Longitudinal three-photon imaging for tracking amyloid plaques and vascular degeneration in a mouse model of Alzheimer’s disease

Significance Vascular abnormalities may contribute to amyloid-beta accumulation and neurotoxicity in Alzheimer’s disease (AD). The ability to monitor vascular degeneration as AD progresses is essential. Three-photon fluorescence microscopy (3PM) enables high-resolution deep tissue imaging with minimal invasiveness and photodamage. Aim This study established a longitudinal 3P imaging pipeline to quantify vascular degeneration and amyloid plaque for-mation in the APP NL-G-F mouse model. Approach 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, density, tortuosity, length, inter-vessel distance) and plaque metrics (radius, nearest plaque-to-vessel distance). Results We imaged the same field over 4 weeks quantifying a decrease in vasculature and increase in amyloid plaque formation with age. Significant decreases in vessel diameter, increases in inter-vessel distance, and alterations in vessel length were observed. Changes in vessel tortuosity, plaque radius, and plaque proximity to vessels were not significant. Conclusions This pipeline tracks vascular remodeling and amyloid pathology 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.