skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Correlative SIP-FISH-Raman-SEM-NanoSIMS links identity, morphology, biochemistry, and physiology of environmental microbes

Journal Article · · ISME Communications

Microscopic and spectroscopic techniques are commonly applied to study microbial cells but are typically used on separate samples, resulting in population-level datasets that are integrated across different cells with little spatial resolution. To address this shortcoming, we developed a workflow that correlates several microscopic and spectroscopic techniques to generate an in depth analysis of individual cells. By combining stable isotope probing (SIP), fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), confocal Raman microspectroscopy (Raman), and nano-scale secondary ion mass spectrometry (NanoSIMS), we illustrate how individual cells can be thoroughly interrogated to obtain information about their taxonomic identity, structure, physiology, and metabolic activity. Analysis of an artificial community demonstrated that our correlative approach was able to resolve the activity of single cells using heavy water SIP in conjunction with Raman and/or NanoSIMS and establish their taxonomy and morphology using FISH and SEM. We then applied this workflow to a sample of yet uncultured multicellular magnetotactic bacteria. In addition to establishing their identity and activity, backscatter electron microscopy (BSE), NanoSIMS, and energy-dispersive X-ray spectroscopy (EDS) were employed to characterize the magnetosomes within the cells. By integrating these techniques, we demonstrate a cohesive approach to thoroughly study environmental microbes at single cell resolution.

Research Organization:
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
Sponsoring Organization:
USDOE
DOE Contract Number:
AC05-76RL01830
OSTI ID:
1883223
Report Number(s):
PNNL-SA-172171
Journal Information:
ISME Communications, Vol. 2
Country of Publication:
United States
Language:
English

References (62)

Next-generation physiology approaches to study microbiome function at single cell level journal February 2020
The 2018 correlative microscopy techniques roadmap journal August 2018
Advances in Correlative Single-Molecule Localization Microscopy and Electron Microscopy journal January 2015
Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell journal June 1978
Visualization of the same PtK2 cytoskeletons by both immunofluorescence and low power electron microscopy journal November 1978
Correlative Light- and Electron Microscopy with chemical tags journal May 2014
Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging journal May 2021
ColorEM: analytical electron microscopy for element-guided identification and imaging of the building blocks of life journal August 2018
Correlation of fluorescence microscopy, electron microscopy, and NanoSIMS stable isotope imaging on a single tissue section journal July 2020
Super-Resolution Correlative Light and Electron Microscopy (SR-CLEM) Reveals Novel Ultrastructural Insights Into Dendritic Cell Podosomes journal August 2018
Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging journal October 2014
Juxtaposed membranes underpin cellular adhesion and display unilateral cell division of multicellular magnetotactic prokaryotes journal April 2020
Subgroup Characteristics of Marine Methane-Oxidizing ANME-2 Archaea and Their Syntrophic Partners as Revealed by Integrated Multimodal Analytical Microscopy journal April 2018
Novel prosthecate bacteria from the candidate phylum Acetothermia journal June 2018
Three-Dimensional Mapping of Soil Chemical Characteristics at Micrometric Scale by Combining 2D SEM-EDX Data and 3D X-Ray CT Images journal September 2015
Correlative Imaging Reveals Holistic View of Soil Microenvironments journal December 2018
Spatially resolved correlative microscopy and microbial identification reveal dynamic depth‐ and mineral‐dependent anabolic activity in salt marsh sediment journal August 2021
Tracking microbial interactions with NanoSIMS journal October 2016
Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function journal August 2007
Proposal to reclassify the proteobacterial classes Deltaproteobacteria and Oligoflexia, and the phylum Thermodesulfobacteria into four phyla reflecting major functional capabilities journal November 2020
Structure, Behavior, Ecology and Diversity of Multicellular Magnetotactic Prokaryotes
  • Keim, Carolina N.; Lopes Martins, Juliana; Lins de Barros, Henrique
  • Magnetoreception and Magnetosomes in Bacteria, p. 103-132 https://doi.org/10.1007/7171_040
book January 2006
‘Candidatus Magnetoglobus multicellularis’, a multicellular, magnetotactic prokaryote from a hypersaline environment
  • Abreu, Fernanda; Martins, Juliana Lopes; Silveira, Thaís Souza
  • International Journal of Systematic and Evolutionary Microbiology, Vol. 57, Issue 6 https://doi.org/10.1099/ijs.0.64857-0
journal June 2007
Cell Adhesion, Multicellular Morphology, and Magnetosome Distribution in the Multicellular Magnetotactic Prokaryote Candidatus Magnetoglobus multicellularis journal April 2013
Magnetotactic Bacteria and Magnetosomes journal November 2008
Formation and function of bacterial organelles journal July 2020
Magnetosome biogenesis in magnetotactic bacteria journal September 2016
Multicellular photo-magnetotactic bacteria: Multicellular photo-magnetotactic bacteria journal September 2010
Unexpected diversity in populations of the many-celled magnetotactic prokaryote journal January 2007
Microscale sulfur cycling in the phototrophic pink berry consortia of the Sippewissett Salt Marsh: Sulfur cycling in pink berries of the Sippewissett Marsh journal February 2014
The Green Berry Consortia of the Sippewissett Salt Marsh: Millimeter-Sized Aggregates of Diazotrophic Unicellular Cyanobacteria journal September 2017
Cable bacteria associated with long-distance electron transport in New England salt marsh sediment journal December 2014
Calcite-accumulating large sulfur bacteria of the genus Achromatium in Sippewissett Salt Marsh journal April 2015
Seasonal Succession and Spatial Patterns of Synechococcus Microdiversity in a Salt Marsh Estuary Revealed through 16S rRNA Gene Oligotyping journal August 2017
Salt marsh sediment diversity: a test of the variability of the rare biosphere among environmental replicates journal June 2012
Mirrored stainless steel substrate provides improved signal for Raman spectroscopy of tissue and cells journal July 2016
Sub-Micrometer-Scale Mapping of Magnetite Crystals and Sulfur Globules in Magnetotactic Bacteria Using Confocal Raman Micro-Spectrometry journal September 2014
Double Labeling of Oligonucleotide Probes for Fluorescence In Situ Hybridization (DOPE-FISH) Improves Signal Intensity and Increases rRNA Accessibility journal December 2009
The Domain-specific Probe EUB338 is Insufficient for the Detection of all Bacteria: Development and Evaluation of a more Comprehensive Probe Set journal September 1999
In Situ Accessibility of Small-Subunit rRNA of Members of the Domains Bacteria, Archaea, and Eucarya to Cy3-Labeled Oligonucleotide Probes journal March 2003
Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms journal January 1993
Phenotypic heterogeneity in metabolic traits among single cells of a rare bacterial species in its natural environment quantified with a combination of flow cell sorting and NanoSIMS journal April 2015
A pipeline for targeted metagenomics of environmental bacteria journal February 2020
NanoSIMS sample preparation decreases isotope enrichment: magnitude, variability and implications for single‐cell rates of microbial activity journal October 2020
The effect of FISH and CARD-FISH on the isotopic composition of 13C- and 15N-labeled Pseudomonas putida cells measured by nanoSIMS journal June 2014
Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques journal May 2008
Raman microspectroscopy for microbiology journal November 2021
Single cell stable isotope probing in microbiology using Raman microspectroscopy journal October 2016
Advancements in the application of NanoSIMS and Raman microspectroscopy to investigate the activity of microbial cells in soils journal August 2015
Influence of Carbon Sources on Quantification of Deuterium Incorporation in Heterotrophic Bacteria: A Raman-Stable Isotope Labeling Approach journal July 2020
Single‐cell determination of iron content in magnetotactic bacteria: implications for the iron biogeochemical cycle journal June 2019
Magnetic iron-sulphur crystals from a magnetotactic microorganism journal January 1990
Ultrastructure, tactic behaviour and potential for sulfate reduction of a novel multicellular magnetotactic prokaryote from North Sea sediments journal June 2009
Characterization and phylogenetic identification of a species of spherical multicellular magnetotactic prokaryotes that produces both magnetite and greigite crystals journal September 2014
Diversity and Characterization of Multicellular Magnetotactic Prokaryotes From Coral Reef Habitats of the Paracel Islands, South China Sea journal September 2018
The transformation of mackinawite into greigite studied by Raman spectroscopy journal March 2011
Iron sulfides from magnetotactic bacteria; structure, composition, and phase transitions journal December 1998
Imaging Intracellular Fluorescent Proteins at Nanometer Resolution journal September 2006
Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) journal August 2006
In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry : Visualizing new proteins in microbes by click chemistry journal April 2014
Individual cell DNA synthesis within natural marine bacterial assemblages as detected by ‘click’ chemistry journal July 2014
d -Amino Acid Chemical Reporters Reveal Peptidoglycan Dynamics of an Intracellular Pathogen journal January 2013
Cell organization and ultrastructure of a magnetotactic multicellular organism journal March 2004