Single-cell studies have important implications in biomedicine. An accurate investigation of biochemical behaviour and status requires a biomolecular probe such as vibrational microscopy. Amongst other approaches, synchrotron infrared microspectroscopy is an appropriate analytical tool for single-cell investigation. However, it is important to understand the precise origin of spectral differences as they are directly related to the cell biochemistry. Beside biomolecular changes, physical properties can interfere in the resulting information, and the two effects need separating. Both cells and nuclei induce Mie scattering effects due to their equivalent size with the probe wavelength. This results in a large modification of the spectra, and its precise contribution has to be determined in order to extract the true spectral information. On this basis, we carried out this study in order to evaluate the exact contribution of cell nuclei to Mie scattering. To this purpose, we isolated whole cancer cell nuclei and obtained, for the first time, their FTIR spectra with good signal to noise ratio. The synchrotron-based FTIR (S-FTIR) spectra of nuclei showed changes in lipids, proteins, and DNA absorptions when compared to spectra of whole lung cancer cells. Importantly, we estimated the Mie scattering properties of single cells and single nuclei spectra and were consequently able to separate optical and chemical properties of single cells and nuclei. This is the first study which sheds new light on the identification of the precise spectral biomarkers of a whole cell and those of the cell nucleus.