{"paper_id":"83b9e6fb-91c3-4e67-8e5d-82db464ff4e7","body_text":"Abstract\nSignificance Vascular abnormalities may contribute to amyloid-beta accumulation and neurotoxicity in Alzheimer’s disease (AD). Monitoring vascular degeneration as AD progresses is essential. Three-photon fluorescence microscopy (3PM) enables high-resolution deep tissue imaging with minimal invasiveness and photodamage.\nAim This proof-of-concept study established a longitudinal 3P imaging pipeline to quantify vascular and amyloid plaque changes in the APPNL-G-F mouse model.\nApproach A cranial window allowed repeated 3P imaging at four-week intervals beginning at five weeks after surgery. Vessels labelled with Texas-Red were segmented using DeepVess, while plaques labelled with methoxy-XO4 were segmented using custom scripts. Quantitative analyses assessed vascular parameters (diameter, tortuosity, length, inter-vessel distance, total volume) and plaque metrics (radius, total volume).\nResults We imaged the same field over 4 weeks quantifying an overall decrease in vasculature and increase in amyloid plaques between two sessions. Significant changes in vessel diameter, inter-vessel distance, as well as alterations in vessel length and plaques radius were observed. Changes in vessel tortuosity were not significant.\nConclusions We demonstrate the potential of three-photon imaging to track vascular and amyloid-related changes in deep cortical structures. It offers a tool for studying the interplay between vascular and amyloid pathologies in AD, supporting future research into disease mechanisms and therapeutic strategies.\nCompeting Interest Statement\nThe authors have declared no competing interest.\nFootnotes\n↵# Co-First Author\nMinor clarifying changes throughout. Figs 5,6 revised. Two new supplemental figures.","source_license":"CC-BY-4.0","license_restricted":false}