Nano-Secondary Ions Mass Spectrometry (nanoSIMS) Coupled with In Situ Hybridization for Ecological Research

This chapter focuses on the application of nano-secondary ions mass spectrometry (nanoSIMS) coupled to in situ hybridization for tracking isotopically labeled cells within complex microbial communities and the importance of nanoSIMS-derived methodologies in analyzing the metabolic and phylogenetic diversity of microorganisms in the environment. The coupling of nanoSIMS and in situ hybridization allows simultaneous quantification of substrate uptake and phylogenetic identification of an individual microbial cell in a single nanoSIMS scan. Halogens such as fluorine, bromine, or iodine are used as markers for the Identification of the cells and are specifically introduced into the cells via in situ hybridization. The broad application of fluorescence in situ hybridization (FISH) on a wide variety of samples offers a big advantage for any combined method that uses FISH or in situ hybridization-related techniques for the phylogenetic identification of single cells. The chapter outlines FISH-based Identification and in situ hybridization using halogen-containing tyramides coupled to nanoSIMS, with focus on the usefulness of both approaches and the problems that can emerge. The first application of the halogen-based identification and nanoSIMS in the natural environment was performed on individual cells of the anaerobic, phototrophic bacteria inhabiting the oligotrophic, meromictic alpine Lake Cadagno. The study focused on quantification of the metabolic activities of three different bacterial species, Chlorobium clathratiforme, Chromatium okenii, andLamprocystis purpurea. Moreover, a remarkable variability in metabolic rates of individual cells of the same species was measured, showing for the first time that a microbial population is a heterogeneous group of physiologically distinct individuals.

[1]  V. Orphan,et al.  Deep-Sea Archaea Fix and Share Nitrogen in Methane-Consuming Microbial Consortia , 2009, Science.

[2]  M. Kuypers,et al.  Co-occurrence of denitrification and nitrogen fixation in a meromictic lake, Lake Cadagno (Switzerland). , 2009, Environmental microbiology.

[3]  V. Orphan,et al.  Patterns of 15N assimilation and growth of methanotrophic ANME-2 archaea and sulfate-reducing bacteria within structured syntrophic consortia revealed by FISH-SIMS. , 2009, Environmental microbiology.

[4]  V. Orphan,et al.  Geobiological investigations using secondary ion mass spectrometry: microanalysis of extant and paleo‐microbial processes , 2009, Geobiology.

[5]  R. Popa,et al.  Fixation and fate of C and N in the cyanobacterium Trichodesmium using nanometer-scale secondary ion mass spectrometry , 2009, Proceedings of the National Academy of Sciences.

[6]  A. Montanari,et al.  A novel symbiosis between chemoautotrophic bacteria and a freshwater cave amphipod , 2009, The ISME Journal.

[7]  Rudolf Amann,et al.  A single-cell view on the ecophysiology of anaerobic phototrophic bacteria , 2008, Proceedings of the National Academy of Sciences.

[8]  V. Orphan,et al.  Micron-scale mapping of sulfur cycling across the oxycline of a cyanobacterial mat: a paired nanoSIMS and CARD-FISH approach , 2008, The ISME Journal.

[9]  R. Amann,et al.  Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques , 2008, Nature Reviews Microbiology.

[10]  D. Relman,et al.  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 , 2008, Applied and Environmental Microbiology.

[11]  Tianlun Li,et al.  Simultaneous analysis of microbial identity and function using NanoSIMS , 2008, Environmental microbiology.

[12]  M. Kuypers.  Sizing Up the Uncultivated Majority , 2007, Science.

[13]  D. Distel,et al.  Quantitative Imaging of Nitrogen Fixation by Individual Bacteria Within Animal Cells , 2007, Science.

[14]  Michael Wagner,et al.  Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function. , 2007, Environmental microbiology.

[15]  K. Nealson,et al.  Carbon and nitrogen fixation and metabolite exchange in and between individual cells of Anabaena oscillarioides , 2007, The ISME Journal.

[16]  Michael Wagner,et al.  Who eats what, where and when? Isotope-labelling experiments are coming of age , 2007, The ISME Journal.

[17]  B. Jørgensen,et al.  The future of single-cell environmental microbiology. , 2007, Environmental microbiology.

[18]  Douglas Benson,et al.  High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry , 2006, Journal of biology.

[19]  Rika Anderson,et al.  Heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Michael Wagner,et al.  Linking microbial community structure with function: fluorescence in situ hybridization-microautoradiography and isotope arrays. , 2006, Current opinion in biotechnology.

[21]  Alain Croisy,et al.  Progress in analytical imaging of the cell by dynamic secondary ion mass spectrometry (SIMS microscopy). , 2005, Biochimica et biophysica acta.

[22]  M. Dumont,et al.  Stable isotope probing — linking microbial identity to function , 2005, Nature Reviews Microbiology.

[23]  C. Lechene,et al.  Measure of carbon and nitrogen stable isotope ratios in cultured cells , 2004, Journal of the American Society for Mass Spectrometry.

[24]  Michael Wagner,et al.  Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes. , 2003, Current opinion in microbiology.

[25]  J. Nielsen,et al.  Quantification of cell-specific substrate uptake by probe-defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization. , 2003, Environmental microbiology.

[26]  F. Stadermann,et al.  Samples of Stars Beyond the Solar System: Silicate Grains in Interplanetary Dust , 2003, Science.

[27]  D. Gaspar,et al.  Exploration of Inorganic C and N Assimilation by Soil Microbes with Time-of-Flight Secondary Ion Mass Spectrometry , 2002, Applied and Environmental Microbiology.

[28]  E. Delong,et al.  Methane-Consuming Archaea Revealed by Directly Coupled Isotopic and Phylogenetic Analysis , 2001, Science.

[29]  N. Winograd,et al.  Imaging with mass spectrometry. , 1999, Chemical reviews.

[30]  J. Fuhrman,et al.  Microbial Desulfurization of a Crude Oil Middle-Distillate Fraction: Analysis of the Extent of Sulfur Removal and the Effect of Removal on Remaining Sulfur , 1999, Applied and Environmental Microbiology.

[31]  M. Tonolla,et al.  In Situ Analysis of Phototrophic Sulfur Bacteria in the Chemocline of Meromictic Lake Cadagno (Switzerland) , 1999, Applied and Environmental Microbiology.

[32]  K. Schleifer,et al.  Combination of Fluorescent In Situ Hybridization and Microautoradiography—a New Tool for Structure-Function Analyses in Microbial Ecology , 1999, Applied and Environmental Microbiology.

[33]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[34]  D A Stahl,et al.  Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology , 1990, Journal of bacteriology.

[35]  E. Delong,et al.  Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. , 1989, Science.