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The original HTML version of this Article was updated shortly after publication because the previous HTML incorrectly linked to a Supplementary Figures file.

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 devel

Impairment of microglial clearance activity contributes to beta-amyloid (Aβ) pathology in Alzheimer's disease (AD). While the transcriptome profile of microglia directs microglial functions, how the microglial transcriptome can be regulated to alleviate AD pathology is largely unknown. Here, we show that injection of interleukin (IL)-33 in an AD transgenic mouse model ameliorates Aβ pathology by reprogramming microglial epigenetic and transcriptomic profiles to induce a microglial subpopulation with enhanced phagocytic activity. These IL-33-responsive microglia (IL-33RMs) express a distinct transcriptome signature that is highlighted by increased major histocompatibility complex class II genes and restored homeostatic signature genes. IL-33-induced remodeling of chromatin accessibility and PU.1 transcription factor binding at the signature genes of IL-33RM control their transcriptome reprogramming. Specifically, disrupting PU.1-DNA interaction abolishes the microglial state transition and Aβ clearance that is induced by IL-33. Thus, we define a PU.1-dependent transcriptional pathway that drives the IL-33-induced functional state transition of microglia, resulting in enhanced Aβ clearance. © 2020 The Author(s) Lau et al. show that interleukin-33 (IL-33) enhances microglial amyloid-beta clearance by inducing a subpopulation of MHC-II+ phagocytic microglia, which is, in turn, controlled by PU.1-dependent transcriptome reprogramming. Thus, the authors reveal an IL-33-PU.1 axis involved in transcriptional regulation that promotes beneficial microglial functions in Alzheimer's disease. © 2020 The Author(s)

Hippocampal synaptic plasticity is important for learning and memory formation. Homeostatic synaptic plasticity is a specific form of synaptic plasticity that is induced upon prolonged changes in neuronal activity to maintain network homeostasis. While astrocytes are important regulators of synaptic transmission and plasticity, it is largely unclear how they interact with neurons to regulate synaptic plasticity at the circuit level. Here, we show that neuronal activity blockade selectively increases the expression and secretion of IL-33 (interleukin-33) by astrocytes in the hippocampal cornu ammonis 1 (CA1) subregion. This IL-33 stimulates an increase in excitatory synapses and neurotransmission through the activation of neuronal IL-33 receptor complex and synaptic recruitment of the scaffold protein PSD-95. We found that acute administration of tetrodotoxin in hippocampal slices or inhibition of hippocampal CA1 excitatory neurons by optogenetic manipulation increases IL- 33 expression in CA1 astrocytes. Furthermore, IL-33 administration in vivo promotes the formation of functional excitatory synapses in hippocampal CA1 neurons, whereas conditional knockout of IL- 33 in CA1 astrocytes decreases the number of excitatory synapses therein. Importantly, blockade of IL-33 and its receptor signaling in vivo by intracerebroventricular administration of its decoy receptor inhibits homeostatic synaptic plasticity in CA1 pyramidal neurons and impairs spatial memory formation in mice. These results collectively reveal an important role of astrocytic IL-33 in mediating the negative-feedback signaling mechanism in homeostatic synaptic plasticity, providing insights into how astrocytes maintain hippocampal network homeostasis. © 2021 National Academy of Sciences. All rights reserved.

Optical deep-brain imaging in vivo at high resolution has remained a great challenge over the decades. Two-photon endomicroscopy provides a minimally invasive approach to image buried brain structures, once it is integrated with a gradient refractive index (GRIN) lens embedded in the brain. However, its imaging resolution and field of view are compromised by the intrinsic aberrations of the GRIN lens. Here, we develop a two-photon endomicroscopy by adding adaptive optics based on direct wavefront sensing, which enables recovery of diffraction-limited resolution in deep-brain imaging. A new precompensation strategy plays a critical role to correct aberrations over large volumes and achieve rapid random-access multiplane imaging. We investigate the neuronal plasticity in the hippocampus, a critical deep brain structure, and reveal the relationship between the somatic and dendritic activity of pyramidal neurons.

Two-photon microscopy has become the method of choice for in vivo brain imaging in neuroscience research during the past decades owing to its inherent sectioning capability and large penetration depth in scattering tißues. By integrating with a gradient refractive index (GRIN) lens that implanted into the brain, two-photon microendoscopy further extends the imaging depth down to subcortical regions. However, the imaging resolution and field of view (FOV) are compromised due to the intrinsic aberrations of the inserted GRIN lens. Here, we developed an adaptive optics (AO) two-photon microendoscopy based on direct wavefront sensing that can measure and correct the aberrations of GRIN lens during in vivo brain imaging. Using our system, the diffraction-limited resolution was restored and the fine structures such as dendritic spines of hippocampal CA1 neurons can be clearly resolved over a much extended FOV. © COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.

Chronic in vivo optical imaging of the spinal cord is an effective way to study the biological processes during and after spinal cord injury (SCI) in mouse models. It normally relies on an implanted spinal chamber to provide continuous optical access to the spinal cord. However, the chronic window consists of multiple layers of transparent materials with various optical properties and irregular thickness, which induce large optical aberration. Therefore, the image quality of multiphoton microscopy as well as the precision of femtosecond laser axotomy were dramatically degraded. In this work, we developed an adaptive optics (AO) microscope system integrating stimulated Raman scattering (SRS) and twophoton excited fluorescence (TPEF). Using our system, the aberrations induced by the spinal cord window were measured and compensated accordingly, enabling both high-resolution imaging and precise laser axotomy of the mouse spinal cord. © COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.

Non-invasive retinal imaging has greatly facilitated the research of eye disease and neurodegenerative disorders in the central nervous system (CNS). Two-photon microscopy is a powerful tool for in vivo imaging of mouse retina because it provides intrinsic optical sectioning capability and the infrared laser is less likely to excite the photoreceptors. However, the dilated mouse eye has large optical aberrations, which must be corrected to achieve high-resolution or even diffraction-limited imaging. Here, we developed an adaptive optics (AO) two-photon microscope for in vivo imaging of retinal neurons through the eyeball of living mouse. We used the two-photon excited fluorescence signal of retina as the guide star to measure and correct the aberration of mouse eye. After AO correction, the fluorescence signal was increased by at least fivefold and the fine structures such as axons of retinal ganglion cells (RGC) were clearly resolved. To take advantage of the non-invasive high-resolution imaging, we demonstrated functional calcium imaging of RGC responding to the light stimulations. © 2020 SPIE.

In vivo fundus imaging offers non-invasive access to neuron structures and biochemical processes in the retina. However, optical aberrations of the eye degrade the imaging resolution and prevent visualization of subcellular retinal structures. We developed an adaptive optics two-photon excitation fluorescence microscopy (AO-TPEFM) system to correct ocular aberrations based on a nonlinear fluorescent guide star and achieved subcellular resolution for in vivo fluorescence imaging of the mouse retina. With accurate wavefront sensing and rapid aberration correction, AO-TPEFM permits structural and functional imaging of the mouse retina with submicron resolution. Specifically, simultaneous functional calcium imaging of neuronal somas and dendrites was demonstrated. Moreover, the time-lapse morphological alteration and dynamics of microglia were characterized in a mouse model of retinal disorder. In addition, precise laser axotomy was achieved, and degeneration of retinal nerve fibres was studied. This high-resolution AO-TPEFM is a promising tool for non-invasive retinal imaging and can facilitate the understanding of a variety of eye disease