Label-free 3-D molecular imaging of living tissues using Raman Spectral Projection Tomography

The ability to image tissues in three-dimensions (3-D) with label-free molecular contrast at mesoscale would be a valuable capability in biology and biomedicine. Here, we introduce Raman spectral projection tomography (RSPT) for volumetric molecular imaging with sub-millimeter spatial resolution. We have developed a RSPT imaging instrument capable of providing 3-D molecular contrast in transparent and semi-transparent samples. A computational pipeline for multivariate reconstruction was established to extract label-free spatial molecular information from Raman projection data. We demonstrate imaging and visualization of phantoms of various complex shapes with label-free molecular contrast. Finally, we apply RSPT as a novel tool for imaging of molecular gradients and extracellular matrix heterogeneities in fixed and live tissue-engineered constructs and explanted native tissues. RSPT imaging opens new possibilities for label-free molecular monitoring of tissues. ### Competing Interest Statement M.S.B and M.A.B.H are inventors on a patent application (WO/2020/254594). All other authors declare that they have no competing interests.