High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry

Background: Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. Results: The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as ^{12}C^{15}N^{-} and ^{13}C^{14}N^{-}, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of ^{14}C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using ^{13}C-oleic acid; to examine nitrogen fixation in bacteria using ^{15}N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using ^{15}N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using ^{15}N-uridine and ^{81}Br of bromodeoxyuridine or ^{14}C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes ^{12}C, ^{16}O, ^{14}N and ^{31}P; and to track a few ^{15}N-labeled donor spleen cells in the lymph nodes of the host mouse. Conclusion: MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments

Correlated nanoscale characterization of a unique complex oxygen-rich stardust grain:Implications for circumstellar dust formation

We report the light to intermediate-mass element abundances as well as the oxygen, magnesium, silicon, and titanium isotope compositions of a unique and unusually large (0.8 µm × 3.75 µm) presolar O-rich grain from the Krymka LL3.2 chondrite. The O-, Al-, and Ti-isotopic compositions are largely compatible with an origin from an asymptotic giant branch (AGB) star of 1.5 solar masses with a metallicity that is 15% higher than the solar metallicity. The grain has an elevated 17O/16O ratio (8.40 ± 0.16 × 10−4) compared to solar, and slightly sub-solar 18O/16O ratio (1.83 ± 0.03 × 10−3). It shows evidence for the presence of initial 26Al, suggesting formation after the first dredge-up, during one of the early third dredge-up (TDU) episodes. Titanium isotopic data indicate condensation of the grain before significant amounts of material from the He-burning shell were admixed to the stellar surface with progressive TDUs. We observed a small excess in 30Si (δ30Si = 41 ± 5‰), which most likely is inherited from the parent star’s initial Si-isotopic composition. For such stars stellar models predict a C/O-ratio < 1 even after the onset of TDU, thus allowing the condensation of O-rich dust. The grain is an unusual complex presolar grain, consisting of an Al-Ca-Ti-oxide core, surrounded by an Mg-Ca-silicate mantle, and resembles the condensation sequence for a cooling gas of solar composition at pressures and dust/gas ratios typically observed for circumstellar envelopes around evolved stars. We also report the first observation of phosphorus in a presolar grain, although the origin of the P-bearing phase remains ambiguous

NUMERICAL UNSTEADY FLOW ANALYSIS OF A TURBINE STAGE WITH EXTREMELY LARGE BLADE LOADS

ABSTRACT This paper presents the detailed numerical analysis including parametric studies on the aerodynamic excitation mechanisms in a turbine stage due to the unsteady stator-rotor interaction. The work is part of the pre-design study of a high pressure subsonic turbine for a rocket engine turbopump. The pressure level in such turbines can be remarkably high (in this case 54 MPa inlet total pressure). Hence, large unsteady rotor blade loads can be expected, which impose difficult design requirements. The parameter studies are performed at midspan with the numerical flow solver UNSFLO, a 2D/Q3D unsteady hybrid Euler/Navier-Stokes solver. Comparisons to 2D and steady 3D results obtained with a fully viscous solver, VOLSOL, are made. The investigated design parameters are the axial gap (~8%-29% of rotor axial chord length) and the stator vane size and count (stator-rotor pitch ratio ~1-2.75). For the nominal case the numerical solution is analyzed regarding the contributions of potential and vortical flow disturbances at the rotor inlet using rotor gust computations. It was found that gust calculations were not capable to capture the complexity of the detected excitation mechanisms, but the possibility to reduce excitations by enforcing cancellation of the vortical and potential effects has been elaborated. The potential excitation mechanism in the present turbine stage is found dominant compared to relatively small and local wake excitation effects. The parameter studies indicate design recommendations for the axial gap and the stator size regarding the unsteady rotor load

Improving magneto-inertial attitude and position estimation by means of magnetic heading observer

International audienceThis paper studies heading estimation jointly with the attitude and position estimation of a rigid body equipped with inertial and magnetic sensors in indoor environment. In contrast with other indoor dead-reckoning approaches, no assumption is made about the nature of the movement or environment layout. Based on a previous paper, an Extended Kalman Filter is designed, which includes inertial sensor biases and magnetic disturbances. A heuristic model of the dynamic of magnetic heading disturbances is then described and added to the observer. The latter is then evaluated in terms of position and heading error on experimental data, showing that in spite of high levels of disturbances, the magnetic field alone can be used to compute heading

A single-cell view on the ecophysiology of anaerobic phototrophic bacteria

Quantitative information on the ecophysiology of individual microorganisms is generally limited because it is difficult to assign specific metabolic activities to identified single cells. Here, we develop and apply a method, Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy (HISH-SIMS), and show that it allows simultaneous phylogenetic identification and quantitation of metabolic activities of single microbial cells in the environment. Using HISH-SIMS, individual cells of the anaerobic, phototropic bacteria Chromatium okenii, Lamprocystis purpurea, and Chlorobium clathratiforme inhabiting the oligotrophic, meromictic Lake Cadagno were analyzed with respect to H13CO3− and 15NH4+ assimilation. Metabolic rates were found to vary greatly between individual cells of the same species, showing that microbial populations in the environment are heterogeneous, being comprised of physiologically distinct individuals. Furthermore, C. okenii, the least abundant species representing ≈0.3% of the total cell number, contributed more than 40% of the total uptake of ammonium and 70% of the total uptake of carbon in the system, thereby emphasizing that numerically inconspicuous microbes can play a significant role in the nitrogen and carbon cycles in the environment. By introducing this quantification method for the ecophysiological roles of individual cells, our study opens a variety of possibilities of research in environmental microbiology, especially by increasing the ability to examine the ecophysiological roles of individual cells, including those of less abundant and less active microbes, and by the capacity to track not only nitrogen and carbon but also phosphorus, sulfur, and other biological element flows within microbial communities

Templated Biomineralization on Self-Assembled Protein Nanofibers Buried in Calcium Oxalate Raphides of <i>Musa</i> spp.

Biological organisms possess an unparalleled ability to control crystallization of biominerals with convoluted internal structures. For example, an occluded organic matrix can interact with the mineral during its formation to control its morphology and structure. Although related matrix proteins that preferentially nucleate minerals have been identified, the mechanisms elucidating the structural and chemical complexity of calcium oxalate biominerals in plants remain unclear. Here, we show that a protein nanofiber (14 kDa) is embedded inside raphide (needle-shaped calcium oxalate) crystals of banana (<i>Musa</i> spp.), and that nanometer-scaled calcium oxalate spheres are arranged along the long axes of this central proteinaceous filament to form laminated structures through an aggregation-based growth mechanism, resulting in the final product of elongated and tapered hexagonal crystals. We further demonstrate that 11 amino acid peptide segments, with hydrophilic and hydrophobic residues rich in proline derived from the C-terminus of this full protein sequence, in vitro self-assemble into fibers and accelerate calcium oxalate nucleation kinetics. Remarkably, elongated and organized microstructures which are similar in appearance to natural raphide crystals are formed, emphasizing interactions between the mineral and self-assembled protein fibers. We anticipate that the present investigation of the structural and morphological complexity of plant calcium oxalate crystals and the underlying mechanisms of their formation will contribute to our understanding not only how plants evolved these sophisticated structures and morphologies for survival and adaptation, but also ultimately provide useful clues about how to maximally sequester calcium ions and/or oxalate in a confined compartment

The capital structure of French corporations : an empirical investigation

Available at INIST (FR), Document Supply Service, under shelf-number : DO 3278 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry

BACKGROUND: Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. RESULTS: The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as (12)C(15)N(- )and (13)C(14)N(-), it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of (14)C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using (13)C-oleic acid; to examine nitrogen fixation in bacteria using (15)N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using (15)N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using (15)N-uridine and (81)Br of bromodeoxyuridine or (14)C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes (12)C, (16)O, (14)N and (31)P; and to track a few (15)N-labeled donor spleen cells in the lymph nodes of the host mouse. CONCLUSION: MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments

Elemental distribution in cephalopod statoliths: NanoSIMS provides new insights into nano-scale structure

We have applied the novel analytical method NanoSIMS to cephalopod statoliths for the first time in order to analyse their chemical microstructure, using a spatial resolution of 400 nm. This technique makes it possible to analyse in situ nano-scale chemical variations between increment layers. In statoliths of the boreoatlantic armhook squid Gonatus fabricii, we found distinct concentration patterns indicating a periodicity in strontium and sodium distributions. Sr and Na show a negative relation, both elements showing alternating patterns where the increments vary in width between approximately 1 and 5 μm. Results suggest, that aragonite deposited during the night is rich in Na and poor in Sr, while aragonite deposited during the day is rich in Sr and poor in Na. This study demonstrates the excellent suitability of NanoSIMS for nano-scale microchemical analyses of aragonite, providing new information on calcification processes and individual life histories. Possible future fields of application include not only cephalopod statoliths, but also virtually all biomineralized tissues in aquatic organisms like fish otoliths, gastropod statoliths, bivalve shells, foraminifers and corals