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Chemical detection, mapping and imaging in three dimensions will help refine our understanding of wood properties and durability. We describe here a pioneering infrared method to create visual 3D images of the chemicals in wood, providing for the first time, spatial and architectural information at the cellular level without liquid extraction or prior fixation. Analysis utilized high-resolution Fourier transform infrared (FTIR) microspectroscopy that obtains infrared light from a synchrotron beamline facility at the University of Wisconsin Synchrotron Radiation Center that is equipped with a unique design for illuminating samples. An advanced detector allows multiple mid-infrared spectra to be collected simultaneously. Chemical images were generated in 3D for cell wall layers of Populus deltoides Bartr. A complete mid-infrared spectrum was obtained for each data point in the 3D image.