In this study we therefore multiplexed TIMS MSI with differential LCO staining for hyperspectral imaging of polymorphic Aβ at the single plaque level. Following MSI acquisition, we performed LCO double staining using q-FTAA and h-FTAA on the same tissue sections (Figure ). Both probes have different emission properties, allowing hyperspectral delineation of differential LCO binding (Figure ). Here, q-FTAA staining was chosen to allow visualization of highly aggregated, mature fibrillary structures within Aβ plaques, while in contrast, immature, protofibrillar Aβ intermediates were visualized through preferential h-FTAA binding as illustrated by crosssectional emission profiles (Figure ) . The two fluorophores were imaged together using a hyperspectral setup, allowing subsequent image segmentation into more and less aggregated Aβ using linear unmixing-based image analysis (Figure ).