自适应光学双光子显微镜在活体小鼠脑成像中的应用研究

荧光成像技术具有强大的标记能力、细胞及亚细胞级的分辨率以及广泛的应用领域等优势，是当今生物学研究中的一项重要的研究手段。利用荧光成像技术，在活体实验动物上，用非侵入式的手段进行观测对于获取真实生理学数据、揭示相关生理学机制而言有着重要意义。但是，在厚组织样本如动物大脑中，样本像差、散射等原因使得光学成像质量受到较大影响，限制了其在该样本中的应用。自适应光学技术是一种可以对样本像差进行测量和矫正，从而有效改善成像质量的技术。所以，本文结合双光子成像技术和自适应光学技术，搭建并优化自适应光学双光子成像系统，利用Cx3Cr1-Tdtomato小鼠，在保留颅骨条件下观察大脑中的小胶质细胞形态，并验证该系统在成像质量提升上的有效性。

本文首先基于Shack-Hartmann波前传感器搭建自适应光学双光子成像系统。并基于MATLAB和显微镜控制软件SCANIMAGE编写自适应光学模块控制和计算程序。在荧光溶液样本和胶原蛋白制片中测量和矫正系统像差和物镜矫正环引起的像差，验证像差测量和矫正的有效性和稳定性。由于基于Shack-Hartmann传感器的波前测量方法对数据质量和像差重建算法较为敏感，本文针对不同的数据质量，应用不同的波前重建算法，并在活体样本中实现样本像差的正确测量和矫正。

为了实现微创活体脑成像，本文利用颅骨光学透明化方法，在保留颅骨的情况下进行活体脑成像。利用搭建的自适应光学双光子成像系统，本文对小鼠大脑中的小胶质细胞和血管进行高分辨荧光成像。实验结果显示，自适应光学像差矫正能够在保留颅骨的情况下有效恢复小胶质细胞及血管的精细形态结构，对于揭示它们在神经发育、损伤反应以及疾病过程中的作用机制至关重要。本文基于自适应光学双光子成像系统，对成像性能表现进行系统评估和优化，在活体小鼠大脑中验证了像差测量矫正的有效性，使荧光成像在活体小鼠大脑中的应用得到拓展，为深入理解小胶质细胞和脑血管在神经系统中的功能及其相互作用提供了重要工具。

Fluorescence imaging technology, known for its powerful labeling capabilities, cellular and subcellular resolution, and wide range of applications, is a crucial research tool in modern biological studies. Observing live, awake experimental animals non-invasively using fluorescence imaging is essential for obtaining accurate physiological data and uncovering related mechanisms. However, the quality of optical imaging in thick tissue like animal brains is significantly affected by factors such as refractive indices mismatch and strong scattering, limiting its application in these samples. Adaptive optics as a technology that is able to measures and corrects sample aberrations, can effectively improve imaging quality. Therefore, this paper integrates two-photon imaging and adaptive optics technologies to construct and optimize an adaptive optics two-photon imaging system. And then using Cx3Cr1-Tdtomato mice, we observed the morphology of microglia in the brain and validated the system's effectiveness in enhancing imaging quality.

This paper begins by constructing an adaptive optics two-photon imaging system based on the Shack-Hartmann wavefront sensor. Control and computational programs for the adaptive optics module were then developed using MATLAB and the microscope control software SCANIMAGE. The system's aberrations, as well as those caused by the objective correction ring, were measured and corrected in fluorescent solution samples and collagen slide to validate the effectiveness and stability of aberration measurement and correction. Given the sensitivity of the wavefront measurement based on the Shack-Hartmann sensor to data quality and aberration reconstruction algorithms, this study applies different wavefront reconstruction algorithms for varying data qualities and achieves accurate measurement and correction of sample aberrations in living samples.

Besides, in order to achieve minimally invasive in vivo brain imaging, this paper utilizes a skull optical clearing technique to perform in vivo brain imaging while retaining the skull. Utilizing the adaptive optics two-photon imaging system constructed in this study, we conducted high-resolution fluorescence imaging of microglia and blood vessels in the mouse brain. The experimental results show that adaptive optics aberration correction can effectively restore the fine morphological structure of microglia and blood vessels under the condition of retaining the skull, which is crucial for revealing their mechanisms of action in neural development, injury response, and disease processes. This paper, based on the adaptive optics two-photon imaging system, systematically assesses and optimizes the imaging performance, and verifies the effectiveness of aberration measurement and correction in the living mouse brain, thus expanding the application of fluorescence imaging in the living mouse brain. It provides an important tool for a deeper understanding of the functions of microglia and blood vessels in the nervous system and their interactions.

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