Marie-Eve Rousseau

Conformation and orientation of proteins in various types of silk fibers produced by Nephila clavipes spiders.

Silk fibers harvested from the web, cocoon, and prey wrapping of the spider Nephila clavipes have been studied by polarized Raman spectromicroscopy. The technique is efficient to differentiate the various types of silk by probing monofilaments produced by the major ampullate (MA), minor ampullate (MI), cylindriform, flagelliform, and aciniform glands. The spectra show that the MA, MI, and cylindriform silks belong to the same structural class and are composed of highly oriented beta-sheets (35-37%) with other slightly oriented secondary structures. Spectral markers of particular motifs involved in the beta-sheets have been identified. The flagelliform silk represents a second, very peculiar structural class. It displays a heterogeneous disordered conformation without any preferential orientation. Such characteristics certainly play a role in the large extensibility of this silk. The aciniform silk represents a third class of silk dominated by moderately oriented beta-sheets (approximately 30%) and alpha-helices (approximately 24%). Such a structure seems important in explaining the high toughness of this silk.

Orientation-insensitive spectra for Raman microspectroscopy.

Separating effects due to molecular conformation from those due to orientation in the spectra of oriented samples obtained by Raman microspectroscopy is a complex issue. To solve this problem, we propose a procedure to calculate an orientation-insensitive spectrum (so-called isotropic spectrum) from polarized spectra obtained by Raman microspectroscopy that is valid for systems that exhibit a uniaxial symmetry. The method has first been tested on highly oriented samples of high-density polyethylene (HDPE). Polarized and isotropic spectra of a highly oriented HDPE cylindrical rod and an isotropic HDPE sample have been compared. The differences in the relative intensities, which occur in the polarized spectra and are due to orientation of the polyethylene chains, are nearly cancelled in the isotropic spectra, showing that the orientation-insensitive spectrum adequately represents the molecular conformation without contributions of orientation. Second, spectra of silk fibroins have been compared in the amide I region for Bombyx mori cocoon silk fibers and methanol-treated regenerated fibroin films. The similarity of the shape of the amide I band of the isotropic spectra indicates that the secondary structure of the fibroins is very close in both samples. These experimental results support the conclusion that the molecular conformation can be efficiently characterized from the intensity and the shape of Raman bands in the orientation-insensitive spectrum.

Orientation and Relaxation in Thick Poly(ethylene terephthalate) Films by Transmission Infrared Linear Dichroism

Infrared spectroscopy is a powerful tool for the study of the orientation in amorphous and semi-crystalline polymers, but it is generally limited to thin samples. In this study, we have used transmission infrared linear dichroism to study the orientation of thick poly(ethylene terephthalate) (PET) films. To overcome the saturation problem of the intense bands of PET, overtones and combination bands in the high-frequency region of the mid-infrared spectrum were used. Using polarization-modulation infrared linear dichroism (PM-IRLD), it was possible to follow in real-time the relaxation of orientation of uniaxially oriented PET films up to 500 μm thick. It was observed that between 30 and 500 μm, the thickness of the films has no effect on the orientation relaxation dynamics. It should, therefore, be possible to use thick films, which are much easier to prepare than thin films, for future infrared studies of the deformation of PET. A very good correlation was also observed for the relaxation curves obtained using high- and low-frequency bands related to gauche and trans conformers in thin and thick films. An example of the application of these high-frequency bands to obtain orientation and structural information is also given in the case of a commercial PET bottle showing a biaxial orientation.

In situ azimuthal rotation device for linear dichroism measurements in scanning transmission x-ray microscopy

A novel miniature rotation device used in conjunction with a scanning transmission x-ray microscope is described. It provides convenient in situ sample rotation to enable measurements of linear dichroism at high spatial resolution. The design, fabrication, and mechanical characterization are presented. This device has been used to generate quantitative maps of the spatial distribution of the orientation of proteins in several different spider and silkworm silks. Specifically, quantitative maps of the dichroic signal at the C 1s-->pi* (amide) transition in longitudinal sections of the silk fibers give information about the spatial orientation, degree of alignment, and spatial distribution of protein peptide bonds. A new approach for analyzing the dichroic signal to extract orientation distributions, in addition to magnitudes of aligned components, is presented and illustrated with results from Nephila clavipes dragline spider silk measured using the in situ rotation device.

Chemical Imaging by Soft X-ray Scanning Transmission X-ray Microscopy

A.P. Hitchcock,* D. Hernandez-Cruz,* J.J. Dynes,* M.-E. Rousseau,** M. Pezolet** * Dept. of Chemistry, McMaster University, Hamilton, ON, L8S 4M1, Canada. ** Departement de Chimie, Universite Laval, Ste Foy, QC, G1K 7P4,Canada The X-ray absorption signal in synchrotron based soft X-ray scanning transmission X-ray microscopy (STXM) is providing quantitative maps of chemical species in many samples, under a wide range of environments (wet, variable temperature, pH, stress, electrochemical control, etc). State-of-the art zone plates provide a spatial resolution of 15 nm [1], while 35 nm resolution is achieved routinely in STXMs at the Advanced Light Source (ALS) and at the National Synchrotron Light Source (NSLS) [2]. With appropriate software [3], sequences of images recorded over a range of photon energies spanning one or more core excitation edges can be inverted by multivariate statistical analysis methods [4] or by pixel-by-pixel spectral fitting to generate quantitative chemical component maps.