Raman imaging was used to illustrate heterogeneity of a single endothelial cell and the vascular wall sample. The spectral analysis allowed for exploring the complexity of the studied systems in three dimensions and defining the size, volume, shape and biochemical composition of cellular organelles. The ability to construct the 3D maps by a method that does not disrupt the spatial integrity of the cell provided a unique insight into biochemical architecture and cellular processes of endothelium and vascular wall. 3D Raman imaging may be considered as a new trend in analytical spectroscopy to be applied in biomedical research. This method has a potential to be used for medical imaging and diagnostic purposes together with other, already established, imaging techniques.