Bias enhanced nucleation (BEN) is the crucial process step to generate epitaxially oriented diamond nuclei on various substrates comprising elemental semiconductors, carbides and non-carbide forming metals. Numerous studies on Si and SiC have shown that the diamond growth immediately starts during the biasing as soon as nuclei have been formed. In contrast, nucleation on Ir requires a stronger ion bombardment under which bulk diamond is etched and 3D crystal growth of the nuclei is suppressed. In the present work, we used high resolution electron energy loss spectroscopy (HR-EELS) to study the vibrational features of the nucleation layers, which were formed by BEN on Ir(001) as compared to the carbon layers formed by BEN on 3C-SiC. It was found that the HR-EEL spectra taken for a BEN layer on iridium with successful diamond nucleation, as indicated by the observation of domains, significantly differs from a carbon nucleation layer without these patterns. The peaks at 300 and 450 meV due to overtones stemming from diamond's optical phonon represent a unique feature correlated with the presence of diamond nuclei. The appearance and strengthening of the corresponding loss lines after annealing is interpreted in terms of a weakly bonded amorphous or highly defective hydrogenated carbon layer that initially covers the nuclei. These findings confirm and complement models for the nucleation process, which had been developed on the base of former observations by various other techniques. The stronger signals from crystalline diamond in the spectra taken from BEN on 3C-SiC confirm that the weaker ion bombardment leaves a more intact diamond crystal at the surface.