetic scattering
interactions in a periodic system. Hence, we will calculate such photonic band diagrams for the
structures that we encounter in the range of butterflies that we study.
We will also look for instances where the band structure influences the fluorescent emission
of pigments found on the wing scales of many species of butterflies [76]. It has been recently
demonstrated that the blue-green coloration found on the wings of Swallowtail (Papilio)
butterflies has directional emission of fluorescence whose direction has been modified by a
photonic crystal slab (PCS) into which the fluorescent pigments have been infused [76]. This is
quite analogous to what has been
demonstrated for organic light emitting
diodes (OLEDs) [77, 78]. In order to
increase the efficacy of coupling the
light out of an organic light emitting
diode (OLED), photonic crystals in
conjunction with distributed Bragg
Reflectors (DBRs, which are 1-D
photonic crystals - see Fig. 15) have
been used. It should come as no surprise
that many of the design elements that the
butterflies use are technologically
Figure 16. SEM of a fractured green scale from Parides
sesostris (left) and cracked bristle of Tomares ballus (right).
relevant [78].
Scale bar is 1m. From ref. 68.
On the one hand, we will study the
photophysics of the natural pigments that have already been infused into the wing scales of
butterflies. On the other hand, we will also perform experiments where we infuse organic
molecules (including polymeric ones) that are widely studied for their optical, and
semiconducting properties, which have found use in a variety of devices ranging from light
emitting diodes to solar cells [79-82]. These materials will be dissolved in a suitable solvent and
infused into the individual wing scales of a variety of butterflies that exhibit color due to the
nano-structures on the wing scales. We will study the emission characteristics which include the
fluorescence spectrum, lifetime and fluorescence polarization, of such molecules infused into the
nanostructures. Such a systematic study should provide us with valuable data to understand how
the photophysical properties (fluorescence emission characteristics and lifetimes) change due to
the fact that the molecules are trapped in a nano-structured environment.
6. Work plan
6.1. Monteiro Lab:
Year 1: Start artificial selection experiment on two replicate lines of B. anynana butterflies.
After first generation, determine heritability for structural scale colors and estimate the number of
generations necessary to produce a significant change in scale color. Continually select butterflies
until end of year, possibly middle of year 2. In between selection bouts, collect high-resolution
SEM and TEM data for all the different wing colors in all the collected Bicyclus species. Send
samples of all the Bicyclus species to Srinivasarao lab for light reflectivity analysis.
Year 2: Continue collecting SEM and TEM data for different Bicyclus species as well as for
B. anynana material from different stages of the selection experiments. Send samples from
selection material to Srinivasarao lab for light measurements and modeling.
14
Year 3: Analyze the correlation between structure and color data between Bicyclus species,
and between the selection lines. Understand which nano-morphologies are primitive and which
are derived, and the extent to which particular morphologies have evolved independently. Help
analyze data for all the parameters collected by the Srinivasarao lab. Write papers.
6.2. Srinivasarao Lab:
Year 1: Build a dedicated instrument that will measure the angle dependent reflectivity of the
wing scales. Take microspectrophotometric data on wing scale specimens sent by Monteiro, in
both reflective and transmission mode, as a function of angle and wavelength. Obtain optical
images of the wings and scales using polarized optical microscopy and confocal laser scanning
microscopy. Perform these with different incoming polarization to determine the polarization
dependent characteristics of the butterfly wing scales.
Year 2: Continue the optical and photophysical measurements for all the available wing
scales. Perform optical modeling calculation and construct chromaticity charts for all the wing
scales for which the optical properties were measured in year 1. Study how the nanomorphologies described in Year 1 correlate with the wing scale colors.
Year 3: Simulate interference/diffraction patterns of the wing scales. Help correlate the
morphological/structural characteristics with the color/optical/photophysical properties in a
phylogenetic context.
7. Anticipated Results
7.1. Artificial selection: We expect the selection experiment to result in a visible change
in the hue of the colors of the wings of Bicyclus anynana within 7-9 generations of selection (1
year). Unpredictable results will be the extent to which all the scales in the same wing surface, or
other wings (hindwings) or surfaces (ventral surface) will show correlated changes. It is possible
that the alterations in hue will be localized just to the region that was placed under selection as
was found for vein pattern selection in Drosophila [83] and eyespot shape in Bicyclus [84]. It is
difficult to predict whether the replicate lines will evolve similar colors via the same morphology,
or alternative morphologies, as this type of artificial selection has never been done before.
7.2. Evolution of color with the genus Bicyclus: Through our survey of scale color
and TEM, SEM measurements for the Bicyclus butterflies, we will determine to what extent the
similarly brown, white, blue, and purple colors shared by several members of the genus have
similar nano-morphologies. We predict that there will be several different nano-morphological
solutions to the same “color” but at this stage we cannot predict whether some morphologies will
more readily evolve into a diverse array or colors relative to other morphologies. This will be a
novel finding for this study. We will also learn which components of a scale’s ultra-structure are
actually likely responsible for the structural color observed, and which components are likely
redundant (relative to color).
7.3. Modeling of optical properties and target-oriented study for potential
applications: For the first time we will obtain systematic optical data on an individual wing
scale that will allow us to understand the relationship between the subwavelength structures and
the observed coloration/pattern on the wings of butterflies. Such data will allow for a
comprehensive modeling of the optical properties of the wings from the point of view of “Color
Science” and “Photonic Crystals”.
8. Broader impacts of the proposed project
Education and Outreach Plan.
15
Monteiro and Srinivasarao are currently building a mutually enriching collaboration between
a Materials and a Biology department, capitalizing on their shared interest in butterfly scales and
providing students with a cross-disciplinary training environment
As one of only three women faculty in the Department of Ecology and Evolutionary Biology
at Yale University, A. Monteiro plays an important role in serving to attract, retain, and mentor
graduate and undergraduate women in science. Since 2003 A. Monteiro has recruited three
graduate women, and ten undergraduate women. She also has a strong tradition of including
undergraduate students on small, self-contained but valuable projects that will contribute to the
understanding of Bicyclus anynana biology, and thus leverage progress among the larger projects
in the lab. She has systematically pursued REU supplements to her grants, and has been awarded
3 of these fellowships in the last three years that have helped recruit over nineteen undergraduates
to work in her lab. Several of these students have gone on to pursue research careers in graduate
school. As an outreach component, the work in the Monteiro lab will be featured as part of a
public exhibit about butterflies at the Natural History Museum of Lisbon, Portugal, that will later
travel through Europe (http://www.borboletasatravesdotempo.com). The work will also be
featured in an upcoming television series about a “new generation of scientists”, produced by the
Portuguese television channel RTP2.
In addition to providing education and training for graduate students and undergraduate
students, Srinivasarao has been quite active in outreach activities some of which are listed below.
In the past four years we have organized 10 high school science teacher workshops and all have
been quite successful. We also go to local schools to lecture at least twice during a semester.
High school Science Teachers Day Workshop dealing with Polymer Science and Optics At
least four workshops have been organized in various cities in conjunction with National ACS
meetings during the period 1997-2000. The workshops were aimed at addressing some of the
missing links based on "Project 2061: Bench Marks in Science Literacy", published by The
American Association of Advancement in Science. Project 2061 describes, in great detail, what
students should know at every level from K-12. It is rather interesting and quite puzzling to find
that the words polymer, macromolecule, biopolymer and plastic do not exist in the index of
Project 2061. The teachers have always liked the workshops and wanted to participate in future
workshops. This activity was funded by Lucent Technologies, Polaroid Corporation, NSF and
Exxon, among others. In 2001, we switched to having workshops at Georgia Tech. There have
been four workshops in the last year at Georgia Tech for local teachers with support from Exxon,
NSF and Georgia Tech. We will continue this effort during the years to come. Also, in
conjunction with NASA, CEISMC has a high school science teachers’ day in which we actively
participate every year and where we discuss issue related to solar power.
We plan to have more workshops aimed at addressing the need for education of high school
science teachers in the area of liquid crystals, optics, color science, polymer science, and energy
conversion from the sun. The workshops will be conducted so that the teachers can use what is
done in the workshop in their classes. At a later stage, this effort will be carried over to other
professional societies that we are members of. We will strive to have at least one lecture for the
general public on energy – this would be done with the blessing of the upper administration at
Georgia Tech.
Collaboration with CEISMC: Research Experiences for Teachers
The Georgia Industrial Fellowships for Teachers (GIFT) program places approximately
85 science, mathematics, and social studies teachers per summer in 8-week business, industry or
laboratory research experiences to provide them with a greater understanding of the use of
science and mathematics in the real world. Approximately 20-30 of these teachers per year spend
their summer participating in research internships at Georgia Tech and Emory. Some GIFT
teachers are also hired to assist faculty in developing K-12 educational materials.
CEISMC recruits the teachers, and the participating faculty member provides a stipend
plus $1,250 program fee (which we have written into this proposal).