# Restoration of deuterium marker for multi-isotope mapping of cellular metabolic activity

## TL;DR

This paper introduces a new method to track cellular metabolic activity using deuterium markers with high precision, enabling detailed analysis of nutrient interactions in complex biological systems.

## Contribution

A novel analytical approach is introduced to restore deuterium fractions from mass interference, enabling accurate multi-isotope tracing at the single-cell level.

## Key findings

- Deuterium incorporation into biomass was quantified using nanoscale Secondary Ion Mass Spectrometry.
- A method was developed to correct for mass-interference in deuterium tracing.
- The approach was successfully applied to a phototrophic symbiotic consortium to study metabolic interplay.

## Abstract

Investigation of cellular metabolic activity with stable-isotope probing (SIP) implies the admittance of an isotope tracer into the metabolic pathway. Incubation with several isotope-markers (multi-isotope tracing) is required to trace nutrient metabolization and elucidate inter-cellular interactions in complex hosts and environmental communities. To cope with the lability of cell nutrition, deuterium in heavy 2H216O water is employed as a substrate-independent general tracer of metabolic activity. However, the spatially-resolved deuterium tracing is hampered by detection limits due to its relatively low ionization yield and mass-interference issues. In the present work, we comprehensively assess the quantitation of deuterium incorporation into biomass employing the outstanding capabilities of nanoscale Secondary Ion Mass Spectrometry facilitating quantitative analysis of metabolic activity with single-cell or subcellular resolution. The effect of ion-probe-induced material relocation on the acquired pattern in 2H enrichment has been considered. Analytical expressions are suggested for the restoration of the deuterium fraction from the unresolved C22H–C21H2 mass-interference. Application of the suggested principle of equal relative assimilation and the multi-isotope tracing with the 2H-marker on a phototrophic symbiotic consortium paves the way to sensing the metabolic interplay among cells, recognition of homeostatic and shifted nutrition, checking for completeness of isotope-labelling and elucidation of nonlabelled substrate contribution.

The online version contains supplementary material available at 10.1038/s41598-025-33762-5.