Francois Hillion

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.

Distribution of magnesium in coral skeleton

Ion micro-probe imaging of the aragonite skeleton of Pavona clavus, a massive reef-building coral, shows that magnesium and strontium are distributed very differently. In contrast to strontium, the distribution of magnesium is strongly correlated with the fine-scale structure of the skeleton and corresponds to the layered organization of aragonite fibers surrounding the centers of calcification, which have up to ten times higher magnesium concentration. This indicates a strong biological control over the magnesium composition of all structural components within the skeleton. Magnesium may be used by the coral to actively control the growth of the different skeletal crystal components.

Carbon and nitrogen assimilation in deep subseafloor microbial cells.

Remarkable numbers of microbial cells have been observed in global shallow to deep subseafloor sediments. Accumulating evidence indicates that deep and ancient sediments harbor living microbial life, where the flux of nutrients and energy are extremely low. However, their physiology and energy requirements remain largely unknown. We used stable isotope tracer incubation and nanometer-scale secondary ion MS to investigate the dynamics of carbon and nitrogen assimilation activities in individual microbial cells from 219-m-deep lower Pleistocene (460,000 y old) sediments from the northwestern Pacific off the Shimokita Peninsula of Japan. Sediment samples were incubated in vitro with 13C- and/or 15N-labeled glucose, pyruvate, acetate, bicarbonate, methane, ammonium, and amino acids. Significant incorporation of 13C and/or 15N and growth occurred in response to glucose, pyruvate, and amino acids (∼76% of total cells), whereas acetate and bicarbonate were incorporated without fostering growth. Among those substrates, a maximum substrate assimilation rate was observed at 67 × 10−18 mol/cell per d with bicarbonate. Neither carbon assimilation nor growth was evident in response to methane. The atomic ratios between nitrogen incorporated from ammonium and the total cellular nitrogen consistently exceeded the ratios of carbon, suggesting that subseafloor microbes preferentially require nitrogen assimilation for the recovery in vitro. Our results showed that the most deeply buried subseafloor sedimentary microbes maintain potentials for metabolic activities and that growth is generally limited by energy but not by the availability of C and N compounds.

Scanning secondary ion analytical microscopy with parallel detection

The secondary ion microscope described here allows to obtain the simultaneous registration of chemical and isotopic distribution maps of several elements composing the sample. The instrument has been specially designed to optimize both sensitivity and selectivity: bombardment with primary Cs+ ions to increase the ionization yields of negative secondary ions, efficient collection of secondary ions at the target surface, matching of the secondary ion beam etendue with the acceptance of the mass spectrometer working at high mass resolution, spectrometer with parallel detection capabilities. The probe diameter can be made as low as 30 nm and ion induced electron images registered at the same time as ion images. Presently, four ion micrographs are obtained simultaneously over a field of view up to 20 × 20 μm2 containing up to 512 × 512 pixels. Examples are shown with an ion probe diameter of 0.1 μm.

A New Radio Frequency Plasma Oxygen Primary Ion Source on Nano Secondary Ion Mass Spectrometry for Improved Lateral Resolution and Detection of Electropositive Elements at Single Cell Level

An important application field of secondary ion mass spectrometry at the nanometer scale (NanoSIMS) is the detection of chemical elements and, in particular, metals at the subcellular level in biological samples. The detection of many trace metals requires an oxygen primary ion source to allow the generation of positive secondary ions with high yield in the NanoSIMS. The duoplasmatron oxygen source is commonly used in this ion microprobe but cannot achieve the same quality of images as the cesium primary ion source used to produce negative secondary ions (C(-), CN(-), S(-), P(-)) due to a larger primary ion beam size. In this paper, a new type of an oxygen ion source using a rf plasma is fitted and characterized on a NanoSIMS50L. The performances of this primary ion source in terms of current density and achievable lateral resolution have been characterized and compared to the conventional duoplasmatron and cesium sources. The new rf plasma oxygen source offered a net improvement in terms of primary beam current density compared to the commonly used duoplasmatron source, which resulted in higher ultimate lateral resolutions down to 37 nm and which provided a 5-45 times higher apparent sensitivity for electropositive elements. Other advantages include a better long-term stability and reduced maintenance. This new rf plasma oxygen primary ion source has been applied to the localization of essential macroelements and trace metals at basal levels in two biological models, cells of Chlamydomonas reinhardtii and Arabidopsis thaliana.

Oxygen isotopic measurements on the Cameca Nanosims 50

Abstract The Cameca Nanosims 50 has been designed to provide for parallel detection along a focal plane over a large mass range but spacing between adjacent mass lines makes it necessary to use miniature electron multipliers (EMs). A new version of a miniature EM, designed to reduce aging effects, has been tested. Means to characterize aging effects are presented. Data are interpreted by simulations with a simple model. Consequences of aging on isotopic measurements in a parallel detection context are examined in the special case of oxygen. Measurements of the most abundant isotope with a Faraday cup (FC) and weak isotopes with EMs are reported. Advantages of FC are emphasized: no dead time correction and no quasi-simultaneous arrivals to account for in a situation where high collection efficiencies (8% for O− for instance) leads to large corrections.