Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders characterized by the conversion of the normal prion protein (PrP(C)) into aggregates of its pathological conformer (PrP(Sc)). The mechanism behind this structural conversion is unclear. We report the identification of disease-related protein structural differences directly within the tissue environment. Utilizing a synchrotron infrared (IR) light source, IR images of protein structure were obtained at a subcellular resolution, revealing regions of decreased alpha-helical content and elevated beta-sheet structure in and around infected neurons in the 263 K scrapie hamster model. PrP(Sc) immunostaining of the same tissue demonstrated that the elevated beta-sheet regions correspond to regions where the misfolded structure of PrP(Sc) is located. No evidence of these structural changes was observed in normal neurons.