Freezing is widely used for preserving different types of cells. Frozen concentrates of lactic acid bacteria (LAB) are extensively used for manufacturing food, probiotic products and for green chemistry and medical applications. However, the freezing and thawing processes cause cell injuries that result in significant cell death. Producing homogeneous bacterial populations with high cryotolerance remains a real challenge. Our objective was to investigate the biochemical and physiological changes in a LAB model at the cell scale following fermentation and freezing in order to identify cellular biomarkers of cryotolerance. Infrared spectra of individual bacteria produced by applying different fermentation and freezing conditions were acquired using synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy to achieve sub-cellular spatial resolution. Fluorescent microscopy was concomitantly assessed, thus making possible to simultaneously analyse the biochemistry and physiological state of a single cell for the first time. Principal component analysis was used to evaluate changes in cell composition, with particular focus on lipids, proteins and polysaccharides. SR-FTIR results indicated that before freezing, freeze-resistant cells grown in a rich medium presented a high content of CH₃ groups from lipid chains, of cell proteins in an α-helix secondary structure and of charged polymers such as teichoic and lipoteichoic acids that constitute the Gram-positive bacterial wall. Moreover, SR-FTIR microspectroscopy made it possible to reveal cell heterogeneity within the cluster of resistant cells, which was ascribed to the diversity of potential substrates in the growth medium. Freezing and thawing processes induced losses of membrane integrity and cell viability in more than 90% of the freeze-sensitive bacterial population. These damages leading to cell death were ascribed to biochemical modification of cell membrane phospholipids, in particular a rigidification of the cytoplasmic membrane following freezing. Furthermore the freeze-resistant cells remained viable after freezing and thawing but a modification of protein secondary structure was detected by SR-FTIR analysis. These results highlighted the potential application of bimodal analysis by SR-FTIR and fluorescence microscopy to increase our knowledge about mechanisms related to cell damage.