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FTIR micro-spectroscopy using synchrotron-based and thermal source-based radiation for probing live bacteria
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Edité par CCSD ; Springer Verlag -
International audience. 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 x 1 µm² was achieved, which allowed spectra acquisition at the single-cell level in the 1800-1300 cm-1 region. The second setup used a demountable liquid micro-chamber with a thermal-source powered FTIR microscope, in transmission geometry, for probing clusters of few thousands of live cells in the mid-IR region (4000-975 cm-1). 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 in 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.
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