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Comparing Raman and NanoSIMS for heavy water labeling of single cells.

George A Schaible1,2, John B Cliff3, Jennifer A Crandall2,4

  • 1Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717.

Biorxiv : the Preprint Server for Biology
|April 1, 2025
PubMed
Summary

Stable isotope probing (SIP) experiments using Raman microspectroscopy and NanoSIMS show comparable results for measuring heavy water (2H2O) incorporation in single cells. This enhances the reliability of microbial metabolic activity studies.

Keywords:
Cellular heterogeneityD2ONanoSIMSRamancell activityheavy watersingle cell microbiologytechnique comparison

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Area of Science:

  • Microbiology
  • Analytical Chemistry
  • Biotechnology

Background:

  • Stable isotope probing (SIP) with Raman microspectroscopy (Raman) and nano-scale secondary ion mass spectrometry (NanoSIMS) are key for studying single-cell metabolism.
  • Direct comparison of isotope incorporation measurements between Raman and NanoSIMS on the same cell is lacking, creating uncertainty in data consistency.
  • This knowledge gap hinders the reliable application of these powerful techniques in microbiome research.

Purpose of the Study:

  • To comparatively analyze isotope incorporation measurements between Raman and NanoSIMS on individual Escherichia coli cells.
  • To assess the comparability of 2H incorporation quantification using correlative Raman and NanoSIMS measurements.
  • To provide a framework for improving cross-technique data consistency in single-cell SIP studies.

Main Methods:

  • Correlative Raman microspectroscopy and NanoSIMS measurements were performed on 543 Escherichia coli cells.
  • Cells were grown in M9 minimal medium with heavy water (2H2O) to label cellular components.
  • Various mass ratios, including 2H/1H and C2 2H/C2 1H, were analyzed to quantify deuterium incorporation.
  • An empirical approach using the 2nd derivative was employed to determine optimal Raman wavenumber ranges.

Main Results:

  • Raman and NanoSIMS demonstrated highly comparable measurements of 2H incorporation in E. coli cells.
  • The 2H/1H ratio offered lower detection limits for comprehensive quantification, while C2 2H/C2 1H showed potential due to lower background and higher count rates.
  • Empirical determination of Raman wavenumber ranges significantly improved data equivalency between the two techniques.

Conclusions:

  • Correlative Raman and NanoSIMS are reliable for quantifying 2H incorporation in single-cell SIP experiments.
  • The choice of mass ratio impacts comparability, with 2H/1H and C2 2H/C2 1H offering distinct advantages.
  • Optimizing Raman data acquisition enhances cross-technique comparability, advancing the application of SIP in microbial research.