单个活细胞多功能高灵敏实时荧光显微成像研究
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摘要
单个活细胞实时检测具有重要的理论意义和实际应用价值,荧光显微成像技术因灵敏度高、特异性好、易实现“可视化”而成为该领域最具发展潜力的方法。然而由于活细胞样品的复杂性,真正适用的方法仍面临挑战。本论文着力搭建一套适用于单个活细胞实时检测和生物单分子研究的多功能、高灵敏、实时荧光显微成像系统,并探索该系统在具体生命科学问题中的应用。
首先,搭建了一套多功能高灵敏实时荧光显微成像系统,并发展相关图像处理方法。利用像增强型CCD实现了实时荧光成像,灵敏度高、速度快、对细胞光损伤小,适合于实时检测活细胞内分子、离子分布和变化;实现了物镜型全内反射荧光成像,提高纵向分辨率、图像信噪比和灵敏度,适合于实时追踪细胞膜附近单分子动态过程;实现了荧光共振能量转移成像,分辨率突破衍射极限达到纳米量级,适合于研究活细胞内分子相互作用或构象变化;扩展了荧光比率、双标记双/三通道、荧光+微分干涉相差、三维层切等多种活细胞实时成像功能。各功能可单独或联合使用,为生命科学研究提供多种先进技术手段。
其次,以胸腺细胞凋亡研究为例探索了该系统的具体应用,并发展相关量化分析方法。首次在单个活细胞水平实时记录了亚硝基谷胱甘肽诱导胸腺细胞凋亡的启动过程,并确定启动时间点;实时记录了不同抑制剂的协作过程,并提示不同的作用机理;实时记录并量化分析了诱导凋亡时细胞质酸化、游离钙离子水平迅速升高和线粒体跨膜电位逐渐丧失的过程,结果揭示了一定的规律性,为凋亡信号转导通路的研究提供了重要线索。
此外,以建立的系统为平台开展生物单分子成像基础研究,优化了实验条件,并发展相关数据分析方法,为活细胞内生物单分子研究打下基础。清晰记录了单个拉直的DNA分子,并为长时间成像优化实验条件;实时记录了单个纳米粒子的运动轨迹,并进行三维追踪,分析其轨迹得到与理论计算吻合的受限布朗运动扩散系数;记录了量子点和荧光分子的荧光共振能量转移现象。
本论文的研究将促进荧光显微成像技术在活细胞生命过程研究的应用。
Real-time detection in single living cells is of great importance in both theory and practical application. Fluorescence microscopic imaging techniques possess high sensitivity and good specificity, as well as easy realization of“visualization”, and become the most potential approaches in this field. However, applicable methods are still facing challenges because of the complexity of living cells. In this thesis, great efforts have been made to establish a real-time multifunctional fluorescence microscopic imaging system with high sensitivity for real-time detection in single living cells and studies on single biomolecules, and to explore its application to specific biological research.
First, a real-time multifunctional fluorescence microscopic imaging system with high sensitivity was set up and relevant image processing methods were developed. An intensified charge-coupled device was employed to realize real-time fluorescence imaging, which was suitable for detecting molecular and ionic distributions and their variations in living cells in real-time with the advantages of high sensitivity, fast acquisition speed and little photodamage of cells. Objective-type total internal reflection fluorescence imaging was introduced into the system to meet the need of tracking single molecules within, on or around cell membranes in real-time, for it greatly improved the vertical resolution, signal to noise ratio in images and sensitivity. To study molecular interactions or molecular conformational changes in living cells, fluorescence resonance energy transfer imaging was realized to break through the restriction of diffraction limit and reach a resolution of nanometer order. Other real-time imaging functions for living cells were also extended, such as fluorescent ratio detection, double-labeling with two- or three-channel detection, fluorescence with differential interference contrast imaging and three-dimensional optical sectioning. Each function mentioned above could be used separately or combined with other functions for complementary advantages, providing multifunctional techniques for biological studies.
Then specific applications of the system were explored, taking studies on thymocyte apoptosis as an example, and relevant quantitative analysis methods were developed. The start-up processes of thymocyte apoptosis induced by S-nitrosoglutathione were recorded in real-time in the level of single living cells for the first time, with the apoptosis initiating time points determined. Real-time studies of the cooperation processes of the inducer with different inhibitors suggested different mechanisms of action. During the apoptosis-inducing course, the decrease of intracellular pH, the rapid increase of intracellular free calcium ion concentration and the gradual loss of mitochondrial membrane potential were also detected in real-time and quantitatively analyzed. The results revealed some regularity, providing valuable clues for apoptotic signaling pathway studies.
Furthermore, some fundamental researches on single biomolecules were carried out based on the system, in which experimental conditions were optimized and relevant data analysis methods were developed, laying a foundation for further application to studies on single biomolecules within living cells. Single stretched DNA molecules were clearly imaged, and experimental conditions for long-term imaging were optimized. The motion trajectories of single nano-scaled particles were recorded in real-time and tracked in three-dimension, and a diffusion coefficient of constrained Brownian motion, which was consistent with theoretical calculation, was obtained by analyzing the trajectories. Fluorescence resonance energy transfer between quantum dots and fluorescent molecules was also detected.
This thesis is supposed to result in, to some extent, promotion of the application of fluorescence microscopic imaging techniques to studies on living cells.
首先,搭建了一套多功能高灵敏实时荧光显微成像系统,并发展相关图像处理方法。利用像增强型CCD实现了实时荧光成像,灵敏度高、速度快、对细胞光损伤小,适合于实时检测活细胞内分子、离子分布和变化;实现了物镜型全内反射荧光成像,提高纵向分辨率、图像信噪比和灵敏度,适合于实时追踪细胞膜附近单分子动态过程;实现了荧光共振能量转移成像,分辨率突破衍射极限达到纳米量级,适合于研究活细胞内分子相互作用或构象变化;扩展了荧光比率、双标记双/三通道、荧光+微分干涉相差、三维层切等多种活细胞实时成像功能。各功能可单独或联合使用,为生命科学研究提供多种先进技术手段。
其次,以胸腺细胞凋亡研究为例探索了该系统的具体应用,并发展相关量化分析方法。首次在单个活细胞水平实时记录了亚硝基谷胱甘肽诱导胸腺细胞凋亡的启动过程,并确定启动时间点;实时记录了不同抑制剂的协作过程,并提示不同的作用机理;实时记录并量化分析了诱导凋亡时细胞质酸化、游离钙离子水平迅速升高和线粒体跨膜电位逐渐丧失的过程,结果揭示了一定的规律性,为凋亡信号转导通路的研究提供了重要线索。
此外,以建立的系统为平台开展生物单分子成像基础研究,优化了实验条件,并发展相关数据分析方法,为活细胞内生物单分子研究打下基础。清晰记录了单个拉直的DNA分子,并为长时间成像优化实验条件;实时记录了单个纳米粒子的运动轨迹,并进行三维追踪,分析其轨迹得到与理论计算吻合的受限布朗运动扩散系数;记录了量子点和荧光分子的荧光共振能量转移现象。
本论文的研究将促进荧光显微成像技术在活细胞生命过程研究的应用。
Real-time detection in single living cells is of great importance in both theory and practical application. Fluorescence microscopic imaging techniques possess high sensitivity and good specificity, as well as easy realization of“visualization”, and become the most potential approaches in this field. However, applicable methods are still facing challenges because of the complexity of living cells. In this thesis, great efforts have been made to establish a real-time multifunctional fluorescence microscopic imaging system with high sensitivity for real-time detection in single living cells and studies on single biomolecules, and to explore its application to specific biological research.
First, a real-time multifunctional fluorescence microscopic imaging system with high sensitivity was set up and relevant image processing methods were developed. An intensified charge-coupled device was employed to realize real-time fluorescence imaging, which was suitable for detecting molecular and ionic distributions and their variations in living cells in real-time with the advantages of high sensitivity, fast acquisition speed and little photodamage of cells. Objective-type total internal reflection fluorescence imaging was introduced into the system to meet the need of tracking single molecules within, on or around cell membranes in real-time, for it greatly improved the vertical resolution, signal to noise ratio in images and sensitivity. To study molecular interactions or molecular conformational changes in living cells, fluorescence resonance energy transfer imaging was realized to break through the restriction of diffraction limit and reach a resolution of nanometer order. Other real-time imaging functions for living cells were also extended, such as fluorescent ratio detection, double-labeling with two- or three-channel detection, fluorescence with differential interference contrast imaging and three-dimensional optical sectioning. Each function mentioned above could be used separately or combined with other functions for complementary advantages, providing multifunctional techniques for biological studies.
Then specific applications of the system were explored, taking studies on thymocyte apoptosis as an example, and relevant quantitative analysis methods were developed. The start-up processes of thymocyte apoptosis induced by S-nitrosoglutathione were recorded in real-time in the level of single living cells for the first time, with the apoptosis initiating time points determined. Real-time studies of the cooperation processes of the inducer with different inhibitors suggested different mechanisms of action. During the apoptosis-inducing course, the decrease of intracellular pH, the rapid increase of intracellular free calcium ion concentration and the gradual loss of mitochondrial membrane potential were also detected in real-time and quantitatively analyzed. The results revealed some regularity, providing valuable clues for apoptotic signaling pathway studies.
Furthermore, some fundamental researches on single biomolecules were carried out based on the system, in which experimental conditions were optimized and relevant data analysis methods were developed, laying a foundation for further application to studies on single biomolecules within living cells. Single stretched DNA molecules were clearly imaged, and experimental conditions for long-term imaging were optimized. The motion trajectories of single nano-scaled particles were recorded in real-time and tracked in three-dimension, and a diffusion coefficient of constrained Brownian motion, which was consistent with theoretical calculation, was obtained by analyzing the trajectories. Fluorescence resonance energy transfer between quantum dots and fluorescent molecules was also detected.
This thesis is supposed to result in, to some extent, promotion of the application of fluorescence microscopic imaging techniques to studies on living cells.
引文
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