NanoSIMS Imaging Research Articles

In situ phenotypic heterogeneity among single cells of the filamentous bacterium Candidatus Microthrix parvicella

Microorganisms in biological wastewater treatment plants require adaptive strategies to deal with rapidly fluctuating environmental conditions. At the population level, the filamentous bacterium Candidatus Microthrix parvicella (Ca. M. parvicella) has been found to fine-tune its gene expression for optimized substrate assimilation. Here we investigated in situ substrate assimilation by single cells of Ca. M. parvicella using nano-scale secondary-ion mass spectrometry (nanoSIMS). NanoSIMS imaging highlighted phenotypic heterogeneity among Ca. M. parvicella cells of the same filament, whereby 13C-oleic acid and 13C-glycerol-3-phosphate assimilation occurred in ≈21–55% of cells, despite non-assimilating cells being intact and alive. In response to alternating aerobic–anoxic regimes, 13C-oleic acid assimilation occurred among subpopulations of Ca. M. parvicella cells (≈3–28% of cells). Furthermore, Ca. M. parvicella cells exhibited two temperature optima for 13C-oleic acid assimilation and associated growth rates. These results suggest that phenotypic heterogeneity among Ca. M. parvicella cells allows the population to adapt rapidly to fluctuating environmental conditions facilitating its widespread occurrence in biological wastewater treatment plants.

702 Multiscale distribution of anticancer platinum(IV) complexes in murine samples revealed by combination of NanoSIMS, LA-ICP-MS and TEM imaging

702 Multiscale distribution of anticancer platinum(IV) complexes in murine samples revealed by combination of NanoSIMS, LA-ICP-MS and TEM imaging

Interactions between natural organic matter, sulfur, arsenic and iron oxides in re-oxidation compounds within riparian wetlands: NanoSIMS and X-ray adsorption spectroscopy evidences

Arsenic (As) is a toxic and ubiquitous element which can be responsible for severe health problems. Recently, Nano-scale Secondary Ions Mass Spectrometry (nanoSIMS) analysis has been used to map organomineral assemblages. Here, we present a method adapted from Belzile et al. (1989) to collect freshly precipitated compounds of the re-oxidation period in a natural wetland environment using a polytetrafluoroethylene (PTFE) sheet scavenger. This method provides information on the bulk samples and on the specific interactions between metals (i.e. As) and the natural organic matter (NOM). Our method allows producing nanoSIMS imaging on natural colloid precipitates, including 75As−, 56Fe16O−, sulfur (32S−) and organic matter (12C14N) and to measure X-ray adsorption of sulfur (S) K-edge. A first statistical treatment on the nanoSIMS images highlights two main colocalizations: (1) 12C14N−, 32S−, 56Fe16O− and 75As−, and (2) 12C14N−, 32S− and 75As−. Principal component analyses (PCAs) support the importance of sulfur in the two main colocalizations firstly evidenced. The first component explains 70% of the variance in the distribution of the elements and is highly correlated with the presence of 32S−. The second component explains 20% of the variance and is highly correlated with the presence of 12C14N−. The X-ray adsorption near edge spectroscopy (XANES) on sulfur speciation provides a quantification of the organic (55%) and inorganic (45%) sulfur compositions. The co-existence of reduced and oxidized S forms might be attributed to a slow NOM kinetic oxidation process. Thus, a direct interaction between As and NOM through sulfur groups might be possible.

Micron-scale D/H heterogeneity in chondrite matrices: A signature of the pristine solar system water?

Organic matter and hydrous silicates are intimately mixed in the matrix of chondrites and in-situ determination of their individual D/H ratios is therefore challenging. Nevertheless, the D/H ratio of each pure component in this mixture should yield a comprehensible signature of the origin and evolution of water and organic matter in our solar system.We measured hydrogen isotope ratios of organic and hydrous silicates in the matrices of two carbonaceous chondrites (Orgueil CI1 and Renazzo CR2) and one unequilibrated ordinary chondrite (Semarkona, LL3.0). A novel protocol was adopted, involving NanoSIMS imaging of H isotopes of monoatomatic (H−) and molecular (OH−) secondary ions collected at the same location. This allowed the most enriched component with respect to D to be identified in the mixture. Using this protocol, we found that in carbonaceous chondrites the isotopically homogeneous hydrous silicates are mixed with D-rich organic matter. The opposite was observed in Semarkona. Hydrous silicates in Semarkona display highly heterogeneous D/H ratios, ranging from 150 to 1800×10−6 (δDSMOW=−40 to 10 600‰). Organic matter in Semarkona does not show such large isotopic variations. This suggests limited isotopic exchange between the two phases during aqueous alteration. Our study greatly expands the range of water isotopic values measured so far in solar system objects. This D-rich water reservoir was sampled by the LL ordinary chondrite parent body and an estimate (≤9%) of its relative contribution to the D/H ratio of water in Oort cloud family comets is proposed.

Evolving insights to understanding mycorrhizas

Evolving insights to understanding mycorrhizas

Correlated Motion and Complex Formation of Lipid-Raft Components Analyzed by High-Resolution Secondary Ion Mass Spectrometry

It is widely believed that certain membrane lipids and membrane-anchored proteins associate to form clusters with emergent function. While extensive data on phase diagrams are available for a range of lipid compositions, these are most commonly visualized by the partitioning of dyes between different phases. We have been developing the use of secondary ion mass spectrometry imaging to directly visualize molecules of interest within supported lipid bilayers (Lozano et al. JACS 2013). Specifically, we want to address the question which neutral membrane components associate with the well-known lipid-raft marker ganglioside GM1, which has a single negative charge, after GM1 is reorganized by an in-plane electric field on a patterned supported bilayer (SLB). NanoSIMS imaging was used to generate molecule-specific concentration profiles of several SLB compositions following electrophoresis: GM1/DOPC, GM1/CHOL/DOPC, and GM1/CHOL/PSM/DOPC. In a control SLB sample without GM1 it is observed that CHOL, PSM, and DOPC do not reorganize by membrane electrophoresis. However, NanoSIMS images clearly illustrate that PSM and CHOL associate with GM1 as it is transported toward the positive electrode while DOPC is displaced in the opposite direction towards the negative electrode. Further analysis of the concentration profiles for the co-diffusing components (GM1/CHOL/PSM) suggest the formation of a 4:2:1 PSM:GM1:CHOL stoichiometric complex. These results demonstrate that both cholesterol and sphingomyelin do tend to associate with GM1 and we might speculate that similar behavior would be observed for GPI or myristolyated proteins.

High-resolution imaging of dietary lipids in cells and tissues by NanoSIMS analysis

Nanoscale secondary ion MS (NanoSIMS) imaging makes it possible to visualize stable isotope-labeled lipids in cells and tissues at 50 nm lateral resolution. Here we report the use of NanoSIMS imaging to visualize lipids in mouse cells and tissues. After administering stable isotope-labeled fatty acids to mice by gavage, NanoSIMS imaging allowed us to visualize neutral lipids in cytosolic lipid droplets in intestinal enterocytes, chylomicrons at the basolateral surface of enterocytes, and lipid droplets in cardiomyocytes and adipocytes. After an injection of stable isotope-enriched triglyceride-rich lipoproteins (TRLs), NanoSIMS imaging documented delivery of lipids to cytosolic lipid droplets in parenchymal cells. Using a combination of backscattered electron (BSE) and NanoSIMS imaging, it was possible to correlate the chemical data provided by NanoSIMS with high-resolution BSE images of cell morphology. This combined imaging approach allowed us to visualize stable isotope-enriched TRLs along the luminal face of heart capillaries and the lipids within heart capillary endothelial cells. We also observed examples of TRLs within the subendothelial spaces of heart capillaries. NanoSIMS imaging provided evidence of defective transport of lipids from the plasma LPs to adipocytes and cardiomyocytes in mice deficient in glycosylphosphatidylinositol-anchored HDL binding protein 1.

Combined SIMS and AFM study of complex structures of streamers on metallic multilayers

The discharge between a platinum electrode and different metallic monolayers on a silicon substrate produced sub‐micrometric structures. The scanning electron microscope images showed the formation of craters with cones, holes, corona, and other exotic structures. The atomic force microscopy images allowed evaluating the depth and height of these complex structures together with the chemical information as determined by NanoSIMS imaging. The plasma impacts locally removed the mono‐layer and the bilayer down to the bare silicon substrate. In order to convert the acquired NanoSIMS images into a true three‐dimensional distribution of the analyzed species, the SIMS images are overlapped with the corresponding atomic force microscopy images of the same probed area. This analysis method allows a visualization of a complex 3D structure and helps understand the formation mechanisms of the streamer impact. Copyright © 2014 John Wiley & Sons, Ltd.

NanoSIMS imaging alteration layers of a leached SON68 glass via a FIB‐made wedged crater

Currently, nuclear wastes are commonly immobilized into glasses because of their long‐term durability. Exposure to water for long periods, however, will eventually corrode the waste form and is the leading potential avenue for radionuclide release into the environment. Because such slow processes cannot be experimentally tested, the prediction of release requires a thorough understanding of the mechanisms governing glass corrosion. In addition, because of the exceptional durability of glass, much of the testing must be performed on high‐surface area powders. A technique that can provide accurate compositional profiles with nanometer scale depth resolution for non‐flat samples would be a major benefit to the field. In this study, NanoSIMS was used to image the cross section of the corrosion layers of a leached SON68 glass sample. A wedged crater was prepared by a focused ion beam instrument to obtain a five‐fold improvement in depth information for NanoSIMS measurements. This improvement allowed us to confirm that the breakdown of the silica glass network is further from the pristine glass than a second dissolution front for boron, another glass former, despite only ~50 nm distance between them. More importantly, NanoSIMS images show that the roughness majorly exists in Si corrosion layer. This novel sample geometry will be a major benefit to efficient NanoSIMS sampling of irregular interfaces at the nanometer scale that would otherwise be obscured within time‐of‐flight secondary ion mass spectroscopy depth profiles. Copyright © 2014 John Wiley & Sons, Ltd.

Reduction of OH contamination in quantification of water contents using NanoSIMS imaging

Quantification of water content is relevant in various topics in geology and planetary sciences. NanoSIMS has capabilities for high spatial resolution imaging and offers opportunities to accurately quantify water contents at fine scale on small surface areas. The main concern using ion microprobe techniques is to estimate and minimize contribution of water contamination, from residual gas in the sample chamber, sticking onto the surface of the sample. Here we tackle a set of sputtering/analytical parameters and we evaluate their relative influence on the OH−/Si− ratio. We demonstrate that a high erosion rate, reached using a primary beam intensity of ~25pA, is sufficient to lower this OH contamination for basaltic glass. This leads us to describe a procedure to correct for OH contamination and thus determine accurate values of OH/Si ratio in order to quantify water contents in silicate materials using NanoSIMS imaging.

Correlated SEM, FIB-SEM, TEM, and NanoSIMS Imaging of Microbes from the Hindgut of a Lower Termite: Methods forIn SituFunctional and Ecological Studies of Uncultivable Microbes

The hindguts of lower termites harbor highly diverse, endemic communities of symbiotic protists, bacteria, and archaea essential to the termite's ability to digest wood. Despite over a century of experimental studies, ecological roles of many of these microbes are unknown, partly because almost none can be cultivated. Many of the protists associate with bacterial symbionts, but hypotheses for their respective roles in nutrient exchange are based on genomes of only two such bacteria. To show how the ecological roles of protists and nutrient transfer with symbiotic bacteria can be elucidated by direct imaging, we combined stable isotope labeling (13C-cellulose) of live termites with analysis of fixed hindgut microbes using correlated scanning electron microscopy, focused ion beam-scanning electron microscopy (FIB-SEM), transmission electron microscopy, and high resolution imaging mass spectrometry (NanoSIMS). We developed methods to prepare whole labeled cells on solid substrates, whole labeled cells milled with a FIB-SEM instrument to reveal cell interiors, and ultramicrotome sections of labeled cells for NanoSIMS imaging of 13C enrichment in protists and associated bacteria. Our results show these methods have the potential to provide direct evidence for nutrient flow and suggest the oxymonad protist Oxymonas dimorpha phagocytoses and enzymatically degrades ingested wood fragments, and may transfer carbon derived from this to its surface bacterial symbionts.

Measuring carbon and N2 fixation in field populations of colonial and free-living unicellular cyanobacteria using nanometer-scale secondary ion mass spectrometry(1).

Unicellular cyanobacteria are now recognized as important to the marine N and C cycles in open ocean gyres, yet there are few direct in situ measurements of their activities. Using a high-resolution nanometer scale secondary ion mass spectrometer (nanoSIMS), single cell N2 and C fixation rates were estimated for unicellular cyanobacteria resembling N2 fixer Crocosphaera watsonii. Crocosphaera watsonii-like cells were observed in the subtropical North Pacific gyre (22°45' N, 158°0' W) as 2 different phenotypes: colonial and free-living. Colonies containing 3-242 cells per colony were observed and cell density in colonies increased with incubation time. Estimated C fixation rates were similarly high in both phenotypes and unexpectedly for unicellular cyanobacteria 85% of the colonial cells incubated during midday were also enriched in (15) N above natural abundance. Highest (15) N enrichment and N2 fixation rates were found in cells incubated overnight where up to 64% of the total daily fixed N in the upper surface waters was attributed to both phenotypes. The colonial cells retained newly fixed C in a sulfur-rich matrix surrounding the cells and often cells of both phenotypes possessed areas (<1nm) of enriched (15) N and (13) C resembling storage granules. The nanoSIMS imaging of the colonial cells also showed evidence for a division of N2 and C fixation activity across the colony where few individual cells (<34%) in a given colony were enriched in both (15) N and (13) C above the colony average. Our results provide new insights into the ecophysiology of unicellular cyanobacteria.

Pulsed 86Sr-labeling and NanoSIMS imaging to study coral biomineralization at ultra-structural length scales

A method to label marine biocarbonates is developed based on a concentration enrichment of a minor stable isotope of a trace element that is a natural component of seawater, resulting in the formation of biocarbonate with corresponding isotopic enrichments. This biocarbonate is subsequently imaged with a NanoSIMS ion microprobe to visualize the locations of the isotopic marker on sub-micrometric length scales, permitting resolution of all ultra-structural details. In this study, a scleractinian coral, Pocillopora damicornis, was labeled 3 times with 86Sr-enhanced seawater for a period of 48 h with 5 days under normal seawater conditions separating each labeling event. Two non-specific cellular stress biomarkers, glutathione-S-transferase activity and porphyrin concentration plus carbonic anhydrase, an enzymatic marker involved in the physiology of carbonate biomineralization, as well as unchanged levels of zooxanthellae photosynthesis efficiency indicate that coral physiological processes are not affected by the 86Sr-enhancement. NanoSIMS images of the 86Sr/44Ca ratio in skeleton formed during the experiment allow for a determination of the average extension rate of the two major ultra-structural components of the coral skeleton: Rapid Accretion Deposits are found to form on average about 4.5 times faster than Thickening Deposits. The method opens up new horizons in the study of biocarbonate formation because it holds the potential to observe growth of calcareous structures such as skeletons, shells, tests, spines formed by a wide range of organisms under essentially unperturbed physiological conditions.

Scanning electron microscopic imaging and nano-secondary ion microprobe analyses of bacteria-like nanoball structures in oncoids from the Ediacaran Boonall Dolomite of Kimberley, northwestern Australia: testing their biogenicity

Nanoball structures have been frequently detected in modern and ancient sedimentary rocks worldwide. They are widely believed to be biological in origin due to their similar morphology to modern bacteria. Abundant, well-preserved nanoball structures from the oncoids of the Boonall Dolomite of Ediacaran age in the Kimberley region, northwestern Australia are being described. Scanning electron microscopic imaging indicates that these minute objects are identical with nanobacteria previously documented elsewhere. However, nano-secondary ion microprobe (NanoSIMS) imaging analysis could not reveal the presence of organic elements such as C, N and S in the nanoball-like structures. Therefore, a biological origin for the Ediacaran nanoball structures seems unlikely. NanoSIMS imaging analysis may well be needed for other occurrences of nanoball structures in order to determine if all of these similar objects are really of biological origin.

Extreme 54Cr-rich nano-oxides in the CI chondrite Orgueil – Implication for a late supernova injection into the solar system

Systematic variations in 54Cr/ 52Cr ratios between meteorite classes ( Trinquier et al., 2007; Qin et al., 2010a) point to large scale spatial and/or temporal isotopic heterogeneity in the solar protoplanetary disk. Two explanations for these variations have been proposed, with important implications for the formation of the Solar System: heterogeneous seeding of the disk with dust from a supernova, or energetic-particle irradiation of dust in the disk. The key to differentiating between them is identification of the carrier(s) of the 54Cr anomalies. Here we report the results of our recent NanoSIMS imaging search for the 54Cr-rich carrier in the acid-resistant residue of the CI chondrite Orgueil. A total of 10 regions with extreme 54Cr-excesses ( δ 54Cr values up to 1500‰) were found. Comparison between SEM, Auger and NanoSIMS analyses showed that these 54Cr-rich regions are associated with one or more sub-micron (typically less than 200 nm) Cr oxide grains, most likely spinels. Because the size of the NanoSIMS primary O − ion beam is larger than the typical grain size on the sample mount, the measured anomalies are lower limits, and we estimate that the actual 54Cr enrichments in three grains are at least 11 times Solar and in one of these may be as high as 50 times Solar. Such compositions strongly favor a Type II supernova origin. The variability in bulk 54Cr/ 52Cr between meteorite classes argues for a heterogeneous distribution of the 54Cr carrier in the solar protoplanetary disk following a late supernova injection event. Such a scenario is also supported by the O-isotopic distribution and variable abundances in different planetary materials of other presolar oxide and silicate grains from supernovae.

Correlated AFM and NanoSIMS imaging to probe cholesterol-induced changes in phase behavior and non-ideal mixing in ternary lipid membranes

Cholesterol is believed to be an important component in compositionally distinct lipid domains in the cellular plasma membrane, which are referred to as lipid rafts. Insight into how cholesterol influences the interactions that contribute to plasma membrane organization can be acquired from model lipid membranes. Here we characterize the lipid mixing and phase behavior exhibited by 15N-dilaurolyphosphatidycholine (15N-DLPC)/deuterated distearoylphosphatiylcholine (D70-DSPC) membranes with various amounts of cholesterol (0, 3, 7, 15 or 19mol%) at room temperature. The microstructures and compositions of individual membrane domains were determined by imaging the same membrane locations with both atomic force microscopy (AFM) and high-resolution secondary ion mass spectrometry (SIMS) performed with a Cameca NanoSIMS 50. As the cholesterol composition increased from 0 to 19mol%, the circular ordered domains became more elongated, and the amount of 15N-DLPC in the gel-phase domains remained constant at 6–7mol%. Individual and micron-sized clusters of nanoscopic domains enriched in D70-DSPC were abundant in the 19mol% cholesterol membrane. AFM imaging showed that these lipid domains had irregular borders, indicating that they were gel-phase domains, and not non-ideally mixed lipid clusters or nanoscopic liquid-ordered domains.

NanoSIMS imaging ofBacillusspores sectioned by focused ion beam

Preparation and sectioning of bacterial spores by focused ion beam and subsequent high resolution secondary ion mass spectrometry analytical imaging is demonstrated. Scanning transmission electron microscopy mode imaging in a scanning electron microscope is used to show that the internal structure of the bacterial spore can be preserved during focused ion beam sectioning and can be imaged without contrast staining. Ion images of the sections show that the internal elemental distributions of the sectioned spores are preserved. A rapid focused ion beam top-sectioning method is demonstrated to yield comparable ion images without the need for sample trenching and section lift-out. The lift-out and thinning method enable correlated transmission electron microscopy and high resolution secondary ion mass spectrometry analyses. The top-cutting method is preferable if only secondary ion mass spectrometry analyses are performed because this method is faster and yields more sample material for analysis; depth of useful sample material is approximately 300 nm for top-cut sections versus approximately 100 nm for electron-transparent sections.

Fixation and fate of C and N in the cyanobacterium Trichodesmium using nanometer-scale secondary ion mass spectrometry

The marine cyanobacterium Trichodesmium is ubiquitous in tropical and subtropical seas and is an important contributor to global N and C cycling. We sought to characterize metabolic uptake patterns in individual Trichodesmium IMS-101 cells by quantitatively imaging (13)C and (15)N uptake with high-resolution secondary ion mass spectrometry (NanoSIMS). Trichodesmium fix both CO(2) and N(2) concurrently during the day and are, thus, faced with a balancing act: the O(2) evolved during photosynthesis inhibits nitrogenase, the key enzyme in N(2) fixation. After performing correlated transmission electron microscopy (TEM) and NanoSIMS analysis on trichome thin-sections, we observed transient inclusion of (15)N and (13)C into discrete subcellular bodies identified as cyanophycin granules. We speculate that Trichodesmium uses these dynamic storage bodies to uncouple CO(2) and N(2) fixation from overall growth dynamics. We also directly quantified both CO(2) and N(2) fixation at the single cell level using NanoSIMS imaging of whole cells in multiple trichomes. Our results indicate maximal CO(2) fixation rates in the morning, compared with maximal N(2) fixation rates in the afternoon, bolstering the argument that segregation of CO(2) and N(2) fixation in Trichodesmium is regulated in part by temporal factors. Spatial separation of N(2) and CO(2) fixation may also have a role in metabolic segregation in Trichodesmium. Our approach in combining stable isotope labeling with NanoSIMS and TEM imaging can be extended to other physiologically relevant elements and processes in other important microbial systems.

Linking Microbial Phylogeny to Metabolic Activity at the Single-Cell Level by Using Enhanced Element Labeling-Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (EL-FISH) and NanoSIMS

To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with (13)C-carbon and (15)N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.

Sub-cellular localisation of a 15N-labelled peptide vector using NanoSIMS imaging

Dynamic SIMS imaging is proposed to map sub-cellular distributions of isotopically labelled, exogenous compounds. NanoSIMS imaging allows the characterisation of the intracellular transport pathways of exogenous molecules, including peptide vectors employed in innovative therapies, using stable isotopes as molecular markers to detect the compound of interest. Shiga toxin B-subunit (STxB) was chosen as a representative peptide vector. The recombinant protein ( 15N-STxB) was synthesised in Escherichia coli using 15NH 4Cl as sole nitrogen source resulting in 15N enrichment in the molecule. Using the NanoSIMS 50 ion microprobe (Cameca), different ion species ( 12C 14N −, 12C 15N −, 31P −) originating from the same sputtered micro volume were simultaneously detected. High mass resolving power enabled the discrimination of 12C 15N − from its polyatomic isobars of mass 27. We imaged the membrane binding and internalisation of 15N-STxB in HeLa cells at spatial resolutions of less than 100 nm. Thus, the use of rare stable isotopes like 15N with dynamic SIMS imaging permits sub-cellular detection of isotopically labelled, exogenous molecules and imaging of their transport pathways at high mass and spatial resolution. Application of stable isotopes as markers can replace the large and chemically complex tags used for fluorescence microscopy, without altering the chemical and physical properties of the molecule.