本文選題：顯微鏡　切入點：光片熒光顯微鏡　出處：《浙江大學》2016年博士論文　論文類型：學位論文

【摘要】：隨著生物研究的發(fā)展,越來越多的活體研究需要以更快的速度和更高的分辨率來觀察生物的動態(tài)活動,特別是在免疫細胞蛋白活動追蹤、線蟲胚胎后期發(fā)育以及其他發(fā)展生物學的研究中,迫切需要高時空分辨率的三維成像技術(shù)。光片熒光顯微成像技術(shù)(Light Sheet Fluorescence Microscopy, LSFM或者Selective Plane Illumination Microscopy, SPIM)通過對光片激發(fā)熒光的寬場成像,該技術(shù)綜合了成像的速度和空間分辨率的優(yōu)勢,同時以極小的光損傷和光漂白,廣泛應(yīng)用于生物樣品的長時間動態(tài)活體三維成像。本文圍繞倒置型LSFM技術(shù),結(jié)合具體的生物應(yīng)用,在顯微鏡的搭建、圖像重建和圖像分析等方面展開相關(guān)的技術(shù)探索工作。本文的主要內(nèi)容和貢獻如下：1.在顯微鏡系統(tǒng)的搭建方面,首先采用數(shù)值孔徑高、低搭配的物鏡組合,設(shè)計并搭建了非對稱雙視角倒置光片熒光顯微鏡系統(tǒng),在實現(xiàn)光片掃描采集成像的同時,也實現(xiàn)了平臺掃描采集成像,并對系統(tǒng)的性能進行了測試。其次,針對熒光蛋白的熒光各向異性(Fluorescence Anisotropy, FA),設(shè)計并搭建了熒光各向異性成像的顯微鏡系統(tǒng),并首次對線蟲胚胎進行長時間的FA三維動態(tài)成像,研究了其肌球蛋白在胚胎發(fā)育過程中的磷酸化過程。2.在圖像重建方面,雙視角光片熒光顯微鏡圖像的三維重建往往耗時巨大,本文通過CUDA編程對重建過程中配準和聯(lián)合去卷積算法進行GPU加速,使其適于對批量大數(shù)據(jù)的重建。同時,針對三維斷層相位顯微鏡的圖像重建,構(gòu)建了新的成像系統(tǒng)模型,提出了一種基于全變差約束的新的重建算法。一方面緩解了缺失錐帶來軸向分辨率偏低的問題,另一方面通過稀疏采樣實現(xiàn)了顯微鏡的快速成像。3.在圖像分析方面,對于LSFM采集的線蟲胚胎的熒光圖像,為了解決在胚胎發(fā)育過程中線蟲因扭曲和盤卷而對后續(xù)分析造成的困難,本文首次提出了對線蟲胚胎圖像的半自動拉直算法,通過對不同時刻的線蟲胚胎圖像的拉直,研究了線蟲在胚胎發(fā)育過程中的形態(tài)變化和神經(jīng)細胞遷移。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：TP391.41

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