大面积高速非线性光学显微成像技术以及应用的研究

发布时间：2018-03-18 22:47

本文选题：非线性光学显微成像技术　切入点：双光子激发荧光　出处：《华中科技大学》2012年博士论文　论文类型：学位论文

【摘要】：基于双光子吸收和二次谐波效应的非线性光学显微成像技术,具有高分辨率,较深的成像深度,三维层析成像能力和低光损伤等优势,为生物学领域的研究提供了有力的工具。但是,常规非线性光学显微镜较小的成像视场,较低的成像速率以及体积庞大、笨重的成像器件制约了其更为广泛的应用。本文即围绕上述问题展开研究。搭建大面积非线性光学显微成像系统,并对该系统的具体应用做了深入的研究。利用搭建的大面积成像系统,对不同时间节点尾静脉注射B16细胞的C57BL/6小鼠离体肺组织,进行了无标记的自发荧光和二次谐波成像。图像的形态学分析和量化分析表明,大面积非线性光学显微成像系统可以成功监测小鼠肺部黑色素瘤转移灶的产生和发展。另外,利用搭建的大面积成像系统,成功实现了完整的小脑和脑干切片的二次谐波成像。研究证明,二次谐波图像不仅可以提供不同脑区高分辨率的形态结构信息,而且可以对小脑不同脑区的神经元密度进行量化。搭建视频速率多通道探测非线性光学显微成像系统。本文解决了多边形转镜的扫描控制,线同步脉冲整形以及电压信号衰减等硬件相关难题,设计了基于LabVIEW平台的多通道数据采集和存储的程序框架,并测试了系统的性能参数。视频速率成像系统的横向和轴向分辨率分别为O.42μm和1.6μm(XLPLN25×WMP/1.05 NA, Olympus),单个视场扫描成像速率30帧/秒,大面积扫描成像速率10帧/秒,轴向扫描成像速率6帧/秒。另外,利用搭建的成像系统对多色标记的细胞进行了多通道成像,并实现了小鼠皮窗中树突细胞的动态监测。研究结果表明,视频速率非线性光学显微成像系统,可以实现快速多探测通道成像,为活体内细胞与细胞相互作用的监测提供了全新的研究工具。为了更好的满足活体成像的需求,对适用于非线性光学显微成像系统的光纤耦合器进行研究。结合光纤化非线性光学显微成像的特点,提出了理想的光纤耦合器应具有的特性：无畸变传输飞秒脉冲激光,较高的荧光收集效率,激发光和荧光通过耦合器时的耦合特性相反便于将激发光和荧光分离。鉴于大模场无截止单模传输光子晶体光纤(LMA-ESM-PCF)具有较大的模场,无截止单模传输和低非线性效应的特点,使用有效折射率方法和弱耦合理论分析LMA-ESM-PCF耦合器激发光和400nm-650nm波段荧光的耦合特性。另外,详细讨论了磨削工艺制作的LMA-ESM-PCF耦合器耦合效率的可调谐特性。研究分析发现,合理的控制西根光纤纤芯片之间的距离和两根光纤之间的水平夹角,可以制作出适用于光纤化非线性光学显微成像系统的LMA-ESM-PCF耦合器。
[Abstract]:Nonlinear optical microscopic imaging technology based on two-photon absorption and second-harmonic effect has the advantages of high resolution, deep imaging depth, three-dimensional tomography ability and low light damage. But the conventional nonlinear optical microscope has smaller imaging field, lower imaging rate and larger volume. The clunky imaging device restricts its wider application. A large area nonlinear optical microscopic imaging system was set up and its specific application was studied. The lung tissue of C57BL / 6 mice injected with B16 cells at different time points was injected into the tail vein of C57BL / 6 mice at different time points by using the large area imaging system. Unlabeled autofluorescence and second harmonic imaging were performed. Morphological and quantitative analysis of the image showed that the large area nonlinear optical microscopic imaging system could successfully monitor the generation and development of metastatic foci of mouse lung melanoma. The second harmonic imaging of the whole cerebellum and brain stem slices has been successfully realized by using a large area imaging system. It is proved that the second harmonic imaging can not only provide high resolution morphological and structural information in different brain regions, Furthermore, the density of neurons in different brain regions of the cerebellum can be quantified. A video rate multi-channel detection nonlinear optical microscopic imaging system is built. In this paper, the hardware problems such as scanning control of polygon mirror, linear synchronous pulse shaping and voltage signal attenuation are solved. The program framework of multi-channel data acquisition and storage based on LabVIEW platform is designed, and the performance parameters of the system are tested. The horizontal and axial resolutions of video rate imaging system are 0. 42 渭 m and 1. 6 渭 m XLPLN25 脳 WMP/1.05 NA, Olympus, and a single field of view scan imaging rate of 30 frames per second. A large area scanning imaging rate of 10 frames per second and an axial scanning rate of 6 frames per second. In addition, the multichannel imaging of multicolor labeled cells was performed using the built imaging system. The dynamic monitoring of dendritic cells in mouse skin window is realized. The results show that the video rate nonlinear optical microscopic imaging system can realize fast multi-channel imaging. It provides a new research tool for the monitoring of cell-cell interaction in vivo. In order to better meet the need of in-vivo imaging, the optical fiber coupler suitable for nonlinear optical microscopic imaging system is studied. The characteristics of the ideal fiber coupler are put forward: the femtosecond pulse laser is transmitted without distortion, and the fluorescence collection efficiency is higher than that of the optical fiber coupler. The coupling characteristics of excitation light and fluorescence passing through coupler facilitate the separation of excitation light and fluorescence. Considering that large mode field photonic crystal fiber (LMA-ESM-PCF) has the characteristics of large mode field, no cut-off single mode transmission and low nonlinear effect. The effective refractive index method and weak coupling theory are used to analyze the coupling characteristics of excitation light and 400nm-650nm fluorescence in LMA-ESM-PCF coupler. In addition, the tunable characteristics of coupling efficiency of LMA-ESM-PCF coupler fabricated by grinding process are discussed in detail. The LMA-ESM-PCF coupler suitable for optical nonlinear optical microscopic imaging system can be made by reasonably controlling the distance between the optical fiber chips and the horizontal angle between the two optical fibers.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.51

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