IR Map of the Human Cell

Classic infrared (IR) microscopy is limited by the diffraction limit, which obscures subcellular heterogeneity, and by the complex overlap of vibrational bands, which complicates precise molecular assignment. This study presents a comprehensive "IR map of the cell" that provides a standardized framework for label-free chemical identification of subcellular compartments across various human cell lines. By integrating Fourier transform infrared (FTIR) spectroscopy with submicron-resolution optical photothermal infrared (OPTIR) microscopy (∼0.3 μm), the cellular landscape was mapped with improved spatial specificity beyond that achievable by conventional FTIR imaging. Advanced chemometric tools were employed to segment the nucleus, cytoplasm, and regions rich in lipids and glycogen, each characterized by a definitive "IR barcode". Furthermore, in silico modeling validated spectral assignments by simulating cellular fingerprints from reference biocompounds, while detailed spectroscopic characterization of subcellular compartments defined marker bands for proteins, lipids, carbohydrates, and nucleic acids. The modeling supported the interpretation that experimental spectra can be approximated as a linear combination of biomolecular classes, helping to constrain spectral band overlap and refine the proposed "IR barcode" for cellular identification. The developed IR map provides a robust, standardized foundation for the label-free interpretation of cellular chemistry. This study demonstrates the utility of IR microscopy as a powerful diagnostic and analytical tool for monitoring metabolic shifts and cellular status at the micrometric level.