A single-molecule imaging technique has been developed to study the hydrophobicity of species formed during aggregation of proteins; specifically, those that form amyloid-like structures, which are the hallmarks of many protein misfolding diseases such as Parkinson’s disease. The technique, named spectral-PAINT (Points Accumulation for Imaging in Nanoscale Topography), provides a spatial resolution far beyond the optical diffraction limit, allowing the hydrophobicity of amyloid-like oligomers (~20 nm species) or elongated fibrils (~1 um in length) to be analysed. Significantly, measurements were able to distinguish structural differences between early and mature aggregated species with distinct hydrophobicity patterns being observed for amyloid-like oligomers as compared to elongated fibrils.