Label‐free and nondestructive mid‐infrared vibrational hyperspectral imaging is an essential tissue analysis tool, providing spatially resolved biochemical information critical to understanding physiological and pathological processes. However, the chemically complex and spatially heterogeneous composition of tissue specimens and the inherently weak interaction of infrared light with biomolecules limit the analytical performance of infrared absorption spectroscopy. Here, an advanced mid‐infrared spectrochemical tissue imaging modality is introduced using metasurfaces that support strong surface‐localized electromagnetic fields to capture quantitative molecular maps of large‐area murine brain tissue sections. The approach leverages polarization‐multiplexed multi‐resonance plasmonic metasurfaces to simultaneously detect various functional biomolecules. The surface‐enhanced mid‐infrared spectral imaging method eliminates the non‐specific effects of bulk tissue morphology on quantitative spectral analysis and improves chemical selectivity. This study shows that metasurface enhancement increases the retrieval of amide I and II bands associated with protein secondary structures. Moreover, it is demonstrated that plasmonic metasurfaces enhance the chemical contrast in infrared images and enable detection of ultrathin tissue regions that are not otherwise visible to conventional mid‐infrared spectral imaging. While this work uses murine brain tissue sections, the chemical imaging method is well‐suited for other tissue types, which broadens its potential impact for translational research and clinical histopathology.
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