基于铱配合物磷光探针的活细胞及斑马鱼成像研究

发布时间：2018-08-04 12:28

【摘要】：磷光铱配合物具有发射寿命长、光稳定性好、发射波长易调节和量子效率高等优异的光物理性质,被广泛应用于化学传感和生物成像等领域。共聚焦激光扫描显微镜和时间分辨光学显微镜是生命科学领域先进的光学成像设备。共聚焦显微镜具有优越的光学切片和三维重建能力,可在体内和体外获得高度清晰的图像。基于磷光铱配合物长的发射寿命,可通过时间分辨光学技术,结合发光寿命成像和时间门成像,有效地消除生物体内短寿命自发荧光的干扰,提高检测信噪比。共聚焦激光扫描显微镜和时间分辨光学显微镜在活细胞成像领域已得到很好的发展,但在活体成像领域报道较少。本论文中,我们首先探究了细胞及线粒体内极性变化,其次我们将基于铱配合物的磷光探针注射进斑马鱼体内,实现斑马鱼活体层次的时间分辨光学成像。主要工作有:1、以对极性敏感的线粒体靶向磷光探针为研究对象,通过共聚焦和发光寿命成像显微镜发现,相比于HL-7702正常细胞,磷光探针在HepG2癌细胞中发射光谱蓝移并且发射寿命变长,表明癌细胞极性比正常细胞小,通过发射寿命不同成功区分了正常细胞与癌细胞,这在癌症诊断方面具有良好的应用前景;接着通过发光寿命成像显微镜监测在细胞凋亡过程中线粒体极性的变化,结果表明在细胞凋亡过程中探针在线粒体中发射寿命逐渐变长,说明线粒体极性变小,并且不同细胞状态下发射寿命不同,从而通过发光寿命成像实现了对活细胞、凋亡细胞和死细胞的区分。2、探究水溶性磷光探针的斑马鱼成像实验,通过考察显微注射时斑马鱼麻醉剂的浓度、注射针头粗细、注射位置、成像时斑马鱼摆放位置以及磷光探针注射体积,一系列不同因素对显微注射及成像结果的影响,得到了最佳注射及成像条件,为斑马鱼成像应用奠定了基础。3、以对温度敏感的水溶性磷光探针为研究对象,首先利用共聚焦和发光寿命成像显微镜检测探针在细胞内对温度的响应性,结果表明随着温度升高探针发射寿命增加,基于细胞内良好的温度响应性,我们将磷光探针通过显微注射进斑马鱼体内,在共聚焦成像结果中发现斑马鱼自身强烈的自发荧光严重干扰了结果的准确性,因此我们利用发光寿命成像显微镜将长寿命的磷光探针从短寿命自发荧光中区分出来。更重要的是利用时间分辨成像技术扣除了斑马鱼自身短寿命自发荧光,提高了检测的信噪比,这是首次利用磷光探针实现在斑马鱼体内的温度响应。
[Abstract]:Iridium phosphorescent complexes have been widely used in chemical sensing and biological imaging for their excellent photophysical properties such as long emission lifetime, good optical stability, easy to adjust emission wavelength and high quantum efficiency. Confocal laser scanning microscope and time-resolved optical microscope are the advanced optical imaging equipment in the field of life science. Confocal microscope has excellent optical slice and three-dimensional reconstruction ability, and can obtain high-definition image in vivo and in vitro. Based on the long emission lifetime of iridium phosphorescent complexes, the interference of short-lived autofluorescence can be effectively eliminated and the signal-to-noise ratio (SNR) can be improved by time-resolved optical technology, combined with light-emitting lifetime imaging and time-gate imaging. Confocal laser scanning microscopy and time-resolved optical microscope have been well developed in the field of living cell imaging, but there are few reports in the field of in vivo imaging. In this thesis, we first investigated the changes of intracellular polarity in cells and mitochondria. Secondly, we injected phosphorescence probes based on iridium complexes into zebrafish to achieve time-resolved optical imaging at the living level of zebrafish. The main work was: 1. The polarity sensitive mitochondrial targeted phosphorescence probe was used as the research object. Confocal and luminescent lifetime imaging microscopy showed that compared with the normal HL-7702 cells, The blue shift of emission spectrum and the longer emission lifetime of the phosphorescence probe in HepG2 cancer cells indicate that the polarity of cancer cells is smaller than that of normal cells. It has a good application prospect in cancer diagnosis, and then monitor the changes of mitochondrial polarity during apoptosis by Luminescent Lifetime Imaging microscope. The results showed that the emission lifetime of the probe in mitochondria gradually became longer during apoptosis, which indicated that the polarity of mitochondria became smaller, and the emission lifetime was different in different cell states. The difference between apoptotic cells and dead cells. 2. The zebrafish imaging experiment with water-soluble phosphorescence probe was carried out. The concentration of anesthetic, the thickness of injection needle and the injection location were investigated. The position of zebrafish and the injection volume of phosphorescence probe during imaging. A series of different factors affect the microinjection and imaging results. The optimum conditions of injection and imaging are obtained. This study laid a foundation for zebrafish imaging application. The temperature sensitive water-soluble phosphorescence probe was used as the research object. Firstly, confocal and luminescent lifetime imaging microscopy was used to detect the temperature response of the probe in the cell. The results showed that the fluorescence probe was microinjected into zebrafish due to its good temperature response. In confocal imaging, it was found that the zebrafish's own strong autofluorescence seriously interfered with the accuracy of the results. Therefore, we used the luminescent lifetime imaging microscope to distinguish the long-lived phosphorescence probe from the short-lived autofluorescence. More importantly, the time-resolved imaging technique is used to deduct the short-lived autofluorescence of zebrafish and improve the signal-to-noise ratio. This is the first time to realize the temperature response in zebrafish by using phosphorescence probe.
【学位授予单位】：南京邮电大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q-33;O657.3

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