FTIR micro-spectroscopy using synchrotron-based and thermal source-based radiation for probing live bacteria.

Fourier transform infrared (FTIR) spectroscopy has proven to be a non-invasive tool to analyse cells without the hurdle of employing exogenous dyes or probes. Nevertheless, the study of single live bacteria in their aqueous environment has long remained a big challenge, due to the strong infrared absorption of water and the small size of bacteria compared to the micron-range infrared wavelengths of the probing photons. To record infrared spectra of bacteria in an aqueous environment, at different spatial resolutions, two setups were developed. A custom-built attenuated total reflection inverted microscope was coupled to a synchrotron-based FTIR spectrometer, using a germanium hemisphere. With such a setup, a projected spot size of 1 × 1 μm was achieved, which allowed spectral acquisition at the single-cell level in the 1800-1300 cm region. The second setup used a demountable liquid micro-chamber with a thermal source-powered FTIR microscope, in transmission geometry, for probing clusters of a few thousands of live cells in the mid-IR region (4000-975 cm). Both setups were applied for studying two strains of a model lactic acid bacterium exhibiting different cryo-resistances. The two approaches allowed the discrimination of both strains and revealed population heterogeneity among bacteria at different spatial resolutions. The multivariate analysis of spectra indicated that the cryo-sensitive cells presented the highest cell heterogeneity and the highest content of proteins with the α-helix structure. Furthermore, the results from clusters of bacterial cells evidenced phosphate and peptidoglycan vibrational bands associated with the cell envelope, as potential markers of resistance to environmental conditions. Graphical Abstract.