倒置型光片荧光显微成像及图像分析技术研究

发布时间：2018-03-22 15:20

本文选题：显微镜　切入点：光片荧光显微镜　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：随着生物研究的发展,越来越多的活体研究需要以更快的速度和更高的分辨率来观察生物的动态活动,特别是在免疫细胞蛋白活动追踪、线虫胚胎后期发育以及其他发展生物学的研究中,迫切需要高时空分辨率的三维成像技术。光片荧光显微成像技术(Light Sheet Fluorescence Microscopy, LSFM或者Selective Plane Illumination Microscopy, SPIM)通过对光片激发荧光的宽场成像,该技术综合了成像的速度和空间分辨率的优势,同时以极小的光损伤和光漂白,广泛应用于生物样品的长时间动态活体三维成像。本文围绕倒置型LSFM技术,结合具体的生物应用,在显微镜的搭建、图像重建和图像分析等方面展开相关的技术探索工作。本文的主要内容和贡献如下：1.在显微镜系统的搭建方面,首先采用数值孔径高、低搭配的物镜组合,设计并搭建了非对称双视角倒置光片荧光显微镜系统,在实现光片扫描采集成像的同时,也实现了平台扫描采集成像,并对系统的性能进行了测试。其次,针对荧光蛋白的荧光各向异性(Fluorescence Anisotropy, FA),设计并搭建了荧光各向异性成像的显微镜系统,并首次对线虫胚胎进行长时间的FA三维动态成像,研究了其肌球蛋白在胚胎发育过程中的磷酸化过程。2.在图像重建方面,双视角光片荧光显微镜图像的三维重建往往耗时巨大,本文通过CUDA编程对重建过程中配准和联合去卷积算法进行GPU加速,使其适于对批量大数据的重建。同时,针对三维断层相位显微镜的图像重建,构建了新的成像系统模型,提出了一种基于全变差约束的新的重建算法。一方面缓解了缺失锥带来轴向分辨率偏低的问题,另一方面通过稀疏采样实现了显微镜的快速成像。3.在图像分析方面,对于LSFM采集的线虫胚胎的荧光图像,为了解决在胚胎发育过程中线虫因扭曲和盘卷而对后续分析造成的困难,本文首次提出了对线虫胚胎图像的半自动拉直算法,通过对不同时刻的线虫胚胎图像的拉直,研究了线虫在胚胎发育过程中的形态变化和神经细胞迁移。
[Abstract]:With the development of biological research, more and more living research needs to observe the dynamic activity of biology with faster speed and higher resolution, especially in the tracking of immunocyte protein activity. There is an urgent need for three-dimensional imaging with high spatial and temporal resolution in the study of post-embryonic development and other developmental biology of nematodes. Photoluminescence imaging techniques such as light Sheet Fluorescence microscopy, LSFM or Selective Plane Illumination microscopy- (SPIMI) excite fluorescence wide-field imaging through photophore. This technique combines the advantages of imaging speed and spatial resolution, at the same time, with minimal light damage and photobleaching, it is widely used in living 3D imaging of biological samples for a long time. Combined with specific biological applications, the related technical exploration work is carried out in the aspects of microscope construction, image reconstruction and image analysis. The main contents and contributions of this paper are as follows: 1. In the construction of microscope system, the numerical aperture is high. The low collocation objective lens combination, designed and built the asymmetrical double angle inverted light slice fluorescence microscope system, not only realized the optical slice scanning acquisition imaging, but also realized the platform scanning acquisition imaging, and carried on the test to the system performance. Aiming at fluorescence anisotropy of fluorescent protein, a fluorescence anisotropic imaging microscope system was designed and set up. For the first time, FA dynamic imaging of nematode embryos was carried out for a long time. The phosphorylation process of myosin in embryonic development was studied. 2. In the aspect of image reconstruction, the 3D reconstruction of fluorescence microscope image with double viewing angle often takes a lot of time. In this paper, the registration and joint deconvolution algorithm are accelerated by CUDA programming to make it suitable for the batch reconstruction of big data. At the same time, a new imaging system model is constructed for the image reconstruction of 3D tomographic phase microscope. In this paper, a new reconstruction algorithm based on total variation constraint is proposed. On the one hand, the problem of low axial resolution caused by missing cone is alleviated, on the other hand, the rapid imaging of microscope by sparse sampling is realized. For the fluorescence images of nematode embryos collected by LSFM, in order to solve the difficulties caused by the twisting and rewinding of nematodes in the course of embryonic development, a semi-automatic straightening algorithm for nematode embryo images is proposed for the first time in this paper. The morphological changes and neuronal migration of nematodes during embryonic development were studied by straightening the embryonic images of nematodes at different times.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TP391.41

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