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Enhanced dark-field hyperspectral imaging (EDF-HSI) integrates advanced spectroscopy and imaging techniques, enabling fast and quantitative insights into the interactions between nanomaterials and organisms at the cellular or invertebrate level. However, the usage of biomolecule-based nanomaterials as contrast agents for EDF-HSI is highly challenging because these nanomaterials usually lack the characteristic optical scattering properties that can be readily distinguished from the scattering signals of organisms. Herein, we report peptide nanoparticles fabricated through covalent self-assembly of dipeptides and genipin for EDF-HSI in vitro and in vivo. Two dipeptides with either C-terminal amidation or not were used as the building blocks for covalent self-assembly. Significantly, the optical scattering intensity of the resulting nanoparticles is enhanced by the amidation. The amidation also enhances the …