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Tuesday, 16 April 2013

Shedding light on silk’s secrets using Raman’s discovery

Maxime Boulet-Audet
Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK. Email: maxime.boulet-audet@zoo.ox.ac.uk

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.
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.

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