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). Monitoring 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 proof-of-concept study established a longitudinal 3P imaging pipeline to quantify vascular and amyloid plaque changes in the APPNL-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, tortuosity, length, inter-vessel distance, total volume) and plaque metrics (radius, total volume).

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.

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. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵# Co-First Author Minor clarifying changes throughout. Figs 5,6 revised. Two new supplemental figures.