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

Sep 29, 2026

Longitudinal three-photon fluorescence microscopy is used to monitor vascular degeneration and amyloid plaque evolution during Alzheimer’s disease progression. A cranial window enables repeated imaging every four weeks from five weeks post-surgery in the APPNL-G-F mouse model. Texas-Red labeled vessels are segmented with DeepVess, while methoxy-XO4 labeled plaques use custom segmentation scripts. Quantification tracks vessel diameter, tortuosity, length, inter-vessel distance, volume, and plaque radius and volume, revealing decreased vasculature alongside increased plaques over time.

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Abstract

Introduction

RESEARCH PAPER Longitudinal three-photon imaging for tracking amyloid plaques and vascular degeneration in a mouse model of Alzheimer’s disease Eline Stas ,† Mengke Yang,† Simon Schultz , and Mary Ann Go* Imperial College London, Department of Bioengineering and Centre for Neurotechnology, London, United Kingdom | ABSTRACT. 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 enables highresolution deep tissue imaging with minimal invasiveness and photodamage. Aim: In this proof-of-concept study, we 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 4-week intervals beginning at 5 weeks after surgery. Vessels labeled with Texas-Red were segmented using DeepVess, whereas plaques labeled 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) .
Results: We imaged the same field over 4 weeks, quantifying an overall decrease in vasculature and an increase in amyloid plaques between two sessions. Significant changes in vessel diameter, inter-vessel distance, and alterations in vessel length and plaque radius were observed. Changes in vessel tortuosity were not significant. Conclusions: 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.
© The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. [DOI: 10.1117/1.JBO.31.1.016004]
Keywords: three-photon microscopy; multicolour; longitudinal; vasculature; amyloid plaques; image segmentation
Paper 250249GR received Aug. 12, 2025; revised Nov. 25, 2025; accepted Dec. 15, 2025; published Jan.
2, 2026 . | | --- | | 1 Introduction
Alzheimer’s disease (AD) is the leading cause of dementia worldwide, impacting the quality of human life and imposing significant healthcare costs.1 A widely accepted theory, the amyloid hypothesis, suggests that cognitive decline in AD patients is partly due to the accumulation of extracellular amyloid beta (Aβ) peptides in the brain.2 This accumulation is believed to trigger a cascade of pathological events, including the formation of intracellular neurofibrillary tangles composed of hyperphosphorylated tau and subsequent neuronal loss.3,4 Nonetheless, although | *Address all correspondence to Mary Ann Go, [m.go@imperial.ac.uk](m.go@imperial.ac.uk)[ ](m.go@imperial.ac.uk)†Co-first author Journal of Biomedical Optics 016004-1 January 2026 • Vol. 31(1) Stas et al.: Longitudinal three-photon imaging for tracking amyloid plaques. . . amyloid pathologies have been the primary focus, emerging studies highlight the vascular system as a major factor in the progression of the disease.5 To meet its high energy demands, the brain relies on a constant supply of oxygen and energy substrates. As the brain has no long-term energy storage, regional cerebral blood flow is precisely regulated to align with the local energy requirements of nervous tissue. Any disruption in this adaptive process or in the delivery of necessary substrates can lead to imbalances in homeostasis, tissue damage, and functional impairment.6 These vascular abnormalities have been shown to not only contribute to Aβ accumulation, but also exacerbate neurotoxicity in AD.7–9 It is believed that the i

FAQs

What was the study’s main goal?

To establish a proof-of-concept longitudinal three-photon (3P) imaging pipeline that quantifies changes in vascular structure and amyloid plaques in a mouse model of Alzheimer’s disease.

How was longitudinal 3P imaging performed in the mouse model?

A cranial window enabled repeated 3P imaging at 4-week intervals starting at 5 weeks after surgery, imaging the same field over the study period.

Which results showed significant changes over time?

Vessel diameter, inter-vessel distance, vessel length, and plaque radius changed significantly, while changes in vessel tortuosity were not significant.

Sep 29, 2026

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