Nanostructural basis of rainbow-like iridescence in common ...

Nanostructural basis of rainbow-like iridescence

in common bronzewing Phaps chalcoptera

feathers

Ming Xiao,1,*

Ali Dhinojwala,1 and Matthew Shawkey

2

1Department of Polymer Science, The University of Akron, Akron, Ohio 44325, USA 2Department of Biology and Integrated Bioscience Program, The University of Akron, Akron, Ohio 44325, USA

*[email protected]

Abstract: Structural colors are common in nature. Generally single

feathers or other integuments contain only one structural color, but those of

the common bronzewing display a consistent color gradient from blue to

red (462-647nm) over the proximo-distal length of individual barbs. We

used optical microscopy and macro- and micro-spectrophotometry to

characterize this color gradient, and transmission electron microscopy to

investigate the nanostructure. Combining optical modeling and

experimental results, we demonstrate that the rainbow-like iridescence is

caused by multilayer interference from organized arrays of melanosome

rods in a keratin matrix and that the color gradient results from subtle shifts

in both diameter and spacing of melanosome rods. This result illustrates

tight developmental control feathers and may provide inspiration for the

design of multi-colored coatings or fibers.

©2014 Optical Society of America

OCIS codes: (230.4170) Multilayers; (240.0310) Thin films; (350.4238) Nanophotonics and

photonic crystals; (330.1690) Color.

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#208397 - $15.00 USD Received 25 Mar 2014; revised 25 May 2014; accepted 28 May 2014; published 6 Jun 2014(C) 2014 OSA 16 June 2014 | Vol. 22, No. 12 | DOI:10.1364/OE.22.014625 | OPTICS EXPRESS 14625

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1. Introduction

Natural structural colors, arising from light scattering by tissues that vary periodically in

refractive index at the nanometer scale, are ubiquitous in many diverse taxa, including plants

[1, 2], molluscs [3], fish [4], insects [5–10], birds [11–17] and mammals [18]. Avian feathers

exhibit varied and complex structural colors for aposematism, sexual display or camouflage


[19]. The mechanisms producing these colors have thus far been identified as: 1) thin-

film/multilayer interference from alternating layers of melanosomes (melanin containing

organelles) and keratin in barbules [11–13], 2) photonic band gaps within the wavelength of

visible light formed by two dimensional packing of melanosomes in a keratin matrix in

barbules [14–16], and 3) coherent scattering from three dimensional quasi-ordered spongy

matrices of keratin and air in barbs [17].

In most cases studied thus far, single feathers have only one structural color that may vary

depending on the incident angle and viewing angle [20–22]. However, the covert feathers of

the common bronzewing (Phaps chalcoptera) contain a color gradient from blue to red over

the proximo-distal length of individual barbs that is present even at a constant illumination

and viewing angle (Fig. 1). This continuous rainbow-like iridescence is distinct from the four

discontinuous colors (blue, green, yellow, brown) distributed in well-defined regions in male

peacock (Pavo muticus) feathers [15], and as far as we are aware has not been described

before. Moreover, it suggests a high degree of nanostructural control during feather

development that may serve as inspiration for the design of multi-colored fibers or coatings.

We therefore investigated the nanostructural basis of this color gradient using optical and

electron microscopy, spectrophotometry and optical modeling.

2. Material and methods

2.1 Barbule macrostructure

An iridescent covert feather of a male common bronzewing (Phaps chalcoptera) was

obtained from the National Museum of Natural History (Washington, D.C. USA). We used a

Leica S8 APO stereomicroscope (Leica Microsystems GmbH, Wetzlar, Germany) to

examine the color variation of barbules along the distal-proximal axis of individual barbs

(Fig. 1)

Fig. 1. a) Stereo light microscope image. Panels b-d) show higher magnification images of

barbules in distal region, middle region, and proximal regions, respectively. Scale bars a) 2

mm, b-d) 200 m .

2.2 Reflectance measurement

2.2.1 Angle-resolved specular reflectance

We visually observed slight change in hue with observation angle in the feather (suggesting

interference effects), so we quantified its iridescent properties using an AvaSpec

spectrometer with a xenon light source (Avantes Inc., Broomfield, USA, beam size ~5 mm)

attached to a custom-built goniometer. Before any spectral measurements were collected, two

calibration steps were done to maximize reflectance and thereby ensure all measurements

were done using the same protocol [23]. First, we made the incident beam ( ) and detector

angle ( ) equal by mounting the feather onto the stage and rotating the stage until the

reflectance intensity reached a maximum. The rotation angle was , shown in Fig. 2.

Second, we tilted the stage on which the feather sat to the angle where reflectance was


maximized. Through these two adjustments, the plane of the color-producing nanostructures

in the barbule was made perpendicular to the bisector line of the angle between incidence and

detection directions [23]. To quantify the effect of incident angle, we measured the specular

reflectance at incident angles varying from 10 to 45 by 5 increments (Fig. 3). Because

the red and blue regions were smaller than the beam size (5 mm) [Figs. 1(a)], we only

performed these measurements on the green portions.

Fig. 2. Schematic for geometry of spectrometer for specular reflectance measurement. is

the angle between the incident beam and the surface normal; similarly, is the angle

between the detector and the surface normal; and is the angle between feather rachis (bold

black line with an arrow) and the plane-of-incidence (yellow plane). The values of and

can be read directly from the spectrometer. The arrow of the feather stands for the distal end

of the feather. is the angle between the stage plane on which the feather lies and the

horizontal plane.

2.2.2 Single barbule normal reflectance

To precisely quantify the color of differently colored sections of the feather, we used a

CRAIC AX10 UV-Visible-NIR microspectrophotometer (MSP) (CRAIC Technologies, Inc,

San Dimas, USA, beam size ~4 m , range 300-800 nm) to measure (once for each) the

normal reflectance of single barbules across the different color regions of the feather (Fig. 4).

We then measured the reflectance of 10 green barbules in the same barb that was then

examined with transmission electron microscopy. We then performed three measurements on

the normal reflectance at p- and s-polarized illumination for barbules with different colors.

2.3 Barbule nanostructure

To investigate the nanostructures of barbules, we prepared ultra-thin cross sections following

the procedure described by Shawkey et al. [24]. We separated one barb containing all colors

from blue to red into three different color regions (blue, green and red) with the help of an

optical microscope. We again cut each color region into two parts to increase the number of

pieces for sampling. We then dehydrated barbs and barbules using 100% ethanol for 20

minutes twice and sequentially infiltrated them with 15%, 50%, 70% and 100% Embed 812

resin. Each infiltration step was performed on a Thermolyne Vari-Mix rocker (Thermo

Scientific, Waltham, USA) for about 24 hours. Next, we placed embedded medium and

samples into block molds and cured them at 60 C overnight. We trimmed the blocks with a

Leica S6 EM-Trim 2 (Leica Microsystems GmbH, Wetzlar, Germany) and cut 80 nm thick

ultrathin sections with an Ultra 45 diamond knife (Diatome Ltd, Biel, Switzerland) on a

Leica UC-6 ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany). Sections were

transferred onto the copper grids that were viewed under a JEM-1230 transmission electron

microscope (JEOL Ltd, Tokyo, Japan). From the obtained TEM images, we measured the

following parameters for the three differently colored regions 100 times via the software

Image J (http://fiji.sc/Fiji): 1) diameters of melanosomes, md ; 2) spacing (edge-to-edge

distance) between melanosome layers, a ; and 3) spacing (center-to-center distance) between


adjacent melanosomes in the same layer, 0d (Table 1). Because the end-to-end spacing (35

15 nm) between melanosome rods in each melanosome layer is small compared to the length

of melanosome rods (870 112 nm), we can assume infinite melanosome cylinders are

evenly spaced (spacing = 0d ) in each melanosome layer and the volume fraction of

melanosomes in each layer can be calculated as 2

0 0( / 2) / / 4mel m m mV d d d d d (Fig. 6).

3. Results and discussion

3.1 Rainbow-like iridescent reflectance

The iridescent color of the bronzewing is limited to the exposed side of the feathers, as

illustrated in Fig. 1(a). The color varies from red to blue along the distal-proximal gradient.

These color differences are shown more clearly in magnified images for barbules in distal,

middle and proximal regions [Figs. 1(b)-1(d)]. The barbules are oriented uniformly. Angle-

resolved specular reflectance of the green region of the feather is shown in Fig. 3, with the

incident angle ranging from 10 to 45 by 5 increments. As the incident angle increased,

the reflectance was blue-shifted (from 531nm to 492 nm) and the intensity decreased. The

range of hues (we define hue as the wavelength of peak reflectance) across the whole feather,

as measured by microspectrophotometer (MSP), is 185 nm [462nm to 647 nm, Fig. 4(d)].

400 450 500 550 600 650 700 7500

5

10

15

20

Re

fle

cta

nce

/ %

Wavelength / nm

10

15

20

25

30

35

40

45

Fig. 3. Specular reflectance spectra for the green region of barbules at incident angles from

10 to 45 .

Fig. 4. Panels a-c) microscopic images of blue, green and red barbules from MSP, respectively

and small black squares in the middle indicate the sampled spot (the length of the black square

is 4 m ). d) Color variation of the barbules measured by MSP, the vertical axis is the

normalized reflectance intensity with arbitrary unit. The color for each curve is based on the

human visual perception according to the standard CIE1931 [25].


3.2 Nanostructure of iridescent barbules

We used TEM to examine the nanostructure of individual barbules. Barbule cross-sections

revealed 6-7 layers of melanosomes arranged parallel to the periphery of the barbule cell

membrane [Figs. 5(a) and S-1]. This multilayer structure is found in all iridescent barbules,

but not in the non-iridescent brown barbules at the very tip of the same feather [Figs. 1(b)

and 5(c)]. Longitudinal sections [Fig. 5(b)] reveal that these melanosomes are cylindrical.

Multilayer structures in red, green and blue barbules are similar (Fig. 12) and the size and

spacing of melanosomes are slightly different. The mean diameter of melanosomes md in

distal barbules is around 10 nm larger than that in middle and proximal barbules, which are

similar to one another (Table 1). The melanosome layer spacing ( a , the keratin layer

thickness) is very similar in distal and middle barbules, but is about 10 nm smaller in

proximal barbules. The spacing between neighboring melanosomes in the same layer (0d ) is

nearly identical, resulting in little variation in volume fraction (packing density, melV ) of

melanosomes in the keratin matrix for each melanosome layer (Table 1).

Fig. 5. TEM images of a) cross section of a red barbule, b) longitudinal section of a green

barbule and c) cross section of a non-iridescent brown barbule. Scale bars: a-c) 500 nm.

Table 1. Spacing and diameter of melanosomes in the barbule nanostructure measured

using TEM results (Errors are standard deviation from 100 measurements)

Group Melanosome

Diameter md /nm Melanosome layer

spacing1 ( a ) /nm

Inner-layer spacing2 of

melanosomes 0d /nm volume fraction3

melV

Distal/Red 92.8 6.4 86.1 14.2 112.7 10.0 0.65 0.12

Middle/Green 83.9 7.4 87.2 15.2 104.7 10.6 0.63 0.12

Proximal/Blue 82.0 6.8 77.4 14.0 101.8 10.2 0.63 0.13 1 distance between edges of neighboring melanosome layers, which is also the thickness of keratin layer 2 distance between centers of neighboring melanosomes in the same layer 3 volume fraction of melanosomes in each melanosome layer

3.3 Multilayer interference modeling

We used a standard multilayer interference model [11, 26, 27] to calculate the theoretical

reflectance for barbules of different colors (see model details in Appendix). In this planar

multilayer system (Fig. 6), the top layer is the thin layer of keratin with thickness 1d 12 5

nm. Below it are layers of alternating melanosomes and keratin. Thickness for all

melanosome layers and keratin layers in individual barbules is consistent (Table 1), therefore

we assume that the thickness of melanosome layer and keratin layer are constant. Complex

refractive index (RI) is described as n n i , where the real component n is related to the

phase velocity and the imaginary part indicates the absorption of the medium. The

refractive index is supposed to dependent on wavelength. The real component ( kern ) for

keratin RI based on by Leertouwer et al. [28], is

2

ker 1.532 5890 /n (1)


Here is wavelength of light in the unit of nm. Keratin is almost transparent and has

negligible absorption [29], thus we assume ker 0 .

Fig. 6. Schematic of multilayer structure in barbules. 1d is the keratin cortex thickness;

md ,

the diameter of melanosomes; a , the spacing between melanosomes layers and 0d , the

spacing between neighboring melanosomes.

The RI for melanin is less well characterized due to its strong absorption [30]. In previous

studies [31], meln was assumed to be 2, and some modeling results have in some cases

confirmed this value [11, 14] while others suggested that it is lower than 2 [6, 8, 32]. Taking

dispersion effect of RI into consideration, we refer to the empirically measured values for a

dense melanin-like substance [32].

Real part:

21.56 36000 /meln (2)

Imaginary part:

1421.62melk e

(3)

In Eqs. (2) and (3), is wavelength of light in nm.

Because the melanosome layer is a mixture of melanosomes and keratin [Fig. 5(a) and

Table 1], the RI for this layer can be corrected by averaging the refractive index of two

components based on various effective medium theories [33]. In this case, the volume

averaging theory, applicable for absorbing materials is used to calculate the average RI of

melanosome layer [34]:

,mel avg eff effn n i (4)

2 2 2[A ] / 2effn A B (5)

2 2 2[ A ] / 2eff A B (6)

The constants A and B can be determined using the following equations:

2 2 2 2

ker ker( ) (1 )( )mel mel mel melA V n V n (7)

ker ker2 2(1 )mel mel mel melB V n V n (8)

In Eqs. (7) and (8), melV is the average melanosome volume fraction in each melanosome

layer (Table 1).


Fig. 7. a) Measured (green line) and modeled spectra (black line) on the average layer thickness for barbules in green region. b-c) The modeled bluest and reddest spectra (black

lines) based on largest and smallest thickness of melanosome and keratin layers in blue and

red barbules; and the blue and red curves are the bluest and reddest spectra measured by MSP, respectively.

The mean values for melanosome layer thickness, keratin layer thickness, and

melanosome inner-layer volume fraction (Table 1) for green barbules were used in modeling

multilayer interference. The model result was compared with average measured spectra of

green barbules on the same barb for TEM imaging (Section 2.2.2). The measured peak

intensity is a relative parameter which is dependent on the white reference, so we standardize

all the intensities in measured and modeled spectra to 1, as has been done in other literature

[16]. Figure 7(a) shows that the modeled peak wavelength, peak width and peak shape match

closely with those in the experimental curve. Since there is still some color variation in the

same color region (e.g. 620~650 nm in red region), the average dimensional values from

TEM images include both (e.g. red) colors with shorter wavelength and longer wavelength.

To further test the contribution of multilayer structure to the color gradient in the feather, we

obtained the theoretical color range (hues for bluest and reddest barbules) by modeling the

color resulting from the smallest layer thickness for blue barbules (75.2 nm for melanosome

layer and 63.4 nm for keratin layer) and the largest layer thickness for red barbules (99.2 nm

for melanosome layer and 100.3 nm for keratin layer). The predicted hues are 477 nm and

638 nm for the bluest and reddest barbules, respectively, Fig. 7(b) which agree quite well

with the color range (462-647 nm) experimentally identified by MSP [Figs. 7(b) and 7(c)].

0 10 20 30 40 50

450

500

550

600

Ma

xim

um

Pe

ak W

ave

len

gth

/ n

m

Angles / degrees

modeled curve

measured green

Fig. 8. Modeled results for angle-resolved reflectance spectrum for middle barbules (green solid line). The black square data points are obtained from experimental spectra in Fig. 3.

The modeling calculations for reflectance as a function of angle predict that hue should

decrease with increasing incident angle, as is observed in our empirical results (Fig. 8).

Taking into consideration the standard deviation of thickness of melanin and keratin layers in

green barbules, we obtain the color range where the measured points for green barbules fall


(Fig. 13). Additionally, s- and p-polarized specular reflectance spectra at normal incident

illumination almost overlap each other and have an identical wavelength of peak reflectance

with unpolarized incidence light for all colored barbules and we only show polarized spectra

for the green barbule (Fig. 9). These results are consistent with TEM images (Fig. 5) where

we have observed the periodicity perpendicular to the thickness direction and not within the

melanosome layers (parallel to the thickness direction). This is also consistent with

polarization dependent iridescence in negative controls with two dimensional photonic

crystal structure in green-winged teal (Anas. carolinensis) [14] and peacock (Pavo cristatus)

[15].

Fig. 9. Reflectance of green barbule measured by MSP using differently polarized input beams. The blue curve is the unpolarized reflectance, and the red, green are the s- and p-

polarization, respectively.

Based on this model, we explored how variation in the nanostructure could potentially

affect color. First, we found that small changes ( 12 nm) in the thickness of keratin cortex

(outmost layer) result in only 7 nm variation in the value of reflectance maximum (Fig.

10). Thus, the outer cortex plays a relatively small role in the color production of the

common bronzewing. Experimentally, it was difficult to precisely measure the thickness of

outmost layer because of the possible shrinkage of the barbule boundaries during embedding.

The result of our model, however, demonstrates that a precise value for the thickness of the

keratin cortex layer is not critical to accurate modeling.

Fig. 10. The hue dependence on the outmost keratin layer (cortex layer) thickness based on multilayer modeling result.

By contrast, the number of melanosome/keratin layers can strongly affect reflected color.

After six layers, the reflectance hue changes little with increasing number of layers. The full

width at half maximum of reflectance peak decreases sharply and its decrement rate


(negative value) with respect to increasing layers plateaus off at around 12 layers (Fig. 11). It

is well know that the peak reflectance intensity increases with the number of layers for a

multilayer system [20] but the increment of intensity for bronzewing feathers declines after 5

layers. The fact that increment rate of intensity is less than 1% after 12 layers indicates that

there is diminishing reward for increasing the number of layers beyond 12. Therefore a sharp

peak with enough reflectance intensity can be produced with about 12 layers of melanin and

keratin (~6 melanosome layers), the number most frequently found in rainbow-like iridescent

bronzewing feathers.

5 10 15 20 25 30-40

-30

-20

-10

0

decrement rate of FWHM

Decre

ment R

ate

of F

WH

M / n

m

Number of Layers

0.0

0.5

1.0

1.5

increment rate of intensity

Incre

me

nt

Ra

te o

f In

ten

sity /

%

Fig. 11. Green solid line is the decrement rate of full width at half maximum (FWHM), which is the derivative of FWHM with respect to layer number and blue dash line is the increment

rate of intensity which is the derivative of intensity with respect to layer number.

4. Conclusion

We report here for the first time a continuous color span ranging from blue to red in a single

feather. We explain by optical modeling and TEM results that this large span (462-647 nm)

is caused by subtle shifts in both spacing and diameter of melanosomes in a multilayer

structure. Although spacing differences in multilayers are common, subtle variation in

melanosome size has never before been reported. This is particularly intriguing because it

necessitates the existence of a mechanism in the developing feather cells to precisely control

(within a few nanometers) the size of the melanosomes. The diameters of melanosomes

increase by 10 nm from blue (proximal) to red (distal) barbules and we hypothesize that this

spatial size control is regulated by decreasing size of melanosomes as feather development

proceeds, since the distal barbules grow earlier than proximal ones [35]. How such fine

control over size and spacing along the barb is achieved is an intriguing question that has

barely been addressed. Maia et al. [36] proposed that depletion attraction of melanosomes

drives arrangement of melanosomes to aggregate into a single one-layer film beneath the

barbule surface during keratinization. However, the more complex and precise structuring

observed here suggests additional forces may also be important in self-assembly of

melanosomes. Whether these forces are subject to perturbation by external stressors, for

example, will suggest whether they may serve as honest indicators of quality, as has been

suggested for other iridescent feathers [36].

Such multilayers of alternating high and low refractive index layers have also been

utilized by many others animals, ranging from nacres of mollusks [3], fish scales [4], beetle

elytra [8], butterfly wing scales [37] to damselfly wing veins [6, 7]. However, use of

multilayer solid melanosome rods in a keratin matrix for color production has only been

reported in the bird of paradise (Parotia lawesii) [13, 38] since seminal early studies [39, 40].

Therefore, this paper may provide new inspiration for the design of multi-colored coatings or


fibers from multilayer of high refractive index nanoparticles in a low refractive index matrix,

which may be used for antireflection or spectral filtering. For example, bio-inspired tunable

colored fibers have been made via multilayer rolling [41], but these are only a single color.

Our results suggest that color gradients can be produced using spaced layers of high

refractive index particles (e.g. melanosome particles) whose size can be tuned to control the

thickness of these layers and whose volume fraction can be manipulated to change the

refractive index of these layers, providing a novel strategy for the design of synthetic

multilayer structures with optical properties.

Appendix

The matrix method for multilayer interference calculation we used was developed by Azzam

and Bashara [26] and has been used in both material science [27] and biological field [11].

First, we need to define three matrices as follows:

Interface matrix for j th interface

( 1)

11

1

j

j j

jj

rI

rt

(9)

In Eq. (9), jr and jt are Fresnel reflection and transmission coefficient.

For p-polorization

1 1

1 1

cos cos

cos cos

j j j j

j

j j j j

n nr

n n

(10)

For s-polorization

1 1

1 1

cos cos

cos cos

j j j j

j

j j j j

n nr

n n

(11)

0 0 1 1 0 0 1 1 j jcos cos cos cosφ cosφ cosφj jn n n n n n (12)

Layer matrix for j th layer

0

0

j

j

ib

j ib

eL

e

(13)

In Eq. (13),

2 cos /j j j jb d n (14)

The scattering matrix including the reflection and transmission properties of multilayer

structure

11 12

01 1 12 2 ( 1) ( 1)

21 22

m m m m m

S SS I L I L I L I

S S

(15)

What we need to obtain from modeling is the reflectivity ,R

2

R r (16)

In Eq. (16), r is the reflection coefficient which can be calculated from the scattering matrix:


11 21/r S S (17)

At last, we average out p- and s- polarization reflectivity to get the modeled reflectance

Fig. 12. TEM images of cross-sections of barbules with different colors under the same

magnification: a) red, b) green and c) blue. Scale bars 100nm

10 20 30 40 50400

450

500

550

600

650

700

Max

imum

Pea

k W

avel

ength

/ n

m

Angle /Degrees

Modeled Green Range

Measured Green

Fig. 13. The modeled color range for barbules in green zone and the experimentally measrued hue for green barbules at different incident angles.

Acknowledgments

Thanks to C. Eliason for help with microspectrophotometry, L. D'Alba for help on TEM

sample preparation and R. Maia for his help with the multilayer interference model. We

thank B. Hsiung, D. Fechyr-Lippens and J. Peteya for helpful comments on earlier versions

of this manuscript. We thank HSFP RGY-0083 (M.D.S), AFOSR FA9550-13-1-0222

(M.D.S) and NSF-DMR-1105370 (A.D.) for funding this research.


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