In Vivo near Non‑Invasive Imaging of the Brain and Retina in Physiological and Pathological states

Abstract

The retina and brain are two of the most important organs for understanding neural function and disease. The retina serves as a highly accessible extension of the central nervous system (CNS), providing direct insight into neuronal, glial, and vascular interactions, while the brain represents the core of cognition and behavior. However, both tissues remain exceptionally challenging to study in vivo because of optical aberrations from the eye lens and strong light scattering by the skull. Non-invasive, longitudinal imaging of these systems is critical, as it allows researchers to monitor neural activity, microvascular dynamics, and pathological processes within their native physiology—something that is not achievable with ex vivo or fixed specimens. Nonlinear optical (NLO) microscopy, particularly when combined with adaptive optics (AO), offers powerful solutions to overcome these barriers by extending imaging depth, reducing phototoxicity, and mitigating tissue aberrations.

In this dissertation, we first optimized the AO-TPEFM system to achieve longitudinal imaging in the silicone oil–induced ocular hypertension (SOHU) glaucoma model at subcellular resolution. Boundary effects caused by the silicone oil droplet impaired wavefront sensing, which we overcame by reducing the laser numerical aperture to minimize interference. This correction enabled high-fidelity AO performance and long term visualization of capillaries, microglia, and retinal ganglion cells (RGCs). Using genetically encoded calcium indicators expressed in Brn3b-Cre mouse lines, we monitored functional RGC activity and achieved single-cell resolution without ambiguity. Moreover, we identified three distinct glaucoma phenotypes with similar intraocular pressure but divergent progression: normal glaucoma, with peripheral RGC loss by 7.5 weeks post-injection (wpi); under glaucoma, with preserved RGCs up to 7.5 wpi; and acute glaucoma, marked by severe RGC and microvascular loss as early as 0.5 wpi. Phenotype-specific analysis further revealed early vascular impairment and microglial responses preceding neuronal degeneration, underscoring the heterogeneity of glaucoma pathology and highlighting the utility of AO-TPEFM for longitudinal retinal studies.

For brain imaging, we systematically compared existing transcranial optical windows using TPEFM. To overcome limitations of skull thinning and optical clearing, we developed a hybrid thinned-clearing (TC) window, which shortened clearing time and provided stronger fluorescence signals. Longitudinal studies revealed that progressive skull regrowth universally degraded imaging quality across all transcranial windows. Using AO-TPEFM and two-photon ALPHA-FSS, we further quantified aberrations and resolution loss with depth: fine structures such as microglial processes dropped below detectable signal-to-background ratios beyond ~100 μm, whereas somata remained resolvable past 300 μm. Importantly, regrowth impaired AO guide-star detection, limiting high-resolution imaging depth.

Date of Award	2025
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Jianan QU (Supervisor)