Label free QCL-based mid-IR reflection microspectroscopy for molecular imaging of biofilms

We present a label-free hyperspectral imaging approach using quantum cascade laser (QCL)-based mid-infrared (mid-IR) reflection microspectroscopy (Spero&reg; QT 340) to visualize and characterize biofilms of <i>Mycobacterium tuberculosis</i> (mc² 6230 AE1601¹ ). This technique facilitates rapid data acquisition (&#8275;1 minute) across the fingerprint region (950–1800 cm^-1), enabling the identification of key biomolecular features without the need for external labels or destructive sample preparation. Spectral absorbance was evaluated at 1650 cm^-1, 1450 cm^-1, 1240 cm^-1, and 1080 cm^-1, corresponding to proteins (amide I), lipids (CH₂ scissoring), phosphodiesters, and polysaccharides, respectively. To address baseline variability arising from sample thickness heterogeneities, z-score normalization was applied, improving the interpretability of chemical maps. The spatial distribution of these biomolecular signatures revealed distinct biochemical compartments within the biofilm matrix, shedding light on potential structural and functional interactions. Lipid-rich domains were frequently segregated from regions enriched in polysaccharides and phosphodiesters, suggesting spatial compartmentalization within the biofilm. These findings highlight the utility of mid-IR reflection microspectroscopy for investigating native microbial architectures while preserving overall sample morphology. Integrating QCL-based imaging with complementary techniques could provide deeper insights into biofilm resilience and mechanisms of antimicrobial resistance. This methodology offers a robust and versatile platform for rapid analysis of microbial specimens across medical, environmental, and industrial applications.