Phasor Analysis of Multiband Deuterium Mid-Infrared Imaging Reveals Spatial Metabolic Organization in Tissues.

Heavy water (D2O) labeling provides a safe and universal approach for probing biosynthesis in mid-infrared (MIR) metabolic imaging. However, current analysis typically simplifies the C-D spectral region into two peaks associated with lipid and protein synthesis, overlooking the broader spectral complexity arising from multiple deuteration sites and chemical environments. Here, we show that in vivo D2O labeling produces multiband C-D spectral features spanning 2000-2300 cm-1, reflecting contributions from distinct d1, d2, and d3 configurations across lipids, proteins, and other biomolecules. To characterize this spectral complexity, we introduce a suite of phasor-based analyses that transform hyperspectral MIR data into a two-dimensional, chemically interpretable representation, enabling model-free dimensionality reduction while preserving spectral mixing relationships. Within this framework, phasor coloring enables rapid, segmentation-free visualization of multiscale metabolic heterogeneity, whereas phasor-ratio analysis identifies localized metabolic niches, including Purkinje cells in the cerebellum, intestinal villi, and tumor invasive margins. Phasor-based segmentation results of the mouse brain are quantitatively compared with the Allen Brain Atlas, showing good agreement with histological architecture. This method presents a rapid and effective way to decipher deuterium-labeled MIR imaging data and visualize the spatial metabolic organization of tissues.