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 H2 18 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.