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

Area of Science:

• Microbiology
• Analytical Chemistry
• Biotechnology

Background:

• Stable isotope probing (SIP) with Raman microspectroscopy and NanoSIMS are key techniques for studying single-cell metabolic activity.
• Direct comparison of isotope incorporation measurements between Raman and NanoSIMS on the same cell is lacking, creating uncertainty in data consistency.

Purpose of the Study:

• To comparatively analyze isotope incorporation measurements between Raman microspectroscopy and NanoSIMS on individual *Escherichia coli* cells.
• To assess the comparability of ²H incorporation quantification using correlative Raman and NanoSIMS measurements.

Main Methods:

• Correlative Raman microspectroscopy and NanoSIMS measurements were performed on 543 *Escherichia coli* cells incubated with heavy water (²H₂O).
• Quantification of ²H incorporation was analyzed using different mass ratios in NanoSIMS (e.g., ²H/¹H, ¹²C²H/¹²C¹H, C₂²H/C₂¹H).
• An empirical approach using the second derivative was employed to determine optimal Raman wavenumber ranges for improved data equivalency.

Main Results:

• Raman microspectroscopy and NanoSIMS demonstrated highly comparable measurements of ²H incorporation.
• The ²H/¹H ratio in NanoSIMS offered lower detection limits for comprehensive quantification, while C-H bond specific ratios showed potential biases.
• Empirical determination of Raman wavenumber ranges improved the cross-technique data equivalency for ²H quantification.

Conclusions:

• Raman microspectroscopy and NanoSIMS provide comparable data for single-cell stable isotope probing, enhancing confidence in microbial metabolism studies.
• The findings offer a framework for optimizing experimental design and data interpretation in SIP studies.
• This work advances SIP methodologies, improving the reliability of microbial metabolism and interaction research.