Raman ¹⁸ O-labeling of bacteria in visible and deep UV-ranges

Raman stable isotope labeling with ² H, ¹³ C or ¹⁵ N has been reported as an elegant approach to investigate cellular metabolic activity, which is of great importance to reveal the functions of microorganisms in native environments. A new strategy termed Raman ¹⁸ O-labeling was developed to probe the metabolic activity of bacteria. Raman ¹⁸ O-labeling refers to the combination of Raman microspectroscopy with ¹⁸ O-labeling using H₂¹⁸ O. At an excitation wavelength of 532 nm, the incorporation of ¹⁸ O into the amide I group of proteins and DNA/RNA bases was observed in Escherichia coli cells, while for an excitation wavelength electronically resonant with DNA or aromatic amino acid absorption at 244 nm ¹⁸ O assimilation was detected using chemometric tools rather than visual inspection. Raman ¹⁸ O-labeling at 532 nm combined with 2D correlation analysis confirmed the assimilation of ¹⁸ O in proteins and nucleic acids and revealed the growth strategy of E. coli cells; they underwent protein synthesis followed by nucleic acid synthesis. Independent cultural replicates at different incubation times corroborated the reproducibility of these results. The variations in spectral features of ¹⁸ O-labeled cells revealed changes in physiological information of cells. Hence, Raman ¹⁸ O-labeling could provide a powerful tool to identify metabolically active bacterial cells.