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 μm2 was achieved, which allowed spectral 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 a 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 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.

中文翻译：

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使用基于同步加速器和基于热源的辐射进行活细菌探测的FTIR显微光谱。

傅里叶变换红外（FTIR）光谱已被证明是分析细胞的一种非侵入性工具，无需使用外源染料或探针。然而，由于水的强烈红外吸收和与探测光子的微米范围红外波长相比细菌较小，长期以来，在水性环境中对单个活细菌的研究一直是一个很大的挑战。为了记录水环境中细菌的红外光谱，以不同的空间分辨率开发了两种设置。使用锗半球将定制的衰减全反射倒置显微镜与基于同步加速器的FTIR光谱仪耦合。通过这种设置，可以实现1×1μm2的投影光斑大小，从而可以在1800-1300 cm-1区域的单细胞水平上进行光谱采集。第二种设置是使用可拆卸的液体微腔室，该腔室带有热源供电的FTIR显微镜，其透射几何形状用于探测中红外区域（4000-975 cm-1）内数千个活细胞的簇。两种设置都用于研究表现出不同抗冻性的两种乳酸菌菌株。两种方法都可以区分两种菌株，并揭示了不同空间分辨率下细菌之间的种群异质性。光谱的多变量分析表明，冷冻敏感细胞表现出最高的细胞异质性和具有α-螺旋结构的蛋白质含量最高。此外，细菌细胞簇的结果证明磷酸化和肽聚糖振动带与细胞包膜有关，作为抵抗环境条件的潜在标志。图形概要。