Single-Cell Raman Spectroscopy of Bacterial Samples

To each tube containing the pellet of non-deuterated or deuterated bacterial suspension, 200 μL of sterilized MilliQ water was added. One microliter was taken and placed on a clean quartz slide. The slide was then placed in the Raman system. A detailed description can be found in the works of these authors [16 (link)]. Briefly, the system allows targeting of bacterial cells thanks to imaging modalitied, and the measurement of single-cell Raman spectra using a confocal arrangement. The beam of a 532 nm, 50 mW laser (Spectra Physics Excelsior 532-50-CDRH) is attenuated and focused by a microscope objective (×100, 0.8 NA, Olympus LMPLFLN) in order to provide a spot size of 1 μm in diameter at the sample. Raman back-scattered light from an individual bacterium is collected by the same objective, filtered from Rayleigh light by a notch filter (NF03-532E, Semrock, New York, USA), and focused into the entrance fiber of a dispersive spectrometer (Hyperflux U1-532, Tornado Spectral systems, Toronto, Canada). The spectrometer featured at −15°C TE-cooled CCD, and spectral resolution of 10 cm^-1 over the band 500–3400 cm^-1. For each analysis point, namely a given configuration in terms of incubation time and D₂O concentration, at least 60 bacteria cells were targeted and the corresponding Raman spectra were acquired. The Raman spectroscope acquisition parameter for each spectrum was tuned to 25 seconds and 250 mW, with our system that is a Lab custom system, not optimized for later industrial purpose.