Nonlinear optical (NLO) microscopy has become an indispensable tool in biological research owing to its several unique advantages over the conventional optical microscopy. First, the multiple nonlinear optical signals enable multimodal imaging with high specificity and selectivity. Each specific nonlinear optical effect can be used to extract different information of the tissue structures and functional properties. Second, nonlinear optical microscopy provides a large penetration depth by using near infrared light of excitation. Furthermore, NLO microscopy has inherent three-dimensional (3D) imaging capabilities and introduces less photobleaching and photo-damage to the specimen. These unique features of NLO microscopy make it an ideal choice for in vivo brain imaging of small animals....[ Read more ]

Nonlinear optical (NLO) microscopy has become an indispensable tool in biological research owing to its several unique advantages over the conventional optical microscopy. First, the multiple nonlinear optical signals enable multimodal imaging with high specificity and selectivity. Each specific nonlinear optical effect can be used to extract different information of the tissue structures and functional properties. Second, nonlinear optical microscopy provides a large penetration depth by using near infrared light of excitation. Furthermore, NLO microscopy has inherent three-dimensional (3D) imaging capabilities and introduces less photobleaching and photo-damage to the specimen. These unique features of NLO microscopy make it an ideal choice for in vivo brain imaging of small animals. However, high-resolution and deep-brain imaging in a minimally invasive manner still remains a great challenge due to the light nature of aberration and scattering, which largely depends on the kinds of surgical preparations for optical access to the brain.

Since the NLO microcopy has such advantages and challenges, my thesis work mainly focuses on advancing the NLO microscopy technologies, and applying them to morphological and functional study of mice brain under physiological and pathological conditions. Firstly, we introduce a near-infrared (NIR) fluorescence two-photon microscopy for in vivo deep-brain imaging in a mouse model of Alzheimer’s disease, which is based on a newly-developed NIR probe of amyloid plaques. Secondly, we develop an adaptive optics (AO) two-photon microscopy for high-resolution brain imaging through the minimally invasive skull window. An optimized AO configuration and an innovative wavefront sensing algorithm are developed, allowing us to perform deep-brain cellular imaging at high resolution. Thirdly, we apply the AO two-photon microscopy to investigate the neuronal and microglial functions under both normal and pathological conditions. Fourthly, we develop a multimodal optical imaging system enabling both mesoscopic and microscopic imaging of fast neural activities and the concurrent vascular responses, which allows us to investigate the interplay of neural activities and vascular hemodynamics under different brain states. Fifthly, we develop an adaptive optics two-photon endomicroscopy for high-resolution in vivo imaging of deep brain, which enables the recovery of diffraction-limited resolution over large imaging volume in deep brain. Using an AO-assisted random-access multiplane imaging technique, we achieve near-simultaneous calcium imaging of separately distributed somata and dendrites in the hippocampus of awake behaving mice.