Infrared phenomics with 2D-COS unveils spatial heterogeneity and chemical evolution in a microbial biofilm

Spatiotemporal mapping of the chemical dynamics of biofilm remains a fundamental challenge in microbial ecology. Here, we developed an infrared phenomics platform that integrating synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy with two-dimensional correlation spectroscopy (2D-COS) to resolve the spatial heterogeneity and dynamic molecular gradients in Escherichia coli biofilms. By coupling hyperspectral imaging (30 μm resolution) with multivariate trajectory analysis, we revealed sequential chemical evolution: ester accumulation (1703 cm⁻¹) was found to precede enrichment of proteins (1653 cm⁻¹) and nucleic acids (1236 cm⁻¹), which was then followed by the deposition of polysaccharide (1055 cm⁻¹). This hierarchical molecular assembly was validated using principal component analysis (PCA) and cluster-based Euclidean distance metrics, thus establishing IR-2D-COS as a novel tool for the spatiotemporal analysis of microbial biofilm dynamics.