Division-Based, Growth Rate Diversity in Bacteria.

To investigate the nature and origins of growth rate diversity in bacteria, we grew Escherichia coli and Bacillus subtilis in liquid minimal media and, after different periods of 15N-labeling, analyzed and imaged isotope distributions in individual cells with Secondary Ion Mass Spectrometry. We find a striking inter- and intra-cellular diversity, even in steady state growth. This is consistent with the strand-dependent, hyperstructure-based hypothesis that a major function of the cell cycle is to generate coherent, growth rate diversity via the semi-conservative pattern of inheritance of strands of DNA and associated macromolecular assemblies. We also propose quantitative, general, measures of growth rate diversity for studies of cell physiology that include antibiotic resistance.

Exploring fungus-plant N transfer in a tripartite ant-plant-fungus mutualism.

Background and Aims: The plant Hirtella physophora, the ant Allomerus decemarticulatus and a fungus, Trimmatostroma sp., form a tripartite association. The ants manipulate both the plant trichomes and the fungus to build galleries under the stems of their host plant used to capture prey. In addition to its structural role, the fungus also improves nutrient uptake by the host plant. But it still remains unclear whether the fungus plays an indirect or a direct role in transferring nutrients to the plant. This study aimed to trace the transfer of N from the fungus to the plant's stem tissue. Methods: Optical microscopy and transmission electron microscopy (TEM) were used to investigate the presence of fungal hyphae in the stem tissues. Then, a 15N-labelling experiment was combined with a nanoscale secondary-ion mass spectrometry (NanoSIMS 50) isotopic imaging approach to trace the movement of added 15N from the fungus to plant tissues. Key Results: The TEM images clearly showed hyphae inside the stem tissue in the cellular compartment. Also, fungal hyphae were seen perforating the wall of the parenchyma cell. The 15N provisioning of the fungus in the galleries resulted in significant enrichment of the 15N signature of the plant's leaves 1 d after the 15N-labelling solution was deposited on the fungus-bearing trap. Finally, NanoSIMS imaging proved that nitrogen was transferred biotrophically from the fungus to the stem tissue. Conclusions: This study provides evidence that the fungi are connected endophytically to an ant-plant system and actively transfer nitrogen from 15N-labelling solution to the plant's stem tissues. Overall, this study underlines how complex the trophic structure of ant-plant interactions is due to the presence of the fungus and provides insight into the possibly important nutritional aspects and tradeoffs involved in myrmecophyte-ant mutualisms.

Combining combing and secondary ion mass spectrometry to study DNA on chips using (13)C and (15)N labeling.

Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA - the combing, imaging by SIMS or CIS method - has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to (13)C-labeling via the detection and quantification of the (13)C (14)N (-) recombinant ion and the use of the (13)C: (12)C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS.

50nm-scale localization of single unmodified, isotopically enriched, proteins in cells.

Imaging single proteins within cells is challenging if the possibility of artefacts due to tagging or to recognition by antibodies is to be avoided. It is generally believed that the biological properties of proteins remain unaltered when (14)N isotopes are replaced with (15)N. (15)N-enriched proteins can be localised by dynamic Secondary Ion Mass Spectrometry (D-SIMS). We describe here a novel imaging analysis algorithm to detect a few (15)N-enriched proteins--and even a single protein--within a cell using D-SIMS. The algorithm distinguishes statistically between a low local increase in (15)N isotopic fraction due to an enriched protein and a stochastic increase due to the background. To determine the number of enriched proteins responsible for the increase in the isotopic fraction, we use sequential D-SIMS images in which we compare the measured isotopic fractions to those expected if 1, 2 or more enriched proteins are present. The number of enriched proteins is the one that gives the best fit between the measured and the expected values. We used our method to localise (15)N-enriched thymine DNA glycosylase (TDG) and retinoid X receptor α (RXRα) proteins delivered to COS-7 cells. We show that both a single TDG and a single RXRα can be detected. After 4 h incubation, both proteins were found mainly in the nucleus; RXRα as a monomer or dimer and TDG only as a monomer. After 7 h, RXRα was found in the nucleus as a monomer, dimer or tetramer, whilst TDG was no longer in the nucleus and instead formed clusters in the cytoplasm. After 24 h, RXRα formed clusters in the cytoplasm, and TDG was no longer detectable. In conclusion, single unmodified proteins in cells can be counted and localised with 50 nm resolution by combining D-SIMS with our method of analysis.

Combed single DNA molecules imaged by secondary ion mass spectrometry.

Studies of replication, recombination, and rearrangements at the level of individual molecules of DNA are often limited by problems of resolution or of perturbations caused by the modifications that are needed for imaging. The Combing-Imaging by Secondary Ion Mass Spectrometry (SIMS) (CIS) method helps solve these problems by combining DNA combing, cesium flooding, and quantitative imaging via the NanoSIMS 50. We show here that CIS can reveal, on the 50 nm scale, individual DNA fibers labeled with different, nonradioactive isotopes and, moreover, that it can quantify these isotopes so as to detect and measure the length of one or more short nucleic acid fragments associated with a longer fiber.

Chemical microscopy of biological samples by dynamic mode secondary ion mass spectrometry.

3D chemical microscopy is one of the emerging applications of secondary ion mass spectrometry (SIMS) in biology. Tissues, cells, extracellular matrices, and polymer films can be imaged at present with a lateral resolution of 50 nm and depth resolution of 1 nm using the latest generation of CAMECA magnetic sector NanoSIMS 50 or with a lower lateral resolution (above 100 nm) using IMS 4f Cameca SIMS equipped with cold stage. Dynamic mode SIMS analysis is performed in ultrahigh vacuum and thus requires specific and careful preparation of biological samples aimed at preserving and minimizing destruction of the original structural and chemical properties of the samples. Here we describe a methodology based on the ultrafast plunge-freezing of biological tissues, preparation of the sample for SIMS analyses and transfer to the SIMS cold stage without interruption of the cold chain during the mounting procedure and subsequent SIMS analyses. Using this strategy, SIMS chemical microscopy can be performed on biological tissue in which unwanted molecular and/or structural reorganization, loss of constituents and chemical modifications are minimized and in which structures are therefore optimally preserved.

Dynamic-SIMS imaging and quantification of inorganic ions in frozen-hydrated plant samples.

We present here SIMS images of the distribution of inorganic cations (Na, K, Mg and Ca) in frozen-hydrated samples of three plant species, ivy, camomile, and flax. The samples were cryofixed using fast plunge-freezing. Stigmatic images were obtained, at 100 K, under dynamic SIMS conditions by fast atom bombarding (FAB). Even though the images obtained with the frozen-hydrated plant samples are still not of upper quality, they show that the method used to prepare these samples preserves existing ionic gradients between the outer and the inner part of the cells, between adjacent cells, including cells with the same type of differentiation, and between tissues. We also describe the quantification of the relative proportions of the ions in the vacuoles of flax. The reasonable accuracy achieved for quantification of the vacuole ion ratios permitted to show (i) that radial gradients of ion ratios in hypocotyls change when the plant is becoming older and (ii) that large differences may exist between adjacent cortical cells of the same type. The role of these substantial differences in vacuole ion balance ratios is a largely unexplored issue in plant physiology.

Appraisal of SIMS applicability to boron studies in plants.

In the search for a new methodological approach applicable to the determination of the still poorly known primary role of boron in plant physiology, we have undertaken to appraise the potential of the SIMS method for the analytical imaging of the boron isotopes, (10)B and (11)B, at physiological concentrations in plants. With our own, CAMECA IMS4F SIMS ion analyser, and using O(2)(+) as primary ions for the detection of B(+) (plus (12)C(+) and (40)Ca(+)) secondary ions, we have been able to map quantitatively the two boron isotopes in control and boron-enriched plants, to evaluate boron concentrations at the level of individual cells and to determine boron isotopic ratios. This provides the opportunity to carry out the simultaneous labeling and imaging of boron, using enrichment with the stable isotopes, (10)B and (11)B. The method has also the potential for the simultaneous, quantitative detection of the boron isotopes and of the borate-binding sites in plant cells.