α-Synuclein (α-Syn) and Tau proteins are neuropathological hallmarks for synucleinopathies and tauopathies, respectively. Co-occurrence of a-Syn and tau aggregates was found in the post-mortem brains of the patients, suggesting a complex interplay between two proteins contributes to the diseases. Here, we explore the polymorphism of α-Syn and Tau cross-seeding with a correlative approach combining fluorescent and super-resolution optical photothermal infrared (O-PTIR) imaging. We first show that hybrid α-Syn-Tau 0N3R pre-formed fibrils (PFFs) form more amyloid-rich structures than α-Syn or a-Syn-Tau mixed PFFs, exhibiting more robust seeding activity and increase phosphorylation of both proteins. The submicron label-free O-PTIR provides insights into distinct amyloid structures of aggregate at subcellular resolution. Our findings demonstrate that α-Syn alone and the hybrid PFFs trigger β-sheets elevation preceding visible aggregate formation. Moreover, we identify a conformational change of β-sheets within aggregates formed by α-Syn and α-Syn-Tau 0N3R hybrid PFFs, corresponding to their enhanced resistance to Protease K resistance. This study underscores distinct patterns of aggregates driven by various PFFs that consist of a-Syn and Tau. It highlights the role of amyloids in strain specificity, marking a significant advance in understanding the molecular basis of cross-seeding between α-Syn and Tau.