Maxime Boulet-Audet
Department of
Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK. Email: maxime.boulet-audet@zoo.ox.ac.uk
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| Maxime Boulet-Audet |
In spite of the glitter and glamour associated
with silk and its excellent properties as a textile fibre, the world raw silk
production has been declining over the years. During 2006 - 11 the world silk
production fell by 14.6% and its trend shows an inverse relationship with the (growth
of) economic well being of silk producing countries. However, developed and
technologically advanced countries recently show renewed interest on silk, as a
‘material for future’. That Oxford University hosts an exclusive centre ‘OxfordSilk Group’ under the leadership of such a famous zoologist as Prof. FritzVollrath, is a glowing example.
Maxime Boulet-Audet is a doctoral researcher at
the University of Oxford- Merton College; within the Oxford Silk group. His
project is supported by a NSERC Doctorate Scholarship from the Canadian
government and by an EPSRC Next Generation Users Studentship from the UK
government. Maxime’s work is to investigate why and how silk proteins have been
optimised for flow processing. His multidisciplinary project introduced novel
integrated rheo-spectroscopic tools to study silk protein structures in
solution dynamically. Specifically, he coupled infrared spectroscopy and small
angle x-ray scattering to rheology to monitor the development of silk’s
multiscale hierarchical structures. In this article Maxime explains the
application of Raman Spectroscopy in elucidating the molecular structure of
silk fibre and in explaining its physical properties.
For millennia, Silk
has been praised by the textile industry for its soft feel on skin. Its
softness comes from its microscopic diameter as well as its incredibly smooth surface.
Recently, it has been found that its size is also responsible for its
remarkable strength[1]. Silk’s fineness however makes it challenging
to study its structure. This is where Sir C.V. Raman’s discovery becomes handy.
The coupling of Raman spectroscopy with confocal
microscopy, Raman spectromicroscopy, is perfectly suited to investigate these
fine fibres. This article describes the vast potential of the technique as well
as its important contribution toward our understanding of silk’s spinning.
Silk’s lustre,
toughness and biocompatibility drove researchers for decades to produce a
comparable analogue. Among those attempts, nitro-cellulose manage to mimic
silk’s lustre, but had the distempering tendency to ignite violently when
brought close to a flame[2]. In fact, silk’s fire retardation
property is often used to tell apart natural and the artificial fibres.
Besides, those failed attempts; efforts directed at mimicking this biological
material have given us valuable textile fibres like nylon and Kevlar which
share some chemical similarities with natural silk. However, silk still have
many secrets to reveal before it can be successfully mimicked.
Even though it
represents the bulk of commercial silk production, ‘mulberry silk’ is only one among
thousands of types of silks produced by arthropods. It is also produced by bees[3], marine barnacles[4] and all spider species[5]. As thinnest silk fibres are only
few hundreds of nanometres, their size can make it challenging to study as few
techniques can probe the structure of a single silk fibre. A technique
particularly well suited for Raman spectromicroscopy as it is only circumscribed
by the diffraction limit of light. Raman spectromicroscopy can extract
information on the molecular structure from an area as small as one square
micrometre (1 µm2). The technique is illustrated in the figure.
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| Raman spectromicroscopy of silk: drawing by Maxime |
To start this
technique requires a monochromatic laser beam. Its shape is defined using a
pinhole before been reflected by the narrow band notch filter. Relying on
destructive interference, the notch filter is designed to reflect only the
wavelength of the laser to the microscope lens. The microscope objective then
focuses the beam onto the probed filament. In addition, the probe area can
directly visualise with a polychromatic light using the same optics.
Most of the photons
directed to the sample will excite its molecules to a virtual energy state
before relaxing back to the fundamental state, emitting back photons of the
same wavelength. This elastic event is described as the Rayleigh Raman
scattering and represents most of the light collected back by the microscope’s
objective. However, a small fraction of the molecules excited to their virtual
energy state will relax to an excited vibrational state instead, emitting
photons with longer wavelength than the laser resulting. The photons scattered
which have lost energy represent the stoke Raman scattering. On the other hand,
the reverse (Anti-stokes) is also possible, but less likely by two orders of
magnitudes. The difference between the incident and emitted light frequency is
thus linked to the vibration energy of the sample’s molecular bonds. As the frequency
measured is related to the type of molecular bond present, we can extract
information on the molecular structure from the frequency difference of the stoke
scattering. Thus the optics of Raman spectrometers aim at separating the stoke
Raman scattering containing the information on the molecular vibration from the
rest of the back scattered light. This is archived using the notch filter which
rejects the Rayleigh scattering, but allows the Raman scattering through. The
confocal plane is then set by another pinhole before the light is dispersed by a
grating acting like a prism. The light is then detected using a high
performance monochrome CCD detector similar to those of professional digital cameras.
The position on the detector can then be directly related to the light’s
wavelength or frequency. The resulting spectra commonly display the light
intensity count as a function of the frequency difference or Raman shift.
Silk Raman spectra contain
a wealth of information with many bands characteristic of samples’ molecular
vibrations. From these spectra we can deduce the protein 3D structure
(conformation) as well as identifying the amino acid side chain. By adding filters
along the beam path, we select the polarisation of light to obtain information
on the molecular orientation in the fibres. For instance, the alignment of the
molecules can thus be quantified and related to the material’s mechanical
properties[6]. In addition, the sample can be
mounted on a stage to be mapped. It then allows the juxtaposition of the
chemical map on the sample’s image. Another advantage of confocal microscopy is
its ability to set the focal plane depth, allowing vertical axis mapping as
well. It can even probe inside transparent containers. This way we can follow
the progression of the molecular structure inside the animal’s silk storage
gland, in the spinning duct and outside once spun, telling us the silk’s
spinning story from beginning to the end[7].
Each type of silk
gives a unique spectrum, which can be used as a unique molecular fingerprint. This
fingerprint can even discriminate between original and counterfeit drugs
without even opening the pack. It can also be used to identify unknown type of
silk or evaluate how closely they are related to one another. There are even differences in silks from the
same animal, as each of the 7 types of silks produced by some spiders has a
unique Raman signature [8]. This is not surprising knowing
that spiders have evolved different types of silks over 400 million years with
particular mechanical properties for specific functions: mobility, predation,
protection and reproduction[9].
For some colourful
samples, a competing phenomenon, fluorescence can potentially mask the Raman
scattering. Fortunately, for the majority of silk samples this does not pose a
major problem. Heat damage can also become an issue when using a powerful laser
on a microscopic area. A longer wavelength can help avoiding both issues, but the
intensity trade-off is important as scattering is proportional to the power 4
of the wavelength (doubling the wavelength decreases the signal by a factor of
16). The scattering intensity dependant on the wavelength also explains why the
sky is blue as red light is less scattered by the atmosphere than blue.
Overall, Raman
spectromicroscopy is perfectly suited to probe silk in all its forms: liquid,
fibre, films or scaffolding. It also offers a tool to monitor its structure
when stress is applied, mechanical or chemical. This technique proved very
valuable for the investigation of the spinning process by revealing the protein
conformation, molecular orientation and composition at any point in the
production process, for any given species. These insights made a significant
contribution to our understanding of the natural silk spinning. Hopefully once
we know enough about Nature’s secrets, the development of artificial silk should
result in comparable analogue.
References
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