The intrinsic variation in the near-edge X-ray absorption fine structure (NEXAFS) spectra of peptides and proteins provide an opportunity to identify and map them in various biological environments, without additional labeling. In principle, with sufficiently accurate spectra, peptides (<50 amino acids) or proteins with unusual sequences (e.g., cysteine-or methionine-rich) should be differentiable from other proteins, since the NEXAFS spectrum of each amino acid is distinct. To evaluate the potential for this approach, we have developed X-SpecSim, a tool for quantitatively predicting the C, N, and O 1s NEXAFS spectra of peptides and proteins from their sequences. Here we present the methodology for predicting such spectra, along with tests of its precision using comparisons to the spectra of various proteins and peptides. The C 1s, N 1s, and O 1s spectra of two novel antimicrobial peptides, Indolicidin (ILPWKWPWWPWRR-NH 2 ) and Sub6 (RWWKIWVIR-WWR-NH 2 ), as well as human serum albumin and fibrinogen are reported and interpreted. The ability to identify, differentiate, and quantitatively map an antimicrobial peptide against a background of protein is demonstrated by a scanning transmission X-ray microscopy study of a mixture of albumin and sub6.

The polarization dependence (linear dichroism) of the C 1s X-ray absorption spectrum of individual multi-walled carbon nanotubes (MWCNTs) is measured using scanning transmission X-ray microscopy. A very strong dichroic effect is found in the C 1s --> pi* transition, with almost complete disappearance of this transition when the electric-field (E)-vector is aligned parallel to high-quality (low-defect) MWCNTs and maximum intensity when the E-vector is orthogonal to the tube axis. In contrast, there is very little dichroism in the C 1s --> sigma* transitions. The origin of this polarization effect is explained. The magnitude of the polarization dependence is found to differ in MWCNTs synthesized by different methods (arc discharge versus chemical vapor deposition). This is ascribed to differences in densities of sp(2)-type defects. The potential for use of this signal to characterize defects in single-carbon-nanotube devices is discussed.

Scanning transmission X-ray microscopy (STXM) has been used to probe the electronic structure of individual multiwall carbon nanotubes by chemical mapping at the nanoscale. Carbon 1s near-edge X-ray absorption fine structure (NEXAFS) spectra of individual structures are shown to be able to differentiate carbon nanotubes from onionlike carbon nanoparticles and to differentiate nanotubes synthesized by different growth methods. Imaging of the very same region by both STXM and transmission electron microscopy is shown to be a very useful and complementary approach.

Calcium silicate hydrates (C-S-H), the primary binding phase in concrete, is the most prominent physiochemical factor controlling the mechanical and chemical properties in the production of concrete. This paper reports the local-binding structure and morphological details of C-S-H as determined by high-resolution X-ray spectromicroscopy. Hydrated tricalcium silicate (C 3 S) was used to determine the properties and role of the outer products (Op) of C 3 S. C-S-H with different molar ratios of Ca/Si, were synthesized (Syn-CSH) to quantitatively evaluate the effect of silicate polymerization on Ca L and Si K edge of C-S-H. Near edge X-ray absorption fine structure (NEXA-FS) spectroscopy of Syn-CSH showed no variation in peak positions and energy separation for CaL III, II edge for the Ca/Si ratios investigated. Compared to Syn-CSH, C 3 S, when hydrated for 17 d, had a similar local structure around Ca. Si K edge NEXAFS analysis on Syn-CSH showed a tendency for the peak positions of both the Si K edge and the peak induced by multiple scattering to shift to higher energy levels. The results also indicated that the distance between the two peaks increased with a decrease of the Ca/Si ratio in Syn-CSH. Silicate polymerization influenced the multiple scattering of distant shell atoms more than the binding energy of the core atoms. Op of C 3 S had a uniform and higher degree of silicate polymerization compared to the core area. The results imply that Op reduces the hydration process of C 3 S into the core area thereby playing a key role on the properties of concrete upon formation.

Nephila clavipes dragline silk microstructure has been investigated by scanning transmission X-ray microscopy (STXM), a technique that allows quantitative mapping of the level of orientation of the peptide groups at high spatial resolution (<50 nm). Maps of the orientation parameter P2 have been derived for spider silk for the first time. Dragline silk presents a very fine microstructure in which small, highly oriented domains (average area of 1800 nm2, thus clearly bigger than individual beta-sheet crystallites) are dispersed in a dominant, moderately oriented matrix with several small unoriented domains. Our results also highlight the orientation of the noncrystalline fraction in silk, which has been underestimated in numerous structural models. No evidence of either a regular lamellar structure or any periodicity along the fiber was observed at this spatial resolution. The surface of fresh spider silk sections consists of a approximately 30-120 nm thick layer of highly oriented protein chains, which was found to vary with the reeling speed, where web building (0.5 cm/s) and lifeline (10 cm/s) spinning speeds were investigated. While the average level of orientation of the protein chains is unaffected by the spinning speed, STXM measurements clearly highlight microstructure differences. The slowpull fiber contains a larger fraction of highly oriented domains, while the protein chains are more homogeneously oriented in the fastpull fiber. In comparison, cocoon silk from the silkworm Bombyx mori presents a narrower orientation distribution. The strength-extensibility combination found in spider dragline silk is associated with its broad orientation distribution of highly interdigitated and unoriented domains.

The spatial distribution of the linear dichroic signal associated with aligned beta-sheets in a microtomed section of a Bombyx mori cocoon silk fiber was derived from scanning transmission X-ray microscopy (STXM). The intense C 1s --> pi(amide) peak at 288.25 eV was found to have negligible dichroic signal in transverse sections but a large dichroic signal in longitudinal sections. This is consistent with other measurements of the orientation of the aligned beta-sheets in silk fibers, in particular with those obtained by polarized Raman microspectroscopy to which our results are compared. When the dichroic signal strength is mapped at better than 100 nm spatial resolution, microscopic variations are found. Although the magnitude of the dichroic signal changes over a fine spatial scale, the direction of the maximum signal at any position does not change. We interpret the spatial variation of the intensity of the dichroic signal as a map of the quality of local orientation of the beta-sheets in the fiber. At sufficiently high magnification and resolution, this technique should image individual beta-sheet crystallites, although the present implementation does not achieve that. A map of the orientation parameter P(2) is derived. The average value of P(2) (-0.20 +/- 0.04) from STXM is smaller than that derived from the analysis of the amide I band in polarized Raman spectra (-0.41 +/- 0.03). This deviation is attributed to the fact that the STXM results also include the signal from unaligned regions of the protein.

Scanning transmission x-ray microscopy ͑STXM͒ has been used to study isolated carbon nanotubes for the first time. STXM and transmission electron microscopy were applied to the same type of nanotubes, providing unique information about their composition, and electronic and structural properties. The carbon 1s near-edge x-ray absorption fine structure spectra show significant differences between multiwall carbon nanotube and carbon nanoparticle contaminants. Pristine and acid treated multiwall carbon nanotubes were also compared, highlighting the potential of the technique to differentiate surface functional groups at the nanoscale.

a b s t r a c tKeywords: TEM (B) NMR Microstructure Ca 3 SiO 5 (D) STXM This paper investigates the atomic and nano-scale structures of a 50-year-old hydrated alite paste. Imaged by TEM, the outer product C-S-H fibers are composed of particles that are 1.5-2 nm thick and several tens of nanometers long. 29 Si NMR shows 47.9% Q 1 and 52.1% Q 2 , with a mean SiO 4 tetrahedron chain length (MCL) of 4.18, indicating a limited degree of polymerization after 50 years' hydration. A Scanning Transmission X-ray Microscopy (STXM) study was conducted on this late-age paste and a 1.5 year old hydrated C 3 S solution. Near Edge X-ray Absorption Fine Structure (NEXAFS) at Ca L 3,2 -edge indicates that Ca 2+ in C-S-H is in an irregular symmetric coordination, which agrees more with the atomic structure of tobermorite than that of jennite. At Si K-edge, multiscattering phenomenon is sensitive to the degree of polymerization, which has the potential to unveil the structure of the SiO 4 4− tetrahedron chain.