The ability to simultaneously image multiple biomolecules in biologically relevant
three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a
computational framework for label-free quantitative volumetric Raman imaging (qVRI). We
apply qVRI to a selection of biological systems: human pluripotent stem cells with their
cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell
culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D
cell culture environment. We demonstrate visualization and quantification of fine details in
cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy