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.