Automated segmentation of microtomography imaging of Egyptian mummies.

Propagation Phase Contrast Synchrotron Microtomography (PPC-SRµCT) is the gold standard for non-invasive and non-destructive access to internal structures of archaeological remains. In this analysis, the virtual specimen needs to be segmented to separate different parts or materials, a process that normally requires considerable human effort. In the Automated SEgmentation of Microtomography Imaging (ASEMI) project, we developed a tool to automatically segment these volumetric images, using manually segmented samples to tune and train a machine learning model. For a set of four specimens of ancient Egyptian animal mummies we achieve an overall accuracy of 94-98% when compared with manually segmented slices, approaching the results of off-the-shelf commercial software using deep learning (97-99%) at much lower complexity. A qualitative analysis of the segmented output shows that our results are close in terms of usability to those from deep learning, justifying the use of these techniques.

An efficient approach to integrated MeV ion imaging.

An ionoluminescence (IL) spectral imaging system, besides the common MeV ion imaging facilities such as µ-PIXE and µ-RBS, is implemented at the Van de Graaff laboratory of Tehran. A versatile processing software is required to handle the large amount of data concurrently collected in µ-IL and common MeV ion imaging measurements through the respective methodologies. The open-source freeware PyMca, with image processing and multivariate analysis capabilities, is employed to simultaneously process common MeV ion imaging and µ-IL data. Herein, the program was adapted to support the OM_DAQ listmode data format. The appropriate performance of the µ-IL data acquisition system is confirmed through a case study. Moreover, the capabilities of the software for simultaneous analysis of µ-PIXE and µ-RBS experimental data are presented.

Towards data format standardization for X-ray absorption spectroscopy.

A working group on data format standardization for X-ray absorption spectroscopy (XAS) has recently formed under the auspices of the International X-ray Absorption Society and the XAFS Commission of the International Union of Crystallography. This group of beamline scientists and XAS practitioners has been tasked to propose data format standards to meet the needs of the world-wide XAS community. In this report, concepts for addressing three XAS data storage needs are presented: a single spectrum interchange format, a hierarchical format for multispectral X-ray experiment, and a relational database format for XAS data libraries.

Ultrahigh vacuum/high-pressure flow reactor for surface x-ray diffraction and grazing incidence small angle x-ray scattering studies close to conditions for industrial catalysis.

A versatile instrument for the in situ study of catalyst surfaces by surface x-ray diffraction and grazing incidence small angle x-ray scattering in a 13 ml flow reactor combined with reaction product analysis by mass spectrometry has been developed. The instrument bridges the so-called "pressure gap" and "materials gap" at the same time, within one experimental setup. It allows for the preparation and study of catalytically active single crystal surfaces and is also equipped with an evaporator for the deposition of thin, pure metal films, necessary for the formation of small metal particles on oxide supports. Reactions can be studied in flow mode and batch mode in a pressure range of 100-1200 mbar and temperatures up to 950 K. The setup provides a unique combination of sample preparation, characterization, and in situ experiments where the structure and reactivity of both single crystals and supported nanoparticles can be simultaneously determined.

Advanced combined application of micro-X-ray diffraction/micro-X-ray fluorescence with conventional techniques for the identification of pictorial materials from Baroque Andalusia paintings.

The process of investigating paintings includes the identification of materials to solve technical and historical art questions, to aid in the deduction of the original appearance, and in the establishment of the chemical and physical conditions for adequate restoration and conservation. In particular, we have focused on the identification of several samples taken from six famous canvases painted by Pedro Atanasio Bocanegra, who created a very special collection depicting the life of San Ignacio, which is located in the church of San Justo y Pastor of Granada, Spain. The characterization of the inorganic and organic compounds of the textiles, preparation layers, and pictorial layers have been carried out using an XRD diffractometer, SEM observations, EDX spectrometry, FT-IR spectrometry (both in reflection and transmission mode), pyrolysis/gas chromatography/mass spectrometry and synchrotron-based micro-X-ray techniques. In this work, the advantages over conventional X-ray diffraction of using combined synchrotron-based micro-X-ray diffraction and micro-X-ray fluorescence in the identification of multi-layer paintings is demonstrated.

Multilayers quantitative X-ray fluorescence analysis applied to easel paintings.

X-ray fluorescence spectrometry (XRF) allows a rapid and simple determination of the elemental composition of a material. As a non-destructive tool, it has been extensively used for analysis in art and archaeology since the early 1970s. Whereas it is commonly used for qualitative analysis, recent efforts have been made to develop quantitative treatment even with portable systems. However, the interpretation of the results obtained with this technique can turn out to be problematic in the case of layered structures such as easel paintings. The use of differential X-ray attenuation enables modelling of the various layers: indeed, the absorption of X-rays through different layers will result in modification of intensity ratio between the different characteristic lines. This work focuses on the possibility to use XRF with the fundamental parameters method to reconstruct the composition and thickness of the layers. This method was tested on several multilayers standards and gives a maximum error of 15% for thicknesses and errors of 10% for concentrations. On a painting test sample that was rather inhomogeneous, the XRF analysis provides an average value. This method was applied in situ to estimate the thickness of the layers a painting from Marco d'Oggiono, pupil of Leonardo da Vinci.

Quantitative X-ray fluorescence analysis of an Egyptian faience pendant and comparison with PIXE.

The X-ray fluorescence (XRF) technique is a common choice in the archaeometric field for in situ investigations with portable instruments. This work shows that XRF portable systems can be used for quantitative analyses using appropriate software, obtaining a similar accuracy to that provided with other techniques such as particle-induced X-ray emission (PIXE), as shown for an Egyptian faience pendant and for two glass standards.

Lactococcus lactis, an efficient cell factory for recombinant protein production and secretion.

The use of Gram-positive bacteria for heterologous protein production proves to be a useful choice due to easy protein secretion and purification. The lactic acid bacterium Lactococcus lactis emerges as an attractive alternative to the Gram-positive model Bacillus subtilis. Here, we review recent work on the expression and secretion systems available for heterologous protein secretion in L. lactis, including promoters, signal peptides and mutant host strains known to overcome some bottlenecks of the process. Among the tools developed in our laboratory, inactivation of HtrA, the unique housekeeping protease at the cell surface, or complementation of the Sec machinery with B. subtilis SecDF accessory protein each result in the increase in heterologous protein yield. Furthermore, our lactococcal expression/secretion system, using both P(Zn)zitR, an expression cassette tightly controlled by environmental zinc, and a consensus signal peptide, SP(Exp4), allows efficient production and secretion of the staphylococcal nuclease, as evidenced by protein yields (protein amount/biomass) comparable to those obtained using NICE or P170 expression systems under similar laboratory conditions. Finally, the toolbox we are developing should contribute to enlarge the use of L. lactis as a protein cell factory.

Synchrotron-based X-ray spectromicroscopy used for the study of an atypical micrometric pigment in 16th century paintings.

Grünewald is a famous German painter of the 16th century, whose celebrity is associated with his unique skill in handling colors. This article presents the analysis of materials used to render a metallic aspect in the Isenhein Altarpiece and the Basel's Crucifixion. Such samples are challenging objects for microanalysis due to both chemical and physical complexity. Their study by synchrotron-based X-ray microscopy techniques was made possible thanks to recent developments carried out at the ID21 beam line (European Synchrotron Radiation Facility, ESRF). A submicron X-ray fluorescence probe revealed the main presence of lead, sulfur, antimony, and calcium. The fluorescence-line interferences (in particular K-lines of sulfur with M-lines of lead, and K-lines of calcium with L-lines of antimony) were resolved with the fitting program, PyMCA. 2D-mapping highlighted the presence of micrometer grains of sulfur and antimony into a lead matrix. XANES measurements were performed at both the sulfur K-edge and the antimony L-edge to refine information from an atomic to a molecular level. Beam stability was a key point in this study to selectively probe micrometer pigment grains, dispersed in the lead matrix. They confirm that the grains are made of stibnite (antimony sulfide), a very atypical pigment. Chemical mapping of sulfides is perfectly correlated with antimony mapping and provides a clear visualization of the stibnite pigments, in addition to their identification. Besides its artistic relevancy, this work aims at illustrating developments of synchrotron X-ray microprobe methods for the chemical characterization and observation of complex and micrometer-scale materials.

Stepwise transition of the tetra-manganese complex of photosystem II to a binuclear Mn2(micro -O)2 complex in response to a temperature jump: a time-resolved structural investigation employing x-ray absorption spectroscopy.

In oxygenic photosynthesis, water is oxidized at a protein-cofactor complex comprising four Mn atoms and, presumably, one calcium. Using multilayers of Photosystem II membrane particles, we investigated the time course of the disassembly of the Mn complex initiated by a temperature jump from 25 degrees C to 47 degrees C and terminated by rapid cooling after distinct heating periods. We monitored polarographically the oxygen-evolution activity, the amount of the Y(D)(ox) radical and of released Mn(2+) by EPR spectroscopy, and the structure of the Mn complex by x-ray absorption spectroscopy (XAS, EXAFS). Using a novel approach to analyze time-resolved EXAFS data, we identify three distinct phases of the disassembly process: (1) Loss of the oxygen-evolution activity and reduction of Y(D)(ox) occur simultaneously (k(1) = 1.0 min(-1)). EXAFS spectra reveal the concomitant loss of an absorber-backscatterer interaction between heavy atoms separated by approximately 3.3 A, possibly related to Ca release. (2) Subsequently, two Mn(III) or Mn(IV) ions seemingly separated by approximately 2.7 A in the native complex are reduced to Mn(II) and released (k(2) = 0.18 min(-1)). The x-ray absorption spectroscopy data is highly suggestive that the two unreleased Mn ions form a di- micro -oxo bridged Mn(III)(2) complex. (3) Finally, the tightly-bound Mn(2)( micro -O)(2) unit is slowly reduced and released (k(3) = 0.014 min(-1)).

First steps towards time-resolved BioXAS at room temperature: state transitions of the manganese complex of oxygenic photosynthesis.

Structural changes and redox transitions at the metal atoms of the active site are essential for the understanding of the catalytic mechanisms of biological metalloenzymes. First steps towards studying these processes by time-resolved X-ray absorption spectroscopy on protein samples (BioXAS) are reported. Photosystem II (PSII) catalyses the light-driven oxidation of bound water molecules at a tetranuclear manganese complex yielding the molecular oxygen of the atmosphere. In this work, first time-resolved XAS results under quasi-physiological conditions (at room temperature, non-crystalline samples) on PSII are presented. Perspectives of time-resolved BioXAS are discussed.

Determination of the iron oxidation state in earth materials using XANES pre-edge information.

Fe K-edge XANES spectra have been measured in more than 35 Fe(II) and Fe(III)-bearing minerals. The separation between the average pre-edge centroid positions for Fe(II) and Fe(III) is 1.4 +/- 0.1 eV. Examination of calculated pre-edge features of mechanical mixtures of phases containing different proportions of Fe(II) and Fe(III) reveals that different trends of pre-edge position vs. pre-edge intensity can be observed, depending on the coordination environment. Both pre-edge parameters have been used to estimate the ferric/ferrous ratio in 12 natural minerals.

Chemical, biochemical and cellular responses in the digestive gland of the mussel Mytilus galloprovincialis from the Spanish Mediterranean coast.

Mussels (Mytilus galloprovincialis) were sampled in March 1996 from five stations along the Western Mediterranean coast (Barcelona, Ebro Delta, Alboraya, Cullera, Denia) corresponding to urban, industrial and agricultural areas. Different biochemical and cellular markers were determined in the mussels in order to assess the effects and/or exposure to pollutants. The cytochrome P450 system, acetylcholinesterase and metallothioneins were among the biochemical markers selected for the study. Histochemical analysis of ß-glucuronidase and catalase activity were performed as marker enzymes for lysosomes and peroxisomes. Chemical analyses indicated that mussels from Barcelona and Denia as highly exposed to polycyclic aromatic hydrocarbons (PAHs)(1.8-2.7 µg g(-1) w.w. against 0.02-0.10 µg g(-1) w.w.), and polychlorinated biphenyls (PCBs)(132-260 ng g(-1) w.w. against 8-24 ng g(-1) w.w.). This was in agreement with changes in lysosome structure and higher number of peroxisomes in those organisms. High levels of metals (particularly Cr and Cu) were recorded in the digestive gland of Alboraya mussels, which also had elevated metallothionein content (28 nmol g(-1) w.w.) in comparison with the other stations (15-20 nmol g(-1) w.w.). Benzo(a)pyrene hydroxylase (BPH) activity indicated Cullera and Barcelona as possibly polluted sites. The results support the usefulness of the biomarker approach to assess and diagnose environmental pollution. The use of a battery of biomarkers at different levels of biological organization coupled with chemical analysis is highlighted.

Direct and Fe(II)-mediated reduction of technetium by Fe(III)-reducing bacteria.

The dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens reduced and precipitated Tc(VII) by two mechanisms. Washed cell suspensions coupled the oxidation of hydrogen to enzymatic reduction of Tc(VII) to Tc(IV), leading to the precipitation of TcO(2) at the periphery of the cell. An indirect, Fe(II)-mediated mechanism was also identified. Acetate, although not utilized efficiently as an electron donor for direct cell-mediated reduction of technetium, supported the reduction of Fe(III), and the Fe(II) formed was able to transfer electrons abiotically to Tc(VII). Tc(VII) reduction was comparatively inefficient via this indirect mechanism when soluble Fe(III) citrate was supplied to the cultures but was enhanced in the presence of solid Fe(III) oxide. The rate of Tc(VII) reduction was optimal, however, when Fe(III) oxide reduction was stimulated by the addition of the humic analog and electron shuttle anthaquinone-2,6-disulfonate, leading to the rapid formation of the Fe(II)-bearing mineral magnetite. Under these conditions, Tc(VII) was reduced and precipitated abiotically on the nanocrystals of biogenic magnetite as TcO(2) and was removed from solution to concentrations below the limit of detection by scintillation counting. Cultures of Fe(III)-reducing bacteria enriched from radionuclide-contaminated sediment using Fe(III) oxide as an electron acceptor in the presence of 25 microM Tc(VII) contained a single Geobacter sp. detected by 16S ribosomal DNA analysis and were also able to reduce and precipitate the radionuclide via biogenic magnetite. Fe(III) reduction was stimulated in aquifer material, resulting in the formation of Fe(II)-containing minerals that were able to reduce and precipitate Tc(VII). These results suggest that Fe(III)-reducing bacteria may play an important role in immobilizing technetium in sediments via direct and indirect mechanisms.

Does the structure of the water-oxidizing photosystem II-manganese complex at room temperature differ from its low-temperature structure? A comparative X-ray absorption study.

Detailed information on room-temperature structure and oxidation state of the Photosystem II (PS II) manganese complex is needed to put mechanistic considerations on solid grounds. Because previously this information had not been available, the tetranuclear manganese complex was investigated by X-ray absorption spectroscopy (XAS) on PS II membrane particles at 290 K. Due to methodical progress (collection of XAS spectra within 10 s or less), significant X-ray radiation damage can be avoided; room-temperature XAS investigations on the PS II in its native membrane environment become feasible. Thus, the ambiguity with respect to the mechanistic relevance of low-temperature XAS results is avoidable. At 290 K as well as at 18 K, the manganese complex in its dark-stable state (S(1)-state) seemingly is a Mn(III)(2)Mn(IV)(2) complex comprising two di-mu(2)-oxo bridged binuclear manganese units characterized by the same Mn-Mn distance of 2.71-2.72 A at both temperatures. Most likely, manganese oxidation states and the protonation state of the bridging oxides are fully temperature independent. Remarkably, at room-temperature manganese-ligand distances of 3.10 and 3.65 A are clearly discernible in the EXAFS spectra. The type of bridging assumed to result in Mn-Mn or Mn-Ca distances around 3.1 A is, possibly, temperature-dependent as suggested by distance lengthening upon cooling by 0.13 A. However, mechanistic proposals on photosynthetic water oxidation, which involve the dimer-of-dimers model [Yachandra, V. K., et al. (1993) Science 260, 675-679] are not invalidated by the presented results.

X-ray Absorption Spectroscopic Studies of the Copper(I) Complexes of Crown Ether Appended Bis{(2-pyridyl)ethyl}amines and Their Dioxygen Adducts.

An X-ray absorption spectroscopic study of the Cu complexes of the bis{(2-pyridyl)ethyl}-appended monoaza crown ether 1, diaza crown ether 2, and diphenylglycoluril diaza basket 3 is reported. Following detailed analysis of the spectra of the crystallographically characterized model compound tetrapyridyl Cu(II) bis(nitrato pyridine) (4), the contributions of the ring atoms of the coordinating pyridine to the EXAFS were simulated using a multiple-scattering approach and the final parameters obtained by restrained refinement. Oxygenation of the Cu(I) complexes resulted in a large increase of the intensity of the major peak in the phase-corrected Fourier transform. This was interpreted as evidence for a &mgr;-eta(2):eta(2)-peroxo coordination mode of the oxygen between the copper ions, which had changed valence from Cu(I) to Cu(II) as judged from the edge position. This oxygen binding mode is reminiscent of that of hemocyanin, but the Cu-Cu distances are significantly shorter in the model than in the enzyme and vary with solvent. The EXAFS of the oxygenated complexes was simulated in a new approach in which, besides the parameters of the pyridine unit, those of an additional multiple scattering unit describing the geometry of the Cu-O(2)-Cu moiety were also refined.