Supplementary material for D’Alba et al. “Colour producing -keratin nanofibres in Blue
Penguin (Eudyptula minor) feathers.”
Detailed Methods
Sample collection
Contour feathers from the blue dorsum of Blue Penguin (Eudyptula minor) specimens were
removed from the ornithological collections of Yale Peabody Museum (YPM# F3883 from New
Zealand and YPM# 96707, a female specimen from South Island, New Zealand) and the San
Diego Natural History Museum (Specimen # 37206 (location of collection unknown) and 39217
(ssp. variabilis from New Zealand)). White belly feathers of Blue Penguin were obtained from
specimen YPM# 96707.
Spectrometry
We taped single feathers to gloss-free black construction paper, and recorded spectral data from
the coloured distal portion of them using an AvaSpec 2048 spectrometer (range 250-880 nm,
Avantes, Broomfield, CO, USA). We collected colour data at normal (0° incident light/0°
measurement) incidence using a bifurcated micron fibre optic probe held by a probe holder
(RPH-1, Avantes) with matte black interior that excluded ambient light. The reading area was
illuminated by a pulsed UV-vis unpolarised light source (AvaLight-XE, Avantes), and all data
were generated relative to a white standard (WS-2, Avantes). We used AvaSoft software
(Avantes) to record and average 20 spectra sequentially, and recorded and averaged
measurements from five haphazardly chosen points on each sample.
Electron microscopy
For both SEM and TEM, barbs from blue and brown feathers were embedded in Epon following
the methods of Shawkey et al. [2]. Barbs and barbules were trimmed from the central rachis,
dehydrated using 100% ethanol (20 min.) twice, and infiltrated with 15, 50, 70 and 100% Epon
(24 h each step). Infiltrated barbs were placed in block moulds and polymerised at 60º C for 16
hours. For SEM, we cut thick (3 µm) longitudinal sections of blocks on a Leica UC-6
ultramicrotome, mounted them on stubs with carbon tape, sputter-coated them with silver and
viewed them on a JEOL JSM7401F SEM. These same sections were also viewed on a Leica
DM2500 light microscope using transmitted light. For TEM, we cut thin (70-100 nm) cross
sections, picked them up on 200 mesh copper Formvar-coated grids and viewed them on a
Tecnai 12 TEM (120 kV, Tecnai Company, Oregon USA).
Infiltration (refractive index-matching) experiment
We placed barbs in glass Petri dishes and coated them with wintergreen (Gaultheria procumbens)
oil (Aura Cacia, Urbana, IA, USA) at 23º C. To facilitate infiltration of oil, we applied slight
pressure to the dorsal surface of barbs with a pair of forceps. To ensure that the pressure itself
did not cause any treatment effects, we repeated the experiment in the absence of oil. Wintergreen
oil has an estimated refractive index of 1.54, close to that of feather -keratin (refractive index 1.58) [3].
Replacing the air (refractive index of 1.0) with an index-matching liquid eliminates light scattering at
fibre-air interfaces and experimentally demonstrates the structural origin of the colour [3].
Small Angle X-ray Scattering (SAXS)
Pinhole transmission SAXS data on single blue penguin barbs from 3 specimens, which were
mounted perpendicular to the X-ray beam, were collected at beamline 8-ID-I of the Advanced
Photon Source, Argonne National Labs. We used a 15 × 15 μm beam (1.68 Å, 7.33 keV, 50 × 0.1
s exposures, sample-detector distance 3.55 m, flux 2.7e9 photons/sec), in order to average over
as few barb cells as possible. Azimuthally averaged scattering profiles were calculated from the
2D SAXS patterns (original image 1340 × 1300 pixels) using the MATLAB-implemented
software, XPCSGUI, provided by 8-ID, at 200 equal dq/q partitions, and with customized masks.
The scattering peaks were deconvoluted from the azimuthal profile using the freely available
peak-fitting software, Fityk (version 0.8.2, http://www.unipress.waw.pl/fityk). We used a Porod
background (q-4 dependence) and Split-Pearson VII functions with a Levenberg-Marquardt least
square method to fit all the scattering features (peaks and shoulders) present in the azimuthal
profiles to extract the peak positions, intensities and widths (R2 = 0.9937). The Porod’s law
describes the asymptotic nature of scattering from two media (-keratin and air) separated by a
sharp interface [4, 5] and is the null expectation for scattering from an unstructured material. The
Pearson VII function is a combination of Gaussian and Lorentzian (Cauchy) type peak profiles
that is generally used to closely approximate X-ray scattering peaks [4, 5], and the split Pearson
VII accommodates any asymmetry in the peak shape. The curve fitting eliminates the anomalous
signal from the scattering off the beam block at small q.
Polydisperse cylindrical form-factor calculations
The cylindrical scattering form factor for a single nanofibre of radius, R = 80 nm and length, 2L
= 3m, and oriented with its axis parallel to the detector plane of the CCD, is given by [4]:
F (q, R, L, ) =
4 sin 2 (qL sin  ) J 12 (qR cos )
,
q 4 R 2 L2 sin 2 cos
(1)
where J1 is the first-order Bessel function of the first-kind, and is the angle between the
cylinder axis and the projection of q in the detector plane that varies from 0 to /2. Further, we
smeared the calculated form factor to account for variation in fibre radii by using a Gaussiandistributed polydispersity of 15% (N = 1000, 10000 fibres) and summing over the resulting
scattering intensities in MATLAB 7.1 R14 (Natwick, MA USA).
SAXS colour predictions
X-rays interact weakly with biological soft matter due to the low electronic contrasts offered by
such media, so SAXS data result from single scattering of photons [6]. We used the SAXS
structural correlation peak to predict the single-scattering optical reflectance of the penguin barb
photonic nanostructure using single-scattering theory [6]. In order to remove the anomalous
scattering signal from the beam block at smaller q, which deviates from Porod’s law, we omitted
the intensities for q < 0.024 nm-1 in the deconvolution or peak-fitting of the first X-ray scattering
peak. Assuming normal incidence illumination and measurement of directly back-scattered light,
the spectral profiles of the scattering wavevector, q, and the wavelength of light,  are simply
related as:
 2
 q
 = 2

nav ,

(6)
where 2/q is the inter-fibre spacing, or the spatial correlation length, nav is the average or
effective refractive index of the nanostructure. The average refractive index was estimated using
the Maxwell-Garnett effective medium approximation [6].
2D Fourier colour prediction analysis
We analyzed cross-sectional TEM images using the Fourier tool for biological nano-optics [7].
This MATLAB-based program uses Fourier analysis to determine whether nanostructures are
sufficiently organised at an appropriate scale to produce colour by coherent light scattering
alone. Subsequent radial analyses incorporating the estimated refractive indices of keratin (RI =
1.58) [8] and air (RI = 1.00) allow the user to obtain a predicted hue. For all analyses, the largest
available square portion of keratin nanofibres and air (>500 pixels) uninterrupted by melanin
granules, cell boundaries or keratin cortex was selected.
Supplementary references
1.
2.
3.
4.
5.
6.
7.
8.
Prum, R. O., Torres, R. H., Williamson, S. & Dyck, J. 1999. Two-dimensional Fourier
analysis of the spongy medullary keratin of structurally coloured feather barbs. Proc.
Roy. Soc. Lond. B 266, 13-22.
Shawkey, M. D., Estes, A. M., Siefferman, L. M. & Hill, G. E. 2003 Nanostructure
predicts intraspecific variation in ultraviolet-blue plumage colours. Proc. Roy. Soc. Lond.
B 270, 1455-1460.
Prum, R. O. 2006 The anatomy, physics and evolution of avian structural color In Bird
Coloration, Volume 1 Mechanisms and Measurements Vol. 1 (eds G. E. Hill & K. J.
McGraw), pp. 295-353. Cambridge: Harvard University Press.
Giacovazzo, C. 2002 Fundamentals of crystallography. Oxford: Oxford University
Press.
Forster, S. et al. 2005 Scattering curves of ordered mesoscopic materials. J. Phys. Chem.
B 109, 1347-1360 (doi:10.1021/jp0467494).
Noh, H. et al. 2010. How Noniridescent Colors Are Generated by Quasi-ordered
Structures of Bird Feathers. Adv. Mat. 22, 2871-2880. (doi: 10.1002/adma.200903699).
Prum, R. O. & Torres, R. H. 2003 A Fourier tool for the analysis of coherent light
scattering by bio-optical nanostructures. Integr. Comp. Biol. 43, 591-602.
Brink, D. J. & van der Berg, N. G. 2004 Structural colours from the feathers of the bird
Bostrychia hagedash. J. Phys. D: Appl. Phys. 37, 813-818.
Supplementary figure captions
Figure S1: SEM (a) and TEM (b-c) images of -keratin nanofibres showing varying bundle
orientations within blue barbs of Eudyptula minor. Cell boundaries are indicated by white
arrows. Scale bars: a) 1 µm, b) 0.5 µm, c) 0.5 µm.
Figure S2: Optical microscope image of a blue barb from Eudyptula minor feather immediately
after being coated with wintergreen oil (a) and after being immersed for an hour with light
pressure applied (b). Scale bar: a-b), 200 µm
Figure S1
Figure S2