Stefan Vogt
Research interests
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Interestsminimum detection limits, X-ray microanalysis, X-Ray Fluorescence
Publications
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4.31Impact points
Submicron hard X-ray fluorescence imaging of synthetic elements.
Analytica chimica acta. 04/2012; 722:21-8.
Synchrotron-based X-ray fluorescence microscopy (XFM) using hard X-rays focused into sub-micron spots is a powerful technique for elemental quantification and mapping, as well as microspectroscopic measurements such as μ-XANES (X-ray absorption near edge structure). We have used XFM to image and sim... [more] Synchrotron-based X-ray fluorescence microscopy (XFM) using hard X-rays focused into sub-micron spots is a powerful technique for elemental quantification and mapping, as well as microspectroscopic measurements such as μ-XANES (X-ray absorption near edge structure). We have used XFM to image and simultaneously quantify the transuranic element plutonium at the L(3) or L(2)-edge as well as Th and lighter biologically essential elements in individual rat pheochromocytoma (PC12) cells after exposure to the long-lived plutonium isotope (242)Pu. Elemental maps demonstrate that plutonium localizes principally in the cytoplasm of the cells and avoids the cell nucleus, which is marked by the highest concentrations of phosphorus and zinc, under the conditions of our experiments. The minimum detection limit under typical acquisition conditions with an incident X-ray energy of 18keV for an average 202μm(2) cell is 1.4fg Pu or 2.9×10(-20)molesPuμm(-2), which is similar to the detection limit of K-edge XFM of transition metals at 10keV. Copper electron microscopy grids were used to avoid interference from gold X-ray emissions, but traces of strontium present in naturally occurring calcium can still interfere with plutonium detection using its L(α) X-ray emission.
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3.42Impact points
Synchrotron radiation induced X-ray emission studies of the antioxidant mechanism of the organoselenium drug ebselen.
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry. 02/2012;
Synchrotron radiation induced X-ray emission (SRIXE) spectroscopy was used to map the cellular uptake of the organoselenium-based antioxidant drug ebselen using differentiated ND15 cells as a neuronal model. The cellular SRIXE spectra, acquired using a hard X-ray microprobe beam (12.8-keV), showed a... [more] Synchrotron radiation induced X-ray emission (SRIXE) spectroscopy was used to map the cellular uptake of the organoselenium-based antioxidant drug ebselen using differentiated ND15 cells as a neuronal model. The cellular SRIXE spectra, acquired using a hard X-ray microprobe beam (12.8-keV), showed a large enhancement of fluorescence at the K(α) line for Se (11.2-keV) following treatment with ebselen (10 μM) at time periods from 60 to 240 min. Drug uptake was quantified and ebselen was shown to induce time-dependent changes in cellular elemental content that were characteristic of oxidative stress with the efflux of K, Cl, and Ca species. The SRIXE cellular Se distribution map revealed that ebselen was predominantly localized to a discreet region of the cell which, by comparison with the K and P elemental maps, is postulated to correspond to the endoplasmic reticulum. On the basis of these findings, it is hypothesized that a major outcome of ebselen redox catalysis is the induction of cellular stress. A mechanism of action of ebselen is proposed that involves the cell responding to drug-induced stress by increasing the expression of antioxidant genes. This hypothesis is supported by the observation that ebselen also regulated the homeostasis of the transition metals Mn, Cu, Fe, and Zn, with increases in transition metal uptake paralleling known induction times for the expression of antioxidant metalloenzymes.
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The unique biogeochemical signature of the marine diazotroph trichodesmium.
Frontiers in microbiology. 01/2012; 3:150.
The elemental composition of phytoplankton can depart from canonical Redfield values under conditions of nutrient limitation or production (e.g., N fixation). Similarly, the trace metal metallome of phytoplankton may be expected to vary as a function of both ambient nutrient concentrations and the b... [more] The elemental composition of phytoplankton can depart from canonical Redfield values under conditions of nutrient limitation or production (e.g., N fixation). Similarly, the trace metal metallome of phytoplankton may be expected to vary as a function of both ambient nutrient concentrations and the biochemical processes of the cell. Diazotrophs such as the colonial cyanobacteria Trichodesmium are likely to have unique metal signatures due to their cell physiology. We present metal (Fe, V, Zn, Ni, Mo, Mn, Cu, Cd) quotas for Trichodesmium collected from the Sargasso Sea which highlight the unique metallome of this organism. The element concentrations of bulk colonies and trichomes sections were analyzed by ICP-MS and synchrotron x-ray fluorescence, respectively. The cells were characterized by low P contents but enrichment in V, Fe, Mo, Ni, and Zn in comparison to other phytoplankton. Vanadium was the most abundant metal in Trichodesmium, and the V quota was up to fourfold higher than the corresponding Fe quota. The stoichiometry of 600C:101N:1P (mol mol(-1)) reflects P-limiting conditions. Iron and V were enriched in contiguous cells of 10 and 50% of Trichodesmium trichomes, respectively. The distribution of Ni differed from other elements, with the highest concentration in the transverse walls between attached cells. We hypothesize that the enrichments of V, Fe, Mo, and Ni are linked to the biochemical requirements for N fixation either directly through enrichment in the N-fixing enzyme nitrogenase or indirectly by the expression of enzymes responsible for the removal of reactive oxygen species. Unintentional uptake of V via P pathways may also be occurring. Overall, the cellular content of trace metals and macronutrients differs significantly from the (extended) Redfield ratio. The Trichodesmium metallome is an example of how physiology and environmental conditions can cause significant deviations from the idealized stoichiometry.
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7.54Impact points
Nanocarriers enhance Doxorubicin uptake in drug-resistant ovarian cancer cells.
Cancer research. 12/2011; 72(3):769-78.
Resistance to anthracyclines and other chemotherapeutics due to P-glycoprotein (pgp)-mediated export is a frequent problem in cancer treatment. Here, we report that iron oxide-titanium dioxide core-shell nanocomposites can serve as efficient carriers for doxorubicin to overcome this common mechanism... [more] Resistance to anthracyclines and other chemotherapeutics due to P-glycoprotein (pgp)-mediated export is a frequent problem in cancer treatment. Here, we report that iron oxide-titanium dioxide core-shell nanocomposites can serve as efficient carriers for doxorubicin to overcome this common mechanism of drug resistance in cancer cells. Doxorubicin nanocarriers (DNC) increased effective drug uptake in drug-resistant ovarian cells. Mechanistically, doxorubicin bound to the TiO(2) surface by a labile bond that was severed upon acidification within cell endosomes. Upon its release, doxorubicin traversed the intracellular milieu and entered the cell nucleus by a route that evaded pgp-mediated drug export. Confocal and X-ray fluorescence microscopy and flow cytometry were used to show the ability of DNCs to modulate transferrin uptake and distribution in cells. Increased transferrin uptake occurred through clathrin-mediated endocytosis, indicating that nanocomposites and DNCs may both interfere with removal of transferrin from cells. Together, our findings show that DNCs not only provide an alternative route of delivery of doxorubicin to pgp-overexpressing cancer cells but also may boost the uptake of transferrin-tagged therapeutic agents.
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3.39Impact points
β-Cell subcellular localization of glucose-stimulated Mn uptake by X-ray fluorescence microscopy: implications for pancreatic MRI.
Contrast media & molecular imaging. 11/2011; 6(6):474-81.
Manganese (Mn) is a calcium (Ca) analog that has long been used as a magnetic resonance imaging (MRI) contrast agent for investigating cardiac tissue functionality, for brain mapping and for neuronal tract tracing studies. Recently, we have extended its use to investigate pancreatic β-cells and show... [more] Manganese (Mn) is a calcium (Ca) analog that has long been used as a magnetic resonance imaging (MRI) contrast agent for investigating cardiac tissue functionality, for brain mapping and for neuronal tract tracing studies. Recently, we have extended its use to investigate pancreatic β-cells and showed that, in the presence of MnCl(2), glucose-activated pancreatic islets yield significant signal enhancement in T(1)-weigheted MR images. In this study, we exploited for the first time the unique capabilities of X-ray fluorescence microscopy (XFM) to both visualize and quantify the metal in pancreatic β-cells at cellular and subcellular levels. MIN-6 insulinoma cells grown in standard tissue culture conditions had only a trace amount of Mn, 1.14 ± 0.03 × 10(-11)µg/µm(2), homogenously distributed across the cell. Exposure to 2 mM glucose and 50 µM MnCl(2) for 20 min resulted in nonglucose-dependent Mn uptake and the overall cell concentration increased to 8.99 ± 2.69 × 10(-11) µg/µm(2). When cells were activated by incubation in 16 mM glucose in the presence of 50 µM MnCl(2), a significant increase in cytoplasmic Mn was measured, reaching 2.57 ± 1.34 × 10(-10) µg/µm(2). A further rise in intracellular concentration was measured following KCl-induced depolarization, with concentrations totaling 1.25 ± 0.33 × 10(-9) and 4.02 ± 0.71 × 10(-10) µg/µm(2) in the cytoplasm and nuclei, respectively. In both activated conditions Mn was prevalent in the cytoplasm and localized primarily in a perinuclear region, possibly corresponding to the Golgi apparatus and involving the secretory pathway. These data are consistent with our previous MRI findings, confirming that Mn can be used as a functional imaging reporter of pancreatic β-cell activation and also provide a basis for understanding how subcellular localization of Mn will impact MRI contrast.
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8.58Impact points
Metabolism of selenite in human lung cancer cells: X-ray absorption and fluorescence studies.
Journal of the American Chemical Society. 09/2011; 133(45):18272-9.
Selenite is an inorganic form of selenium that has a cytotoxic effect against several human cancer cell lines: one or more selenite metabolites are considered to be responsible for its toxicity. X-ray absorption spectroscopy was used to monitor Se speciation in A549 human lung cancer cells incubated... [more] Selenite is an inorganic form of selenium that has a cytotoxic effect against several human cancer cell lines: one or more selenite metabolites are considered to be responsible for its toxicity. X-ray absorption spectroscopy was used to monitor Se speciation in A549 human lung cancer cells incubated with selenite over 72 h. As anticipated, selenodiglutathione and elemental Se both comprised a large proportion of Se in the cells between 4 and 72 h after treatment, which is in accordance with the reductive metabolism of selenite in the presence of glutathione and glutathione reductase/NADPH system. Selenocystine was also present in the cells but was only detected as a significant component between 24 and 48 h concomitant with a decrease in the proportion of selenocysteine and the viability of the cells. The change in speciation from the selenol, selenocysteine, to the diselenide, selenocystine, is indicative of a change in the redox status of the cells to a more oxidizing environment, likely brought about by metabolites of selenite. X-ray fluorescence microscopy of single cells treated with selenite for 24 h revealed a punctate distribution of Se in the cytoplasm. The accumulation of Se was associated with a greater than 2-fold increase in Cu, which was colocalized with Se. Selenium K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy revealed Se-Se and Se-S bonding, but not Se-Cu bonding, despite the spatial association of Se and Cu. Microprobe X-ray absorption near-edge structure spectroscopy (μ-XANES) showed that the highly localized Se species was mostly elemental Se.
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8.20Impact points
High-resolution imaging of selenium in kidneys: a localized selenium pool associated with glutathione peroxidase 3.
Antioxidants & redox signaling. 08/2011; 16(3):185-92.
Recent advances in quantitative methods and sensitive imaging techniques of trace elements provide opportunities to uncover and explain their biological roles. In particular, the distribution of selenium in tissues and cells under both physiological and pathological conditions remains unknown. In th... [more] Recent advances in quantitative methods and sensitive imaging techniques of trace elements provide opportunities to uncover and explain their biological roles. In particular, the distribution of selenium in tissues and cells under both physiological and pathological conditions remains unknown. In this work, we applied high-resolution synchrotron X-ray fluorescence microscopy (XFM) to map selenium distribution in mouse liver and kidney. Liver showed a uniform selenium distribution that was dependent on selenocysteine tRNA([Ser]Sec) and dietary selenium. In contrast, kidney selenium had both uniformly distributed and highly localized components, the latter visualized as thin circular structures surrounding proximal tubules. Other parts of the kidney, such as glomeruli and distal tubules, only manifested the uniformly distributed selenium pattern that co-localized with sulfur. We found that proximal tubule selenium localized to the basement membrane. It was preserved in Selenoprotein P knockout mice, but was completely eliminated in glutathione peroxidase 3 (GPx3) knockout mice, indicating that this selenium represented GPx3. We further imaged kidneys of another model organism, the naked mole rat, which showed a diminished uniformly distributed selenium pool, but preserved the circular proximal tubule signal. We applied XFM to image selenium in mammalian tissues and identified a highly localized pool of this trace element at the basement membrane of kidneys that was associated with GPx3. XFM allowed us to define and explain the tissue topography of selenium in mammalian kidneys at submicron resolution.
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3.67Impact points
Selectivity in biomineralization of barium and strontium.
Journal of structural biology. 08/2011; 176(2):192-202.
The desmid green alga Closterium moniliferum belongs to a small number of organisms that form barite (BaSO(4)) or celestite (SrSO(4)) biominerals. The ability to sequester Sr in the presence of an excess of Ca is of considerable interest for the remediation of (90)Sr from the environment and nuclear... [more] The desmid green alga Closterium moniliferum belongs to a small number of organisms that form barite (BaSO(4)) or celestite (SrSO(4)) biominerals. The ability to sequester Sr in the presence of an excess of Ca is of considerable interest for the remediation of (90)Sr from the environment and nuclear waste. While most cells dynamically regulate the concentration of the second messenger Ca(2+) in the cytosol and various organelles, transport proteins rarely discriminate strongly between Ca, Sr, and Ba. Herein, we investigate how these ions are trafficked in C. moniliferum and how precipitation of (Ba,Sr)SO(4) crystals occurs in the terminal vacuoles. Towards this goal, we simultaneously visualize intracellular dynamics of multiple elements using X-ray fluorescence microscopy (XFM) of cryo-fixed/freeze-dried samples. We correlate the resulting elemental maps with ultrastructural information gleaned from freeze-fracture cryo-SEM of frozen-hydrated cells and use micro X-ray absorption near edge structure (micro-XANES) to determine sulfur speciation. We find that the kinetics of Sr uptake and efflux depend on external Ca concentrations, and Sr, Ba, and Ca show similar intracellular localization. A highly ion-selective cross-membrane transport step is not evident. Based on elevated levels of sulfate detected in the terminal vacuoles, we propose a "sulfate trap" model, where the presence of dissolved barium leads to preferential precipitation of (Ba,Sr)SO(4) due to its low solubility relative to SrSO(4) and CaSO(4). Engineering the sulfate concentration in the vacuole may thus be the most direct way to increase the Sr sequestered per cell, an important consideration in using desmids for phytoremediation of (90)Sr.
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4.66Impact points
Direct determination of the intracellular oxidation state of plutonium.
Inorganic chemistry. 08/2011; 50(16):7591-7.
Microprobe X-ray absorption near edge structure (μ-XANES) measurements were used to determine directly, for the first time, the oxidation state of intracellular plutonium in individual 0.1-μm(2) areas within single rat pheochromocytoma cells (PC12). The living cells were incubated in vitro for 3 h i... [more] Microprobe X-ray absorption near edge structure (μ-XANES) measurements were used to determine directly, for the first time, the oxidation state of intracellular plutonium in individual 0.1-μm(2) areas within single rat pheochromocytoma cells (PC12). The living cells were incubated in vitro for 3 h in the presence of Pu added to the media in different oxidation states (Pu(III), Pu(IV), and Pu(VI)) and in different chemical forms. Regardless of the initial oxidation state or chemical form of Pu presented to the cells, the XANES spectra of the intracellular Pu deposits were always consistent with tetravalent Pu even though the intracellular milieu is generally reducing.
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1.84Impact points
Plutonium uptake and distribution in mammalian cells: molecular vs. polymeric plutonium.
International journal of radiation biology. 07/2011; 87(10):1023-32.
To study the cellular responses to molecular and polymeric forms of plutonium using PC12 cells derived from a rat pheochromocytoma. Serum starved PC12 cells were exposed to polymeric and molecular forms of plutonium for 3 h. Cells were washed with 10 mM ethylene glycol tetraacetic acid (EGTA), 100 m... [more] To study the cellular responses to molecular and polymeric forms of plutonium using PC12 cells derived from a rat pheochromocytoma. Serum starved PC12 cells were exposed to polymeric and molecular forms of plutonium for 3 h. Cells were washed with 10 mM ethylene glycol tetraacetic acid (EGTA), 100 mM NaCl at pH 7.4 to remove surface sorbed plutonium. Localization of plutonium in individual cell was quantitatively analyzed by synchrotron X-ray fluorescence (XRF) microscopy. Molecular plutonium complexes introduced to cell growth media in the form of nitrilotriacetic acid (NTA), citrate, or transferrin complexes were taken up by PC12 cells, and mostly colocalized with iron within the cells. Aged polymeric plutonium prepared separately was not internalized by PC12 cells but it was always found on the cell surface as big agglomerates; however, polymeric plutonium formed in situ was mostly found within the cells as agglomerates. PC12 cells can differentiate molecular and polymeric forms of plutonium. Molecular plutonium is taken up by PC12 cells and mostly co-localizes with iron but aged polymeric plutonium is not internalized by the cells.
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16.06Impact points
An iron-dependent and transferrin-mediated cellular uptake pathway for plutonium.
Nature chemical biology. 06/2011; 7(8):560-5.
Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small-angle X-ray scattering, receptor binding assays and synchrotron X-ray fluorescence microscopy, we find that rat adrenal gland (PC12) cells can acquire plutoniu... [more] Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small-angle X-ray scattering, receptor binding assays and synchrotron X-ray fluorescence microscopy, we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway--receptor-mediated endocytosis of the iron transport protein serum transferrin; however, only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small-angle scattering show that only transferrin with plutonium bound in the protein's C-terminal lobe (C-lobe) and iron bound in the N-terminal lobe (N-lobe) (Pu(C)Fe(N)Tf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal-binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin's two lobes act to restrict, but not eliminate, cellular Pu uptake.
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0.92Impact points
Past and future work on radiobiology mega-studies: a case study at Argonne National Laboratory.
Health physics. 06/2011; 100(6):613-21.
Between 1952 and 1992, more than 200 large radiobiology studies were conducted in research institutes throughout Europe, North America, and Japan to determine the effects of external irradiation and internal emitters on the lifespan and tissue toxicity development in animals. At Argonne National Lab... [more] Between 1952 and 1992, more than 200 large radiobiology studies were conducted in research institutes throughout Europe, North America, and Japan to determine the effects of external irradiation and internal emitters on the lifespan and tissue toxicity development in animals. At Argonne National Laboratory, 22 external beam studies were conducted on nearly 700 beagle dogs and 50,000 mice between 1969 and 1992. These studies helped to characterize the effects of neutron and gamma irradiation on lifespan, tumorigenesis, and mutagenesis across a range of doses and dosing patterns. The records and tissues collected at Argonne during that time period have been carefully preserved and redisseminated. Using these archived data, ongoing statistical work has been done and continues to characterize quality of radiation, dose, dose rate, tissue, and gender-specific differences in the radiation responses of exposed animals. The ongoing application of newly-developed molecular biology techniques to the archived tissues has revealed gene-specific mutation rates following exposure to ionizing irradiation. The original and ongoing work with this tissue archive is presented here as a case study of a more general trend in the radiobiology megastudies. These experiments helped form the modern understanding of radiation responses in animals and continue to inform development of new radiation models. Recent archival efforts have facilitated open access to the data and materials produced by these studies, and so a unique opportunity exists to expand this continued research.
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3.84Impact points
Quantitative comparison of preparation methodologies for X-ray fluorescence microscopy of brain tissue.
Analytical and bioanalytical chemistry. 04/2011; 401(3):853-64.
X-ray fluorescence microscopy (XFM) facilitates high-sensitivity quantitative imaging of trace metals at high spatial resolution over large sample areas and can be applied to a diverse range of biological samples. Accurate determination of elemental content from recorded spectra requires proper cali... [more] X-ray fluorescence microscopy (XFM) facilitates high-sensitivity quantitative imaging of trace metals at high spatial resolution over large sample areas and can be applied to a diverse range of biological samples. Accurate determination of elemental content from recorded spectra requires proper calibration of the XFM instrument under the relevant operating conditions. Here, we describe the manufacture, characterization, and utilization of multi-element thin-film reference foils for use in calibration of XFM measurements of biological and other specimens. We have used these internal standards to assess the two-dimensional distribution of trace metals in a thin tissue section of a rat hippocampus. The data used in this study was acquired at the XFM beamline of the Australian Synchrotron using a new 384-element array detector (Maia) and at beamline 2-ID-E at the Advanced Photon Source. Post-processing of samples by different fixation techniques was investigated, with the conclusion that differences in solvent type and sample handling can significantly alter elemental content. The present study highlights the quantitative capability, high statistical power, and versatility of the XFM technique for mapping trace metals in biological samples, e.g., brain tissue samples in order to help understand neurological processes, especially when implemented in conjunction with a high-performance detector such as Maia.
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4.77Impact points
Selective sequestration of strontium in desmid green algae by biogenic co-precipitation with barite.
ChemSusChem. 04/2011; 4(4):470-3.
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5.74Impact points
Increased brain iron coincides with early plaque formation in a mouse model of Alzheimer's disease.
NeuroImage. 03/2011; 55(1):32-8.
Elevated brain iron content, which has been observed in late-stage human Alzheimer's disease, is a potential target for early diagnosis. However, the time course for iron accumulation is currently unclear. Using the PSAPP mouse model of amyloid plaque formation, we conducted a time course study ... [more] Elevated brain iron content, which has been observed in late-stage human Alzheimer's disease, is a potential target for early diagnosis. However, the time course for iron accumulation is currently unclear. Using the PSAPP mouse model of amyloid plaque formation, we conducted a time course study of metal ion content and distribution [iron (Fe), copper (Cu), and zinc (Zn)] in the cortex and hippocampus using X-ray fluorescence microscopy (XFM). We found that iron in the cortex was 34% higher than age-matched controls at an early stage, corresponding to the commencement of plaque formation. The elevated iron was not associated with the amyloid plaques. Interestingly, none of the metal ions were elevated in the amyloid plaques until the latest time point (56 weeks), where only the Zn content was significantly elevated by 38%. Since neuropathological changes in human Alzheimer's disease are presumed to occur years before the first cognitive symptoms appear, quantification of brain iron content could be a powerful marker for early diagnosis of Alzheimer's disease.
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5.33Impact points
Reduced utilization of selenium by naked mole rats due to a specific defect in GPx1 expression.
The Journal of biological chemistry. 03/2011; 286(19):17005-14.
Naked mole rat (MR) Heterocephalus glaber is a rodent model of delayed aging because of its unusually long life span (>28 years). It is also not known to develop cancer. In the current work, tissue imaging by x-ray fluorescence microscopy and direct analyses of trace elements revealed low levels ... [more] Naked mole rat (MR) Heterocephalus glaber is a rodent model of delayed aging because of its unusually long life span (>28 years). It is also not known to develop cancer. In the current work, tissue imaging by x-ray fluorescence microscopy and direct analyses of trace elements revealed low levels of selenium in the MR liver and kidney, whereas MR and mouse brains had similar selenium levels. This effect was not explained by uniform selenium deficiency because methionine sulfoxide reductase activities were similar in mice and MR. However, glutathione peroxidase activity was an order of magnitude lower in MR liver and kidney than in mouse tissues. In addition, metabolic labeling of MR cells with (75)Se revealed a loss of the abundant glutathione peroxidase 1 (GPx1) band, whereas other selenoproteins were preserved. To characterize the MR selenoproteome, we sequenced its liver transcriptome. Gene reconstruction revealed standard selenoprotein sequences except for GPx1, which had an early stop codon, and SelP, which had low selenocysteine content. When expressed in HEK 293 cells, MR GPx1 was present in low levels, and its expression could be rescued neither by removing the early stop codon nor by replacing its SECIS element. In addition, GPx1 mRNA was present in lower levels in MR liver than in mouse liver. To determine if GPx1 deficiency could account for the reduced selenium content, we analyzed GPx1 knock-out mice and found reduced selenium levels in their livers and kidneys. Thus, MR is characterized by the reduced utilization of selenium due to a specific defect in GPx1 expression.
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9.43Impact points
Radiation damage in protein crystals is reduced with a micron-sized X-ray beam.
Proceedings of the National Academy of Sciences of the United States of America. 03/2011; 108(15):6127-32.
Radiation damage is a major limitation in crystallography of biological macromolecules, even for cryocooled samples, and is particularly acute in microdiffraction. For the X-ray energies most commonly used for protein crystallography at synchrotron sources, photoelectrons are the predominant source ... [more] Radiation damage is a major limitation in crystallography of biological macromolecules, even for cryocooled samples, and is particularly acute in microdiffraction. For the X-ray energies most commonly used for protein crystallography at synchrotron sources, photoelectrons are the predominant source of radiation damage. If the beam size is small relative to the photoelectron path length, then the photoelectron may escape the beam footprint, resulting in less damage in the illuminated volume. Thus, it may be possible to exploit this phenomenon to reduce radiation-induced damage during data measurement for techniques such as diffraction, spectroscopy, and imaging that use X-rays to probe both crystalline and noncrystalline biological samples. In a systematic and direct experimental demonstration of reduced radiation damage in protein crystals with small beams, damage was measured as a function of micron-sized X-ray beams of decreasing dimensions. The damage rate normalized for dose was reduced by a factor of three from the largest (15.6 μm) to the smallest (0.84 μm) X-ray beam used. Radiation-induced damage to protein crystals was also mapped parallel and perpendicular to the polarization direction of an incident 1-μm X-ray beam. Damage was greatest at the beam center and decreased monotonically to zero at a distance of about 4 μm, establishing the range of photoelectrons. The observed damage is less anisotropic than photoelectron emission probability, consistent with photoelectron trajectory simulations. These experimental results provide the basis for data collection protocols to mitigate with micron-sized X-ray beams the effects of radiation damage.
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9.43Impact points
Calcium-dependent copper redistributions in neuronal cells revealed by a fluorescent copper sensor and X-ray fluorescence microscopy.
Proceedings of the National Academy of Sciences of the United States of America. 03/2011; 108(15):5980-5.
Dynamic fluxes of s-block metals like potassium, sodium, and calcium are of broad importance in cell signaling. In contrast, the concept of mobile transition metals triggered by cell activation remains insufficiently explored, in large part because metals like copper and iron are typically studied a... [more] Dynamic fluxes of s-block metals like potassium, sodium, and calcium are of broad importance in cell signaling. In contrast, the concept of mobile transition metals triggered by cell activation remains insufficiently explored, in large part because metals like copper and iron are typically studied as static cellular nutrients and there are a lack of direct, selective methods for monitoring their distributions in living cells. To help meet this need, we now report Coppersensor-3 (CS3), a bright small-molecule fluorescent probe that offers the unique capability to image labile copper pools in living cells at endogenous, basal levels. We use this chemical tool in conjunction with synchotron-based microprobe X-ray fluorescence microscopy (XRFM) to discover that neuronal cells move significant pools of copper from their cell bodies to peripheral processes upon their activation. Moreover, further CS3 and XRFM imaging experiments show that these dynamic copper redistributions are dependent on calcium release, establishing a link between mobile copper and major cell signaling pathways. By providing a small-molecule fluorophore that is selective and sensitive enough to image labile copper pools in living cells under basal conditions, CS3 opens opportunities for discovering and elucidating functions of copper in living systems.
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3.23Impact points
Uptake, distribution, and speciation of selenoamino acids by human cancer cells: X-ray absorption and fluorescence methods.
Biochemistry. 02/2011; 50(10):1641-50.
Selenium compounds exhibit chemopreventative properties at supranutritional doses, but the efficacy of selenium supplementation in cancer prevention is dependent on the chemical speciation of the selenium supplement and its metabolites. The uptake, speciation, and distribution of the common selenoam... [more] Selenium compounds exhibit chemopreventative properties at supranutritional doses, but the efficacy of selenium supplementation in cancer prevention is dependent on the chemical speciation of the selenium supplement and its metabolites. The uptake, speciation, and distribution of the common selenoamino acid supplements, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys), in A549 human lung cancer cells were investigated using X-ray absorption and fluorescence spectroscopies. X-ray absorption spectroscopy of bulk cell pellets treated with the selenoamino acids for 24 h showed that while selenium was found exclusively in carbon-bound forms in SeMet-treated cells, a diselenide component was identified in MeSeCys-treated cells in addition to the carbon-bound selenium species. X-ray fluorescence microscopy of single cells showed that selenium accumulated with sulfur in the perinuclear region of SeMet-treated cells after 24 h, but microprobe selenium X-ray absorption near-edge spectroscopy in this region indicated that selenium was carbon-bound rather than sulfur-bound. X-ray absorption and X-ray fluorescence studies both showed that the selenium content of MeSeCys-treated cells was much lower than that of SeMet-treated cells. Selenium was distributed homogeneously throughout the MeSeCys-treated cells.
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4.41Impact points
Elemental profiling of single bacterial cells as a function of copper exposure and growth phase.
PloS one. 01/2011; 6(6):e21255.
The elemental composition of single cells of Nitrosomonas europaea 19718 was studied via synchrotron X-ray fluorescence microscopy (XFM) as a function of inhibition by divalent copper (Cu(II)) and batch growth phase. Based on XFM, the intracellular Cu concentrations in exponential phase cultures of ... [more] The elemental composition of single cells of Nitrosomonas europaea 19718 was studied via synchrotron X-ray fluorescence microscopy (XFM) as a function of inhibition by divalent copper (Cu(II)) and batch growth phase. Based on XFM, the intracellular Cu concentrations in exponential phase cultures of N. europaea exposed to Cu(II) were statistically higher than in stationary phase cultures at the 95% confidence interval (α = 0.05). However, the impact of Cu inferred from specific oxygen uptake rate (sOUR) measurements at the two physiological states was statistically not dissimilar at the Cu(II) doses tested, except at 1000 µM Cu(II), at which exponential phase cultures were significantly more inhibited. Furthermore, the elemental composition in uninhibited exponential and stationary phase N. europaea cultures was similar. Notably, the molar fractions of Cu and Fe, relative to other elements in N. europaea cultures were statistically higher than those recently reported in Pseudomonas fluorescens possibly owing to the preponderance of metal cofactor rich catalytic enzymes (such as ammonia monooxygenase) and electron transport mechanisms in N. europaea.
Following (2)
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Ian Mcnulty
Argonne National Laboratory -
Jeanette Maier
Università degli studi di Milano -
Andrea Notargiacomo
CNR - Consiglio Nazionale delle Ricerche
Topics (1)
