1700nm波段多光子显微成像系统的搭建和优化及其在活体小鼠大脑深层成像中的应用

发布时间：2018-05-01 03:36

本文选题：多光子 + 显微系统　；参考：《深圳大学》2017年硕士论文

【摘要】：多光子显微成像技术因其大的成像深度、高的空间分辨率、小的光毒性,以及三维结构、功能成像的能力,成为诸多研究领域常用的成像手段。最近,1700nm激发的光子成像已经成为深层生物组织成像极具前景的新方案。例如,科学家首次在不破坏小鼠大脑皮层的条件下,可以直接对皮层下结构进行多光子成像。把握这一发展趋势,本论文演示了我们在该波段多光子成像取得的实验进展。本文首先介绍我们基于该成像方案搭建的1700nm波段显微成像系统及系统的主要参数。显微系统的最大视场为280μm×290μm,系统三个维度的最小分辨率分别为:X向0.668μm,Y向0.656μm,Z向2.83μm。接着,我们定量的比较了常用的镓砷磷(GaAsP)光电倍增管(PMT)和砷化镓(GaAs)光电倍增管(PMT)对不同波段信号光探测能力的强弱,得出在712nm波长处,GaAsP和GaAs PMT具有相同的信号探测灵敏度;在小于712nm的波长处,GaAsP PMT的信号探测灵敏度更高;在大于712nm的波长处,GaAs PMT的信号探测灵敏度更高。另外,我们也定量分析了物镜的浸润介质对成像信号的影响。结果显示,对于二次谐波成像,重水浸润使得信号增强了8.3倍;对于三光子成像,重水浸润使得信号增强了24.2倍;对于三次谐波成像,重水浸润使得信号增强了23.9倍。这些测量结果跟我们基于重水和水的吸收谱数据算得的理论结果符合的很好。接下来,在上述系统建造、优化的基础上,文章介绍了我们利用该显微成像系统在活体小鼠的大脑中的深层成像结果。其中,在Texas red染色过的小鼠大脑三光子荧光成像中实现了1700μm的成像深度,超过该波段深层成像已报道过的最大深度。最后,我们总结了多光子深层成像实验中现存的问题以及进一步优化的可行性。
[Abstract]:Because of its large imaging depth, high spatial resolution, small phototoxicity, and the ability of 3D structure and functional imaging, multiphoton microscopic imaging technology has become a common imaging method in many research fields. Recently, photonic imaging excited at 1700 nm has become a promising new scheme for deep tissue imaging. For example, for the first time, scientists can perform multiphoton imaging of subcortical structures directly without destroying the mouse cerebral cortex. To grasp this trend, this paper demonstrates our experimental progress in multiphoton imaging in this band. In this paper, we first introduce the main parameters of 1700nm band microscopic imaging system based on this imaging scheme. The maximum field of view of the system is 280 渭 m 脳 290 渭 m. The minimum resolution of the three dimensions of the system is: X, 0.668 渭 m, Y, 0.656 渭 m, Z, 2.83 渭 m, respectively. Then, we quantitatively compare the intensity and weakness of the common photomultiplier tube (PMTT) and the photomultiplier tube (PMTT) to the signal light detection ability of different bands. It is concluded that GaAsP and GaAs PMT have the same signal detection sensitivity at 712nm wavelength. The signal detection sensitivity of GaAsP PMT is higher than that of 712nm, and that of GaAs PMT is higher than that of 712nm. In addition, we also quantitatively analyze the effect of the objective lens wetting medium on the imaging signal. The results show that for the second harmonic imaging, heavy water infiltration increases the signal by 8.3 times; for three-photon imaging, the signal is enhanced by 24.2 times; for the third harmonic imaging, the signal is enhanced by 23.9 times. These measurements agree well with our theoretical results based on absorption spectra of heavy water and water. Then, on the basis of the above system construction and optimization, we present the deep imaging results of the microimaging system in the brain of living mice. Among them, the imaging depth of 1700 渭 m is achieved in the three-photon fluorescence imaging of mouse brain stained by Texas red, which exceeds the maximum depth reported in the deep imaging of this band. Finally, we summarize the existing problems in multiphoton deep imaging experiments and the feasibility of further optimization.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q42;TN152

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