No abstract

As synchrotron light sources and optics deliver greater photon flux on samples, X-ray-induced photo-chemistry is increasingly encountered in X-ray absorption spectroscopy (XAS) experiments. The resulting problems are particularly pronounced for biological XAS experiments. This is because biological samples are very often quite dilute and therefore require signal averaging to achieve adequate signal-to-noise ratios, with correspondingly greater exposures to the X-ray beam. This paper reviews the origins of photo-reduction and photooxidation, the impact that they can have on active site structure, and the methods that can be used to provide relief from X-ray-induced photo-chemical artifacts.

Understanding biochemical mechanisms and changes associated with disease conditions and, therefore, development of improved clinical treatments, is relying increasingly on various biochemical mapping and imaging techniques on tissue sections. However, it is essential to be able to ascertain whether the sampling used provides the full biochemical information relevant to the disease and is free from artefacts. A multi-modal micro-spectroscopic approach, including FTIR imaging and PIXE elemental mapping, has been used to study the molecular and elemental profile within cryofixed and formalin-fixed murine brain tissue sections. The results provide strong evidence that amino acids, carbohydrates, lipids, phosphates, proteins and ions, such as Cl(-) and K(+), leach from tissue sections into the aqueous fixative medium during formalin fixation of the sections. Large changes in the concentrations and distributions of most of these components are also observed by washing in PBS even for short periods. The most likely source of the chemical species lost during fixation is the extra-cellular and intra-cellular fluid of tissues. The results highlight that, at best, analysis of formalin-fixed tissues gives only part of the complete biochemical "picture" of a tissue sample. Further, this investigation has highlighted that significant lipid peroxidation/oxidation may occur during formalin fixation and that the use of standard histological fixation reagents can result in significant and differential metal contamination of different regions of tissue sections. While a consistent and reproducible fixation method may be suitable for diagnostic purposes, the findings of this study strongly question the use of formalin fixation prior to spectroscopic studies of the molecular and elemental composition of biological samples, if the primary purpose is mechanistic studies of disease pathogenesis.

A family of dinuclear cobalt complexes with bridging bis(dioxolene) ligands derived from 3,3,3',3'-tetramethyl-1,1'-spirobis(indane-5,5',6,6'-tetrol) (spiroH4) and ancillary ligands based on tris(2-pyridylmethyl)amine (tpa) has been synthesized and characterized. The bis(dioxolene) bridging ligand is redox-active and accessible in the (spiro(cat-cat))(4-), (spiro(SQ-cat))(3-), and (spiro(SQ-SQ))(2-) forms, (cat = catecholate, SQ = semiquinonate). Variation of the ancillary ligand (Mentpa; n = 0-3) by successive methylation of the 6-position of the pyridine rings influences the redox state of the complex, governing the distribution of electrons between the cobalt centers and the bridging ligands. Pure samples of salts of the complexes [Co2(spiro)(tpa)2](2+) (1), [Co2(spiro)(Metpa)2](2+) (2), [Co2(spiro)(Me2tpa)2](2+) (3), [Co2(spiro)(Me3tpa)2](2+) (4), [Co2(spiro)(tpa)2](3+) (5), and [Co2(spiro)(tpa)2](4+) (6) have been isolated, and 1, 4, and 6 have been characterized by single crystal X-ray diffraction. Studies in the solid and solution states using multiple techniques reveal temperature invariant redox states for 1, 2, and 4-6 and provide clear evidence for four different charge distributions: 1 and 2 are Co(III)-(spiro(cat-cat))-Co(III), 4 is Co(II)-(spiro(SQ-SQ))-Co(II), 5 is Co(III)-(spiro(SQ-cat))-Co(III), and 6 is Co(III)-(spiro(SQ-SQ))-Co(III). Of the six complexes, only 3 shows evidence of temperature dependence of the charge distribution, displaying a rare thermally induced two-step valence tautomeric transition from the Co(III)-(spiro(cat-cat))-Co(III) form to Co(II)-(spiro(SQ-cat))-Co(III) and then to Co(II)-(spiro(SQ-SQ))-Co(II) in both solid and solution states. This is the first time a two-step valence tautomeric (VT) transition has been observed in solution. Partial photoinduction of the VT transition is also possible in the solid. Magnetic and spectroscopic studies of 5 and 6 reveal that spiroconjugation of the bis(dioxolene) ligand allows electronic interaction across the spiro bridge, suggesting that thermally activated vibronic coupling between the two cobalt-dioxolene moieties plays a key role in the two-step transition evident for 3.

Synchrotron XPS was used to investigate a series of chemically synthesised, atomically precise gold clusters Au(n)(PPh3)y (n = 8, 9 and 101, y depending on the cluster size) immobilized on anatase (titania) nanoparticles. Effects of post-deposition treatments were investigated by comparison of untreated samples with analogues that have been heat treated at 200 °C in O2, or in O2 followed by H2 atmosphere. XPS data shows that the phosphine ligands are oxidised upon heat treatment in O2. From the position of the Au 4f(7/2) peak it can be concluded that the clusters partially agglomerate immediately upon deposition. Heating in oxygen, and subsequently in hydrogen, leads to further agglomeration of the gold clusters. It is found that the pre-treatment plays a crucial role in the removal of ligands and agglomeration of the clusters.

The EXAFS and resonance Raman spectra on the HNO-myoglobin adduct, 1, are consistent with the presence of HNO bound to a heme center. The three-dimensional structure about the heme center of 1 obtained from multiple-scattering (MS) analysis of the EXAFS of the heme protein yielded an Fe-N-O bond angle of 131 degrees and an Fe-N bond length of 1.82 A, which compare well with published values for model complexes containing RNO ligands. Resonance Raman spectra identified the nu(N=O) stretch at 1385 cm-1 (confirmed by 15N labeling), which corresponds well with those reported for small molecule HNO complexes. The wavelength of the nu(Fe-N) at 636 cm-1 of 1 is significantly higher than those of MbIINO and MbIIINO (554 and 595 cm-1, respectively). The XAFS, XANES, and resonance Raman data are all consistent with the structure deduced from the NMR experiments, providing more detail on the bonding between HNO and the metal center.

An anti-metastatic drug, NAMI-A ((ImH)[Ru(III) Cl4 (Im)(dmso)]; Im=imidazole, dmso=S-bound dimethylsulfoxide), and a cytotoxic drug, KP1019 ((IndH)[Ru(III) Cl4 (Ind)2 ]; Ind=indazole), are two Ru-based anticancer drugs in human clinical trials. Their reactivities under biologically relevant conditions, including aqueous buffers, protein solutions or gels (e.g, albumin, transferrin and collagen), undiluted blood serum, cell-culture medium and human liver (HepG2) cancer cells, were studied by Ru K-edge X-ray absorption spectroscopy (XAS). These XAS data were fitted from linear combinations of spectra of well-characterised Ru compounds. The absence of XAS data from the parent drugs in these fits points to profound changes in the coordination environments of Ru(III) . The fits point to the presence of Ru(IV/III) clusters and binding of Ru(III) to S-donor groups, amine/imine and carboxylato groups of proteins. Cellular uptake of KP1019 is approximately 20-fold higher than that of NAMI-A under the same conditions, but it diminishes drastically after the decomposition of KP1019 in cell-culture media, which indicate that the parent complex is taken in by cells through passive diffusion.

The lanthanoid hafnates Ln 2 Hf 2 O 7 (Ln = La, Pr, Nd, Sm−Tm) were studied using a combination of synchrotron X-ray and neutron powder diffraction together with X-ray absorption and Raman spectroscopy. Spectroscopic methods revealed a gradual increase in disorder from the ideal pyrochlore structure to the defect fluorite structure as the size of the Ln cation increases. The line shape of the Hf L 3 -edge X-ray absorption near edge spectra which is sensitive to the local coordination environment changed with increasing disorder. The general line shape of the O K-edge XANES and Raman spectra also indicated an increase in disorder across the Ln 2 Hf 2 O 7 series. Differences in the diffraction and spectroscopy analysis reflect the greater sensitivity of the spectroscopy techniques to local ordering.