双光子荧光显微镜扫描控制与成像系统研究

	双光子荧光显微镜扫描控制与成像系统研究
其他题名	Study of Scanning Control and Imaging System of Two Photon Fluorescence Microscopy
	刘云
学位类型	硕士
导师	熊大曦
	2015-10
学位授予单位	中国科学院大学
学位专业	光学工程
关键词	双光子共聚焦激光扫描共振扫描畸变校正三维成像
摘要	双光子荧光显微镜（Two Photon Fluorescence Microscopy）是研究活体生物组织和细胞结构的有效技术手段和必备的大型科学仪器，在生物医学领域得到了广泛应用。该系统应用双光子吸收原理来激发样品荧光，荧光激发概率随着远离焦平面而成幂指数下降，所以只会激发焦平面附近很小的一个区域，具有很高的成像分辨率和信噪比；同时在纵向上也只有很薄的一层荧光样品被激发，那么在Z轴方向移动焦平面可以进行层切扫描，从而实现三维成像。由于使用近红外光激发，更容易穿透样品，成像深度可达1mm。双光子荧光显微镜显微镜，在细胞生物学、神经生物学、脑科学等生物及医学领域具有广阔的应用。本文首先从硬件电路设计，控制算法设计，以及软件界面设计三个方面去论述了双光子激光扫描显微技术，然后针对扫描系统的畸变设计了校正算法，并从仿真数据和实验结果两方面去展示了工作的进展，完成了原理样机的搭建以及测试。主要工作包括以下几个部分：首先，设计并搭建出了双光子激光扫描显微系统的硬件控制系统。双光子荧光显微镜是一种激光扫描显微系统，扫描控制与信号采集是整个系统的关键技术之一。本文研究了基于数据采集卡的硬件控制方案，通过数据采集卡对扫描系统的控制，并且采集光电倍增管(PMT)获得的信号，实现图像的重建。其次，编写了控制软件以及人机交互界面。为了实现对系统各硬件资源的协调调度，采用LabVIEW作为编程语言完成了控制软件的编写，能够实现对扫描模块、CCD模块、激光模块、三维平台、物镜扫描等模块的统一控制，并且采集PMT获取的信号，通过在程序中嵌入matlab脚本代码来实现扫描与采集的精确匹配以及畸变校正工作，实时得到正确的样品图像，512X512像素图像成像速度达到每秒30帧。并且，利用搭建完整的双光子显微系统对小牛肺动脉内皮细胞（bovine pulmonary artery endothelial, BPAE）进行扫描，重建出二维图像。同时由于双光子激光扫描显微镜能对样品进行深层成像，并且得到优异的焦平面信号，利用压电驱动器在Z方向进行层切扫描，得到不同深度的样品信息，将这些层切图像进行重建，得到了样品的三维图像。最后，完成了共振扫描畸变的校正算法设计。为了获得更快的成像速度，双光子显微系统采用了共振振镜作为X方向的扫描振镜。但是共振式的扫描带来了成像的畸变，为了评估畸变，使用系统对一块条纹等宽的朗奇光栅成像，通过对边缘条纹和中间条纹宽度的对比得到了系统的畸变函数。基于系统的畸变函数，采用曲线拟合的方法对图像进行校正，最后得到了无畸变的图像。在文章的最后，对所做的研究内容进行了总结和分析，并对双光子荧光显微镜进行了展望。
其他摘要	Two photon fluorescence microscopy (TPFM) is an effective tool in the study of living tissues and cell structures. The system applies two photon absorption principle to excite the sample fluorescence. The fluorescence excitation probability decreases with the distance away from the focal plane, so that only a very small area of the focal plane is excitated, which has a high imaging resolution and SNR (Signal Noise Ratio). Two photon fluorescence microscopy, in the cell biology, neuroscience, brain science and other biological and medical fields have broad application. In this paper, there are three aspects to discuss TPFM--- control project design (hardware and algorithm), software interface design, and distortion correction algorithm design. The system is demonstrated from the simulation data and experimental results. Principle prototype and its testing are completed. The main work includes the following parts: First, the hardware control system of the TPFM is designed and built. TPFM is a laser scanning microscope system. Scanning control system and signal acquisition are the key technologies. In this paper, the hardware control module is built based on data acquisition card, and the signal is acquired by the photo multiplier tube (PMT). Secondly, the control program and human-computer interaction (HCI) are written. In order to comply with the system, the LabVIEW is used to build the control software. It can realize the unified control of the scanning module, CCD module, laser module, 3D platform, Objective scanner and other modules. The MATLAB script is embedded in the program to process image signal. As the same time, bovine pulmonary artery endothelial (BPAE) cell was scanned to image, and the 2D image of BPAE was reconstructed. Because TPFM can image deeply at the sample, and obtain thin focal plane signal，it can obtain the different depth information of the sample by using 压电驱动器 in the Z direction to scan. The images of these layers were reconstructed to obtain 3D image of the sample; Finally, the correction algorithm of the resonant scanning distortion is designed. Due to nonlinearity of resonant scanners' speed, images are distorted in laser scanning imaging systems based on resonant scanner. An efficient algorithm to correct the distortion is developed, by fitting the distortion curve. The core of correction algorithm is to restore the image using distortion function; At the end of the paper, the research contents are summarized and analyzed, and the prospect of two photon fluorescence microscopy is presented.
语种	中文
文献类型	学位论文
条目标识符	http://ir.ciomp.ac.cn/handle/181722/49319
专题	中科院长春光机所知识产出
推荐引用方式 GB/T 7714	刘云. 双光子荧光显微镜扫描控制与成像系统研究[D]. 中国科学院大学,2015.

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