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背景:光敏剂亚细胞分布研究多采用激光共聚焦显微镜来开展。激光共聚焦显微成像系统能够获得细节清晰的细胞断层荧光图像,可以进行精确的光敏剂亚细胞定位。但其样品准备过程复杂,费用不菲,而且对于荧光效率较低的光敏剂探测难度很大。目的:应用带像增强器的冷电感耦合装置(intensifiedcharge-coupledde-vice,ICCD)的超高灵敏度荧光显微成像系统及共聚焦显微成像进行光敏剂细胞内分布的对比研究,深入探讨光动力疗法的损伤机制,为拓展其在临床康复治疗领域的适应证奠定实验基础。设计:非随机非对照的实验研究。地点、材料和干预:实验地点为北京理工大学光电工程系实验室。传代培养内皮细胞、食管癌细胞和肺癌细胞,将不同浓度血卟啉单甲醚(hematoporphrinmonomethylether,HMME)与细胞共同孵育不同时间。采用荧光显微镜及ICCD组成的荧光显微成像系统采集不同浓度及不同孵育时间条件下HMME的荧光图像,并采用计算机图像处理技术进行图像增强、滤波后计算其细胞浆与细胞核的平均荧光强度比值。同时应用激光共聚焦显微镜图像采集进行对比。主要观察指标:不同荧光显微成像系统采集到的细胞的光敏剂荧光图像的细节特点及胞浆与胞核区域光强比值的对比观察。结果:HMME浓度为5mg/L时,荧光显微镜采集到HMME的荧光图?
Background: The research of photosensitizer subcellular distribution is mostly carried out by laser confocal microscopy. Confocal laser scanning microscopy imaging system can obtain a clear detail of the cell fluorescence images, can be precise photosensitizer subcellular localization. However, the sample preparation process is complicated and costly, and it is very difficult to detect photosensitizers with lower fluorescence efficiency. OBJECTIVE: To compare the intracellular distribution of photosensitizers by ultra-high-sensitivity fluorescence microscopy with intensifiedcharge-coupledde-vice (ICCD) and confocal microscopy, and to investigate the effects of photodynamic Therapy injury mechanism, to expand its clinical rehabilitation in the field of indications to lay the foundation for the experiment. Design: A Non-randomized, Uncontrolled Experimental Study. Location, Materials and Interventions: The lab is located in the Laboratory of Optoelectronics Engineering of Beijing Institute of Technology. The endothelial cells, esophageal cancer cells and lung cancer cells were subcultured, and different concentrations of hematoporphyrinmonomethylether (HMME) were incubated with cells for different times. Fluorescence microscope and ICCD were used to collect fluorescence images of HMME under different concentrations and different incubation time. The image intensities of HMME were calculated using computer image processing. The average fluorescence intensity ratio of cytoplasm to nucleus was calculated after filtering. At the same time the application of laser confocal microscope image acquisition for comparison. MAIN OUTCOME MEASURES: The details of the fluorescence images of the photosensitizers collected by different fluorescence microscopy imaging systems and the comparison of light intensity ratios between cytoplasm and nucleus. Results: When the concentration of HMME was 5mg / L, the fluorescence spectrum of HMME was collected by fluorescence microscopy.