As with Alzheimer’s Disease (AD), the pathological hallmark of Cerebral Amyloid Angiopathy (CAA) is the formation of plaques composed of amyloid-β (Aβ) fibrils. It is widely hypothesized that structural variants of fibrils, termed polymorphs, contribute to the pathophysiology of CAA and AD. However, there is no clear understanding of how their molecular structure induces cytotoxic activity. The objective of this application is to employ novel chemical imaging and electrochemical sensing methods to directly monitor the structural dynamics, aberrant interactions, and toxic activities of different Aβ fibril polymorphs. The central hypothesis is that toxic polymorphs promote cytotoxicity by exhibiting faster growth kinetics, disrupting cellular membranes, and inducing higher levels of oxidative stress via reactive oxidative species (ROS) generation. This hypothesis will be tested by pursuing three specific aims: 1) Characterize the molecular structures and growth mechanisms of Aβ fibrils using Raman spectroscopy; 2) Employ novel stimulated Raman chemical imaging methods to directly visualize the interactions between Aβ fibril polymorphs and living cells; and 3) Use electrochemical sensing to directly assess levels of reactive oxygen species (ROS) induced by Aβ fibril polymorphs. Under Aims 1 and 2, a novel methodology pioneered by the Punihaole group called Raman Chemical Imaging, will be used to directly monitor how different fibril polymorphs grow, structurally evolve, and alter the fluidity, integrity, and chemical composition of cellular membranes. In Aim 3, oxidative stress induced by different fibril polymorphs will be monitored. This will be accomplished using fast electrochemical measurements to measure acute and chronic changes in the concentration of ROS generated by the cells. Synergistic coupling of these novel methods is innovative since they together provide structural and chemical information on time scales required to link molecular-level interactions between fibril polymorphs and cellular components with cellular responses, including the production of ROS and rapid release of cytotoxic markers. The proposed work is significant because it builds the foundation of a broader research program that will produce a holistic understanding of how the molecular structure of amyloid fibrils underlies the pathophysiology and clinical symptoms of patients with CAA and AD. Ultimately, the insights obtained will guide treatment strategies and the rational design of drugs to limit the formation of toxic strains of Aβ fibrils, inhibit their aberrant interactions with cells, mitigate oxidative damage, and potentially reverse the loss of cortical tissue and atrophy associated with dementia.